Quantifying the effect of grilling and roasting on the eating quality of lamb leg musclesAl Moadhen H - 2022
AbstractLamb eating quality was measured using consumers to develop a cut by cook method eating quality program for
sheepmeat. The current protocol for cooking and serving roast lamb legs does not allow for the differentiation of muscles.
Thus, there is no comparison of grilled and roasted lamb leg muscles. Previous work conducted in beef has shown that
there is a difference in consumer sensory scores for grilled and roasted m. semimembranosus (Watson et al. 2008). This
differentiation has allowed the beef industry to predict the eating quality of individual cuts, for each prescribed cooking
method. This study aimed to quantify the effect of cooking method on the eating quality of three different muscles:
m.semimembranosus (topside); m.biceps femoris (outside flat); and m.rectus femoris, vastus lateralis and vastus
intermedius (knuckle). It was hypothesised that the outside flat, topside and knuckle will have different consumer eating
quality when cooked using grill and roast.
Eating quality samples were collected from the 2018 drop of information nucleus flock lambs at the University of
New England as part of larger study. Sixty carcasses were fabricated into consumer sensory samples for the grill and roast
cook methods. The outside, topside and knuckle from one leg were portioned into grill samples, while the opposite leg
was portioned into a single roast consisting of topside and outside, with the knuckle removed. Leg (left/right) was rotated
between carcasses for grill and roast cook methods. Sensory testing was completed using untrained consumer sensory
panels who scored lamb for tenderness (T), juiciness (J), flavour (F) and overall liking (OL) (Watson et al. 2008). Ten
consumers tested each sample. Data were analysed using a linear model in R (R Core Team 2020), with muscle and cook
method as fixed effects, and the interaction between muscle and cook method, to estimate the variance in eating quality
score.
Immune sensing of food allergens promotes aversive behaviourEsther B. Florsheim - 2023
AbstractIn addition to its canonical function in protecting from pathogens, the immune system can also
promote behavioural alterations1–3. The scope and mechanisms of behavioural modifications by
the immune system are not yet well understood. Using a mouse food allergy model, here we show
that allergic sensitization drives antigen-specific behavioural aversion. Allergen ingestion
activates brain areas involved in the response to aversive stimuli, including the nucleus of tractus
solitarius, parabrachial nucleus, and central amygdala. Food aversion requires IgE antibodies and
mast cells but precedes the development of gut allergic inflammation. The ability of allergenspecific IgE and mast cells to promote aversion requires leukotrienes and growth and
differentiation factor 15 (GDF15). In addition to allergen-induced aversion, we find that
lipopolysaccharide-induced inflammation also resulted in IgE-dependent aversive behaviour.
These findings thus point to antigen-specific behavioural modifications that likely evolved to
promote niche selection to avoid unfavourable environments.
Allergies are a class of inflammatory diseases that have increased in prevalence over recent
decades4
. Allergic diseases such as atopic dermatitis, food allergies, asthma, and drug
hypersensitivities seem to be directly linked to industrialization and urban lifestyles5–7
. The
physiological roles for these allergic responses, however, remain enigmatic. Type 2 immunity,
which includes Th2 lymphocytes, IgE antibodies, and innate immune cells (e.g., mast cells,
eosinophils, and type 2 innate lymphoid cells), mediates allergic responses. When chronic or
excessive, allergic responses become detrimental, and potentially lethal8
. Allergic responses
appear to play an important role in host defence against noxious substances, including venoms,
hematophagous fluids, xenobiotics, and irritants9–12. Indeed, a common feature of allergic
responses is the exacerbation of defensive neuronal reflexes like sneezing, itching, and vomiting,
which expel harmful substances from the body13. In addition to these reflexes, avoidance behaviour
was shown to be induced in allergic responses14–16, which suggests that type 2 immunity might
limit exposure to detrimental stimuli, acting as an efficient defence strategy to prevent further
damage. However, the mechanisms by which type 2 responses promote behavioural outputs have
yet to be established.
To examine the impact of allergic sensitization on avoidance behaviour, we sensitized mice
with subcutaneous injections of ovalbumin (OVA) and the adjuvant aluminium hydroxide (alum)
on days 0 and 7 (Fig. 1a). Control mice received alum only. Mice were then acclimatized to home
cages equipped with two lickometers (i.e., spouts that automatically detect licks) connected to
water bottles. During the acclimation period, mice showed no increased preference for any of the
water bottles (Extended Data Fig. 1a, b). We then randomly switched the content of one of the
bottles to an OVA solution. Control mice from both BALB/c and C57BL/6 backgrounds showed
an increased preference for the OVA solution compared to water (Fig 1b and Extended Data Fig.
1c), suggesting that the OVA solution is appetitive for mice. In contrast, sensitized mice showed
a decreased preference to the OVA solution in a dose-dependent manner on the first day of
preference test (Fig. 1b and Extended Data Fig 1d, e). This change in OVA preference by sensitized
mice occurred as early as 10 min after providing the test bottles (Extended Data Fig. 1f). Notably,
aversion to the OVA solution persisted for at least 48 weeks after allergic sensitization (Fig. 1d)
and it was specific to OVA, since control and sensitized mice showed comparable preference to a
solution containing bovine serum albumin (BSA) (Fig. 1e). We next found that the transient
receptor potential cation channel subfamily M member 5 (TRPM5), required for taste transduction
in chemosensory cells17, was dispensable for the development of allergen aversion (Fig. 1f). Oral
sensitization with cholera toxin, a commonly used adjuvant to induce experimental food allergy,
also promoted aversion to OVA (Extended Data Fig. 1g). Together, our data indicate that
immunization towards a protein can generate specific food aversions, which is consistent with
previous observations14,18. Since we found that mice naturally preferred OVA alone, we did not
add sucrose to the OVA solution, as prior studies did, to minimize behavioural and metabolic
effects19. Thus, allergic immunization induces long-lasting and antigen-specific avoidance
behaviour independent on the protein taste.
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Aversive responses to unpleasant stimuli were previously shown to induce brain activation
within the nucleus of tractus solitarius (NTS), lateral external parabrachial nucleus (lePBN), and
central amygdala (CeA)20–22. To determine whether the ingestion of allergens can activate these
brain areas, we orally challenged control and sensitized mice with OVA and 90 minutes later
collected their brains to test for neuronal activation using cFos as a marker (Fig. 1g). We found
that one allergen challenge was enough to induce NTS, lePBN, and CeA activation in sensitized
mice as compared with controls (Fig. 1h, i). There was no difference in cFos staining of the lateral
hypothalamus or the paraventricular nucleus of the hypothalamus (LH and PVN, respectively;
Extended Data Fig. 1h, i). Our results demonstrate that sensitization with OVA leads to
prototypical neuronal activation in the central nervous system. These brain regions correlate with
aversive behaviour toward the sensitized protein and are likely triggered as a defence for limiting
allergen intake.
Increased antigen-specific IgE is a hallmark of allergic sensitization and widely used for
clinical diagnosis of allergies23,24. As total and OVA-specific IgE antibodies are increased in the
circulation two weeks after the first allergen sensitization (Fig. 2a), we examined whether these
antibodies could mitigate the avoidance to allergen using IgE-deficient mice. The sensitization to
OVA in IgE KO mice did not result in decreased preference to OVA solution compared with their
sensitized littermate WT controls (Fig. 2b). Instead, IgE KO mice continued preferring the OVA
solution over water (Fig. 2c). Consistently, sensitized mice that are deficient in the high affinity
receptor for IgE, FceRI, showed similar preference to OVA as the WT unsensitized controls
(Extended Data Fig. 2a). As expected, production of IgE in sensitized FceRI KO was comparable
to that of WT mice (Extended Data Fig. 2b). By using chimeric mice, we found that aversion to
OVA was also dependent on hematopoietic cells expressing FceRI (Fig. 2d). The increased OVA
preference observed in sensitized WT mice reconstituted with FceRI KO hematopoietic cells (Fig.
2e) was likely due to downstream effects of IgE signalling as the serum levels of OVA-specific
IgE were comparable with the WT mice reconstituted with WT hematopoietic cells (Fig. 2f).
Because IL-4 is required for IgE production and the development of type 2 immune
responses25,26, we next tested the role of IL-4 signalling through IL-4Ra in the induction of
avoidance behaviour to OVA. We used a one-bottle assay for seven days to quantify the
consumption of OVA solution when water was not provided. We found that OVA/alum sensitized
IL-4Ra KO mice did not decrease their consumption of OVA (Extended Data Fig. 2c), suggesting
a role for IL-4 signalling in the development of food aversion. Eosinophils are duodenum-resident
cells that have previously been implicated in the initiation of type 2 responses27. To determine the
role of eosinophils in avoidance behaviour, we used eosinophil-deficient mice, Gata1D. These
mice showed decreased consumption of OVA when sensitized (Extended Data Fig. 2c), suggesting
that eosinophils might be dispensable for induction of allergen aversion. We found that sensitized
Gata1D, but not IL-4Ra KO, responded to systemic allergen challenge by inducing anaphylaxis
(Extended Data Fig. 2d) and showed increased levels of IgE antibodies (Extended Data Fig. 2e).
Interestingly, the IgE-dependent induction of allergen aversion was not limited to allergic
sensitization, as mice sensitized with OVA and lipopolysaccharide (LPS) – an established nonavailable under aCC-BY-NC-ND 4.0 International license.
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4
allergic inflammatory stimulus – also showed decreased preference to OVA, which required IgE
(Extended Data Fig. 2f, g). Indeed, OVA/LPS sensitized WT mice had low but increased levels of
IgE (Extended Data Fig. 2h), which might be sufficient to promote avoidance to a conditioned
antigen. Finally, the lack of avoidance behaviour in OVA/alum sensitized IgE-deficient mice was
not due to an intrinsic inability to induce aversion because they showed strong aversion to a bitter
compound, denatonium benzoate (DB) (Extended Data Fig. 2i). These data show that avoidance
behaviour induced by both allergic and non-allergic immunological stimuli depends on IgE
antibodies.
Our findings so far point to a protective role for IgE antibodies. However, these antibodies
are known to be detrimental and drive allergic symptoms upon exposure to food allergens28,29. We
hypothesize that, at early stages, IgE promotes allergic defences such as avoidance behaviour, but
upon chronic exposure to allergens, IgE leads to disease30. To better define the role of IgE on gut
allergic inflammation, we orally challenged mice with OVA 5 times after sensitization as an
experimental model of food allergy (Extended Data Fig. 3a). We challenged mice systemically on
day 14 to induce anaphylaxis. We found that IgE antibodies are not required for the induction of
systemic anaphylaxis (Extended Data Fig. 3b), as reported31. This is likely due to OVA-specific
IgG1 antibodies that are increased by allergic sensitization in an IgE-independent way (Extended
Data Fig. 3c). As previously shown32, we found that IgE is essential to accelerate gut motility
(Extended Data Fig. 3d) and diarrhoea (data not shown) in response to acute allergen exposure in
the gastrointestinal tract. In the intestinal tissue, the accumulation of mast cells in the epithelial
and lamina propria compartments, as well as lamina propria eosinophils, was partially dependent
on IgE in sensitized and orally challenged mice (Extended Data Fig. 3f). These data demonstrate
that IgE endorses pathological processes upon chronic stimulation with allergen, promoting
increased GI peristalsis and inflammatory cellular infiltrates in the allergic small intestines.
Mast cells are major gut-resident immune cells implicated in allergies, anaphylaxis,
inflammatory bowel disease, and abdominal pain32–34. To address the role of mast cells in the
development of avoidance behaviour to allergens, we sensitized RMB mice with OVA/alum. We
depleted mast cells in these mice with 3 injections of diphtheria toxin, as previously described35
prior to the preference test (Fig. 2g). Despite being sensitized with OVA/alum, mast cell depletedRMB mice showed higher consumption of OVA solution (Fig. 2h) and OVA preference (Fig. 2i)
compared with control mice. The systemic levels of IgE antibodies were equally increased in mast
cell-depleted mice compared with sensitized WT (Fig. 2j), suggesting that mast cells can drive
avoidance through IgE sensing of allergens.
Diverse mast-cell derived mediators have been implicated in neuronal excitation in the
gastrointestinal tract during intestinal anaphylaxis13,36. Given the rapid change of behaviour
observed, we hypothesized that the responsible mediators must either be pre-formed or synthesized
de novo after IgE-dependent cross-linking37. Histamine and serotonin, released upon mast cell
degranulation, are two pre-formed mediators with known roles in mediating itch, pain, diarrhoea,
and visceral malaise32,38–42. We tested the role of the histamine receptors H1 and H2 using the
inhibitors loratadine and famotidine. Acute pre-treatment with both drugs did not affect OVA
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preference in control or allergic sensitized mice (Fig. 3a), suggesting that histamine might not
contribute to aversion to food allergens. Similarly, blockade of serotonin synthesis through 5 days
of pre-treatment with parachlorophenylalanine led to only a mild and variable effect on the
aversion (Fig. 3a). We found no effect of pre-treatment with the serotonin receptor 5-HT3
antagonist, ondansetron, as compared to vehicle-treated controls (Extended Data Fig. 4a). Mast
cell-nociceptor circuits are well described in the skin, lung, and gastrointestinal tract and are
proposed to contribute to inflammation and pain perception. Two mediators well-known for these
interactions are substance P and CGRP43,44, which we tested to determine their possible role in
mediating the aversive response. Using pharmacological (substance P receptor inhibitor,
aprepitant) and genetic approaches (substance P KO mice), we found that substance P did not
affect aversive responses to OVA after allergic sensitization (Extended Data Fig. 4 a, b). We also
found that sensitized mice treated with a CGRP receptor inhibitor (BIBN4096) developed aversive
behaviour towards OVA comparable to vehicle-treated mice (Extended Data Fig. 4c). Mast cells
can release proteases that activate submucosal neurons through cleavage of PAR245, however, pretreatment with the PAR2 inhibitory peptide FSLLRY-NH2 had no effect on the aversive response
of sensitized mice (data not shown). So far, our results indicate that histamine, serotonin, substance
P, CGRP, and PAR-2 are not required for aversion to food allergens. It is possible, however, that
we were not able to fully suppress the action of some of these mediators due to timing and dosing
issues.
In addition to pre-formed substances, mast cells produce arachidonic acid-derived lipid
mediators in minutes after IgE-mediated degranulation46–48. Prostaglandins and leukotrienes are
known to be produced by mast cells and have profound effects on behaviour through actions on
nociceptors and vagal neurons49–53. Prostaglandins and leukotrienes are generated by a series of
enzymatic steps controlled by rate-limiting cyclooxygenase (COX) and lipoxygenase (LOX)
enzymes, respectively. Although COX1/2 inhibition with indomethacin had no effect on the
magnitude of the aversive response (Extended Data Fig. 4d), aLOX5 inhibition by pre-treatment
with zileuton significantly increased the preference for OVA in sensitized mice, while not
impacting OVA preference in controls (Fig. 3b). Using organ homogenates from BALB/c mice
sensitized and challenged with intragastric OVA, we determined that Alox5 expression was
transcriptionally induced proximally to distally across the gastrointestinal tract, with the highest
induction found in the duodenum (Fig. 3c), and in the epithelium (Extended Data Fig. 4e). This
pattern of expression was compared to that of intestinal mast cell expansion (Extended Data Fig.
3e and Extended Data Fig. 6b), and detection of Alox5 transcripts was largely lost in the duodenum
of sensitized IgE KO and mast cell-depleted mice (Fig. 3d). Tuft cells are epithelial cells that can
be activated by type 2 inflammation and have the capacity to produce leukotrienes, and this
partially depends on TRPM5 signalling17. Because TRPM5 KO mice did not display a reduced
magnitude of aversion (Fig. 1f), these cells may not be the major source of leukotrienes in this
model. Instead, by analysing a previously published single-cell RNA-seq data set of intestinal
immune cells in sensitized and challenged BALB/c mice54, we found that mast cells and basophils,
but not other immune cells, expressed all the transcriptional machinery necessary for leukotriene
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synthesis (Fig. 3e). Only mast cells appear to express Ltc4s, the enzyme necessary to produce
cysteinyl leukotrienes. Taken together, these data suggest that leukotrienes, likely produced by
gastrointestinal mast cells, drive avoidance behaviour to food allergens.
Leukotrienes are known to mediate unfavourable sensations through actions on dorsal-root
ganglion nociceptors and gut innervating vagal neurons. Indeed, leukotrienes can activate
duodenal vagal neurons through Cysltr2 receptors, which are associated with nausea, and
nonpeptidergic nociceptors can be active via similar mechanisms leading to itch40,49. Following
this logic, we probed a role for gut-innervating vagal neurons by conducting subdiaphragmatic
vagotomy (Fig. 3f). Successful subdiaphragmatic vagotomy was confirmed by injecting fluorogold
intraperitoneally and finding the absence of dye in the dorsal motor nucleus of the vagus (Extended
Data Fig. 4f). However, vagotomy had no significant effect on either the preference to OVA in
control mice or the development of avoidance in sensitized mice (Fig. 3g). We also sought to
examine the role of TRPV1-positive neurons by treating mice with resiniferatoxin (Extended Data
Fig. 4g), a potent capsaicin analogue commonly used to ablate these fibres. We confirmed
successful ablation by lack of behavioural response and temperature drop to i.p.-injected capsaicin
(data not shown). Ablation of these neurons slightly decreased the baseline preference of control
mice to OVA (Extended Data Fig. 4h). Because gustatory sensory fibres lack TRPV1 expression55,
these results suggest that TRPV1+ sensory neurons might control the perceived valence of dietary
proteins. Overall, these findings suggest that vagal neuronal afferents individually may not play a
dominant role in allergen aversion and that instead mast cell-derived signals such as leukotrienes
might be sensed through other pathways.
We next examined whether a humoral pathway is involved in allergen aversion. The area
postrema is a sensory circumventricular organ with renowned roles in mediating nausea in the
context of noxious stimuli56. Growth and differentiation factor 15 (GDF15) is a TGFb superfamily
cytokine produced during conditions of inflammation and cell stress that acts on the area postrema
and NTS by binding to its receptor, GFRAL, to mediate conditioned flavour aversion and
anorexia57,58 . Serum GDF15 levels were, in fact, induced upon intragastric food allergen challenge
(Fig. 4a), and this induction was amplified with the increasing number of challenges (Extended
Data Fig. 5a). Increase in GDF15 depended strongly on the mouse background strain, with
C57BL/6 mice displaying no elevation in GDF15 whereas BALB/c displayed a robust elevation
of approximately 3-fold after 6 challenges (Extended Data Fig. 5b). This induction was entirely
dependent on IgE- and Fcer1a-expressing cells and could be partially prevented by pre-treating
mice with zileuton (aLOX5 inhibitor) prior to each challenge (Fig. 4b, Extended Data Fig. 5c-e),
suggesting that mast cell production of leukotrienes may be involved in GDF15 secretion.
Using organ homogenates from sensitized mice challenged intragastrically with OVA, we
found that GDF15 was induced on the transcriptional level mainly in the duodenum and colon
(Extended Data Fig. 5f). GDF15 induction in these tissues was dependent on IgE antibodies and
mast cells (Extended Data Fig. 5g). Using RNAscope in situ hybridization with probes specific for
GDF15, Fcer1a, and EPCAM, we demonstrated that GDF15 was induced by the colon after
intragastric OVA challenges, with a lower magnitude of induction in the small intestine (Fig. 4c,
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Extended Data Fig. 6a-c). This was surprising, as mast cell expansion occurred in a proximal-todistal pattern across the small and large intestine. In fact, GDF15 transcripts showed little (<1%)
overlap with FceR1a transcripts, and instead colocalized primarily (93%) with EPCAM+ cells
(Extended Data Fig. 6d). These GDF15+ epithelial cells were found to form close contact with
mast cells and were primarily found in the colonic crypt/transit amplifying zone. Interestingly,
similar colonic induction patterns were previously described upon treatment of mice with
metformin59. Hence, food allergen ingestion induces colonic epithelial GDF15 transcription
through IgE and mast cells.
To determine whether GDF15 was able to impact the aversive response to OVA in our
behavioural paradigm, and whether mast cells were necessary for these effects, we sensitized WT
and littermate control RMB mice and depleted mast cells with diphtheria toxin as described (Fig.
2g). Mast cell-depleted mice were then injected with increasing doses of recombinant GDF15
immediately prior to each day of OVA preference testing. We found that GDF15 rescued allergen
aversion in mast cell-depleted mice, consistent with GDF15 being downstream of mast cell
activation (Fig. 4d). This aversive response was dose dependent, leading to a partial aversive
response at 0.01 mg/kg and one comparable with sensitized BALB/c mice at 0.1 mg/kg. The doses
necessary to drive OVA aversion were higher than that induced by intragastric OVA challenge
(Fig. 4 a, b). Thus, we sought to directly test whether GDF15 was necessary for the aversive
response to OVA using anti-GDF15 neutralizing antibody60. We sensitized WT mice with
OVA/alum and treated them 5 h prior to each day of OVA preference testing with isotype control
or anti-GDF15 antibody (Extended Data Fig. 7a). We found GDF15 neutralization had no effect
on the first day of two-bottle preference testing in sensitized mice (data not shown). On day 2 of
the preference test, however, blocking GDF15 led to increased consumption of OVA in allergic
sensitized mice relative to isotype control treatment (Fig. 4e, f). The increase in OVA preference
could not be explained by differences in antibody titters, as the levels of total and OVA-specific
IgE as well as OVA-specific IgG1 were similarly induced in isotype and anti-GDF15 treated
groups (Extended Data Fig. 7b). Neutralization of GDF15 decreased preference to OVA in
sensitized mice within 60 min and lasted throughout the 3 hours of the second trial (Extended Data
Fig. 7c). GDF15 appears to be necessary for allergen aversion, yet it is incapable of driving
aversion alone at concentrations comparable to the allergen challenge,suggesting that other signals
may synergize with GDF15 to promote aversion. Leukotrienes are potential mediators that could
explain this effect. Furthermore, our finding that GDF15 – but not IgE, mast cells, or leukotrienes
– is dispensable for the first day of aversion suggests that leukotrienes may act through an acute
pathway that GDF15 later sustains. Thus, we here describe an unexpected mast cell epithelial
circuit that generates allergen aversion through a leukotriene- and GDF15-dependent mechanism.
Together, these findings demonstrate that immune sensing of allergens leads to the
generation of aversive behaviour. We suggest that avoidance behaviour is a defence strategy aimed
at minimizing harmful effects of exposure to harmful substances, including allergens. Our findings
suggest that detection of allergens by IgE antibodies expands sensory capacity of the nervous
system and provides a mechanism to evaluate the quality of the food and other environmental
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factors. This conclusion adds to a growing body of evidence indicating that the immune detection
of noxious stimuli is an important source of sensory information that drives corresponding
behaviours61. It also adds to the growing evidence of by-directional functional interactions between
the immune and nervous systems62,63.
Neuroimmune cortical organoids overexpressing C4A exhibit multiple schizophrenia endophenotypesMorgan M. Stanton - 2023
AbstractElevated expression of the complement component 4A (C4A) protein has been linked to
an increased risk of schizophrenia (SCZ). However, there are few human models available to
study the mechanisms by which C4A contributes to the development of SCZ. In this study, we
established a C4A overexpressing neuroimmune cortical organoid (NICO) model, which
includes mature neuronal cells, astrocytes, and functional microglia. The C4A NICO model
recapitulated several neuroimmune endophenotypes observed in SCZ patients, including
modulation of inflammatory genes and increased cytokine secretion. C4A expression also
increased microglia-mediated synaptic uptake in the NICO model, supporting the hypothesis that
synapse and brain volume loss in SCZ patients may be due to excessive microglial pruning. Our
results highlight the role of C4A in the immunogenetic risk factors for SCZ and provide a human
model for phenotypic discovery and validation of immunomodulating therapies.
Host cell transcriptomic response to the multidrug-resistant Mycobacterium tuberculosis clonal outbreak Beijing strain reveals its pathogenic featuresPinidphon Prombutara - 2022
AbstractThe upsurge of multidrug-resistant infections has rendered tuberculosis the principal cause of death among infectious diseases. A clonal outbreak multidrug-resistant triggering strain of Mycobacterium tuberculosis was identified in Kanchanaburi Province, labelled “MKR superspreader,” which was found to subsequently spread to other regions, as revealed by prior epidemiological reports in Thailand. Herein, we showed that the MKR displayed a higher growth rate upon infection into host macrophages in comparison with the H37Rv reference strain. To further elucidate MKR’s biology, we utilized RNA-Seq and differential gene expression analyses to identify host factors involved in the intracellular viability of the MKR. A set of host genes function in the cellular response to lipid pathway was found to be uniquely up-regulated in host macrophages infected with the MKR, but not those infected with H37Rv. Within this set of genes, the IL-36 cytokines which regulate host cell cholesterol metabolism and resistance against mycobacteria attracted our interest, as our previous study revealed that the MKR elevated genes associated with cholesterol breakdown during its growth inside host macrophages. Indeed, when comparing macrophages infected with the MKR to H37Rv-infected cells, our RNA-Seq data showed that the expression ratio of IL-36RN, the negative regulator of the IL-36 pathway, to that of IL-36G was greater in macrophages infected with the MKR. Furthermore, the MKR’s intracellular survival and increased intracellular cholesterol level in the MKR-infected macrophages were diminished with decreased IL-36RN expression. Overall, our results indicated that IL-36RN could serve as a new target against this emerging multidrug-resistant M. tuberculosis strain.
Functional integration of a semi-synthetic azido-queuosine derivative into translation and a tRNA modification circuitLarissa Bessler - 2022
AbstractSubstitution of the queuine nucleobase precursor preQ1 by an azide-containing derivative (azido-propyl-preQ1) led to incorporation of this clickable chemical entity into tRNA via transglycosylation in vitro as well as in vivo in Escherichia coli, Schizosaccharomyces pombe and human cells. The resulting semi-synthetic RNA modification, here termed Q-L1, was present in tRNAs on actively translating ribosomes, indicating functional integration into aminoacylation and recruitment to the ribosome. The azide moiety of Q-L1 facilitates analytics via click conjugation of a fluorescent dye, or of biotin for affinity purification. Combining the latter with RNAseq showed that TGT maintained its native tRNA substrate specificity in S. pombe cells. The semi-synthetic tRNA modification Q-L1 was also functional in tRNA maturation, in effectively replacing the natural queuosine in its stimulation of further modification of tRNAAsp with 5-methylcytosine at position 38 by the tRNA methyltransferase Dnmt2 in S. pombe. This is the first demonstrated in vivo integration of a synthetic moiety into an RNA modification circuit, where one RNA modification stimulates another. In summary, the scarcity of queuosinylation sites in cellular RNA, makes our synthetic q/Q system a ‘minimally invasive’ system for placement of a non-natural, clickable nucleobase within the total cellular RNA.
Imp interacts with Lin28 to regulate adult stem cell proliferation in the Drosophila intestinePerinthottathil Sreejith - 2022
AbstractStem cells are essential for the development and long-term maintenance of tissues and organisms. Preserving tissue homeostasis requires exquisite control of all aspects of stem cell function: cell potency, proliferation, fate decision and differentiation. RNA binding proteins (RBPs) are essential components of the regulatory network that control gene expression in stem cells to maintain self-renewal and long-term homeostasis in adult tissues. While the function of many RBPs may have been characterized in various stem cell populations, how these interact and are organized in genetic networks remains largely elusive. In this report, we show that the conserved RNA binding protein IGF2 mRNA binding protein (Imp) is expressed in intestinal stem cells (ISCs) and progenitors in the adult Drosophila midgut. We demonstrate that Imp is required cell autonomously to maintain stem cell proliferative activity under normal epithelial turnover and in response to tissue damage. Mechanistically, we show that Imp cooperates and directly interacts with Lin28, another highly conserved RBP, to regulate ISC proliferation. We found that both proteins bind to and control the InR mRNA, a critical regulator of ISC self-renewal. Altogether, our data suggests that Imp and Lin28 are part of a larger gene regulatory network controlling gene expression in ISCs and required to maintain epithelial homeostasis.
Mitochondrial complex I dysfunction alters the balance of soluble and membrane-bound TNF during chronic experimental colitisAinize Peña-Cearra - 2022
AbstractInflammatory bowel disease (IBD) is a complex, chronic, relapsing and heterogeneous disease induced by environmental, genomic, microbial and immunological factors. MCJ is a mitochondrial protein that regulates the metabolic status of macrophages and their response to translocated bacteria. Previously, an acute murine model of DSS-induced colitis showed increased disease severity due to MCJ deficiency. Unexpectedly, we now show that MCJ-deficient mice have augmented tumor necrosis factor α converting enzyme (TACE) activity in the context of chronic inflammation. This adaptative change likely affects the balance between soluble and transmembrane TNF and supports the association of the soluble form and a milder phenotype. Interestingly, the general shifts in microbial composition previously observed during acute inflammation were absent in the chronic model of inflammation in MCJ-deficient mice. However, the lack of the mitochondrial protein resulted in increased alpha diversity and the reduction in critical microbial members associated with inflammation, such as Ruminococcus gnavus, which could be associated with TACE activity. These results provide evidence of the dynamic metabolic adaptation of the colon tissue to chronic inflammatory changes mediated by the control of mitochondrial function.
Tissue remodeling by an opportunistic pathogen triggers allergic inflammationKaren Agaronyan - 2022
AbstractDifferent effector arms of the immune system are optimized to protect from different classes of pathogens. In some cases, pathogens manipulate the host immune system to promote the wrong type of effector response—a phenomenon known as immune deviation. Typically, immune deviation helps pathogens to avoid destructive immune responses. Here, we report on a type of immune deviation whereby an opportunistic pathogen, Pseudomonas aeruginosa (P. aeruginosa), induces the type 2 immune response resulting in mucin production that is used as an energy source by the pathogen. Specifically, P. aeruginosa-secreted toxin, LasB, processed and activated epithelial amphiregulin to induce type 2 inflammation and mucin production. This “niche remodeling” by P. aeruginosa promoted colonization and, as a by-product, allergic sensitization. Our study thus reveals a type of bacterial immune deviation by increasing nutrient supply. It also uncovers a mechanism of allergic sensitization by a bacterial virulence factor.
Cross-tissue and generation predictability of relative Wolbachia densities in the mosquito Aedes aegyptiAustin J. Mejia - 2022
AbstractBackground
The insect endosymbiotic bacterium Wolbachia is being deployed in field populations of the mosquito Aedes aegypti for biological control. This microbe prevents the replication of human disease-causing viruses inside the vector, including dengue, Zika and chikungunya. Relative Wolbachia densities may in part predict the strength of this ‘viral blocking’ effect. Additionally, Wolbachia densities may affect the strength of the reproductive manipulations it induces, including cytoplasmic incompatibility (CI), maternal inheritance rates or induced fitness effects in the insect host. High rates of CI and maternal inheritance and low rates of fitness effects are also key to the successful spreading of Wolbachia through vector populations and its successful use in biocontrol. The factors that control Wolbachia densities are not completely understood.
Methods
We used quantitative PCR-based methods to estimate relative density of the Wolbachia wAlbB strain in both the somatic and reproductive tissues of adult male and female mosquitoes, as well as in eggs. Using correlation analyses, we assessed whether densities in one tissue predict those in others within the same individual, but also across generations.
Results
We found little relationship among the relative Wolbachia densities of different tissues in the same host. The results also show that there was very little relationship between Wolbachia densities in parents and those in offspring, both in the same and different tissues. The one exception was with ovary–egg relationships, where there was a strong positive association. Relative Wolbachia densities in reproductive tissues were always greater than those in the somatic tissues. Additionally, the densities were consistent in females over their lifetime regardless of tissue, whereas they were generally higher and more variable in males, particularly in the testes.
Conclusions
Our results indicate that either stochastic processes or local tissue-based physiologies are more likely factors dictating Wolbachia densities in Ae. aegypti individuals, rather than shared embryonic environments or heritable genetic effects of the mosquito genome. These findings have implications for understanding how relative Wolbachia densities may evolve and/or be maintained over the long term in Ae. aegypti.
A versatile transposon-based technology to generate loss- and gain-of-function phenotypes in the mouse liverAnna Georgina Kopasz - 2022
AbstractUnderstanding the contribution of gene function in distinct organ systems to the pathogenesis of human diseases in biomedical research requires modifying gene expression through the generation of gain- and loss-of-function phenotypes in model organisms, for instance, the mouse. However, methods to modify both germline and somatic genomes have important limitations that prevent easy, strong, and stable expression of transgenes. For instance, while the liver is remarkably easy to target, nucleic acids introduced to modify the genome of hepatocytes are rapidly lost, or the transgene expression they mediate becomes inhibited due to the action of effector pathways for the elimination of exogenous DNA. Novel methods are required to overcome these challenges, and here we develop a somatic gene delivery technology enabling long-lasting high-level transgene expression in the entire hepatocyte population of mice.
Results
We exploit the fumarylacetoacetate hydrolase (Fah) gene correction-induced regeneration in Fah-deficient livers, to demonstrate that such approach stabilizes luciferase expression more than 5000-fold above the level detected in WT animals, following plasmid DNA introduction complemented by transposon-mediated chromosomal gene transfer. Building on this advancement, we created a versatile technology platform for performing gene function analysis in vivo in the mouse liver. Our technology allows the tag-free expression of proteins of interest and silencing of any arbitrary gene in the mouse genome. This was achieved by applying the HADHA/B endogenous bidirectional promoter capable of driving well-balanced bidirectional expression and by optimizing in vivo intronic artificial microRNA-based gene silencing. We demonstrated the particular usefulness of the technology in cancer research by creating a p53-silenced and hRas G12V-overexpressing tumor model.
Conclusions
We developed a versatile technology platform for in vivo somatic genome editing in the mouse liver, which meets multiple requirements for long-lasting high-level transgene expression. We believe that this technology will contribute to the development of a more accurate new generation of tools for gene function analysis in mice.
Oxidative Stress and X-ray Exposure Levels-Dependent Survival and Metabolic Changes in Murine HSPCsMelis Karabulutoglu - 2021
AbstractHaematopoietic bone marrow cells are amongst the most sensitive to ionizing radiation (IR), initially resulting in cell death or genotoxicity that may later lead to leukaemia development, most frequently Acute Myeloid Leukaemia (AML). The target cells for radiation-induced Acute Myeloid Leukaemia (rAML) are believed to lie in the haematopoietic stem and progenitor cell (HSPC) compartment. Using the inbred strain CBA/Ca as a murine model of rAML, progress has been made in understanding the underlying mechanisms, characterisation of target cell population and responses to IR. Complex regulatory systems maintain haematopoietic homeostasis which may act to modulate the risk of rAML. However, little is currently known about the role of metabolic factors and diet in these regulatory systems and modification of the risk of AML development. This study characterises cellular proliferative and clonogenic potential as well as metabolic changes within murine HSPCs under oxidative stress and X-ray exposure. Ambient oxygen (normoxia; 20.8% O2) levels were found to increase irradiated HSPC-stress, stimulating proliferative activity compared to low oxygen (3% O2) levels. IR exposure has a negative influence on the proliferative capability of HSPCs in a dose-dependent manner (0–2 Gy) and this is more pronounced under a normoxic state. One Gy x-irradiated HSPCs cultured under normoxic conditions displayed a significant increase in oxygen consumption compared to those cultured under low O2 conditions and to unirradiated HSPCs. Furthermore, mitochondrial analyses revealed a significant increase in mitochondrial DNA (mtDNA) content, mitochondrial mass and membrane potential in a dose-dependent manner under normoxic conditions. Our results demonstrate that both IR and normoxia act as stressors for HSPCs, leading to significant metabolic deregulation and mitochondrial dysfunctionality which may affect long term risks such as leukaemia.
Evolution of a novel cell type in Dictyostelia required gene duplication of a cudA-like transcription factorKoryu Kin - 2021
AbstractThe evolution of novel cell types has been proposed to result from duplication of gene regulatory networks, but proven examples are rare. In addition to stalk cells and spores that make up the fruiting bodies of three major groups of Dictyostelia, those in group 4 additionally evolved basal disc and cup cells that respectively anchor the stalk to the substratum and the spore mass to the stalk. We noted a putative group-4-specific duplication of a cudA-like transcription factor (TF) in a comparative analysis of group-representative genomes. Using increased taxon sampling, we here confirmed that this TF, cdl1, duplicated into cdl1a and cdl1b in the common ancestor to group 4. cdl1a, but not cdl1b, showed signatures of positive selection, indicative of functional innovation. Deletion of cdl1a in Dictyostelium discoideum resulted in fruiting bodies with sagging spore heads that lacked the supporting cup cells and expression of cup-specific genes. Deletion of cdl1b resulted in thinner fruiting body stalks, while a cdl1b−cdl1a− double knockout showed more severe stalk defects, suggesting an ancestral role of cdl1 in stalk formation. This was confirmed in a closely related non-group 4 species, Polysphondylium violaceum, where cdl1 knockout caused defective stalk formation. These data indicate that the group-specific duplication of cdl1 and subsequent diversification of cdl1a played a pivotal role in the evolution of a novel somatic cell type in group 4 Dictyostelia.
Effect of Periodized Resistance Training on Skeletal Muscle During Androgen Deprivation Therapy for Prostate Cancer: A Pilot Randomized TrialJacqueline K. Dawson, PhD - 2021
AbstractPurpose: Prostate cancer survivors (PCS) receive androgen deprivation therapy (ADT) as treatment for recurrent cancer, yet ADT is associated with loss of skeletal muscle and physical function. Resistance training can counter both muscle and physical function loss; however, an understanding of the molecular responses of skeletal muscle to resistance training during ADT is still undefined. This sub-analysis of the original randomized, controlled pilot trial investigated effects of 12 weeks of periodized resistance training on mRNA expression of the anabolic genes IGF-1, myogenin, PGC-1α4 and the catabolic genes myostatin and MuRF-1 in skeletal muscle of PCS on ADT. Secondary aims investigated if changes in lean mass and physical function correlated with changes in mRNA expression. Methods: PCS on ADT (n=17) were randomized to 12 weeks of supervised resistance training (EXE, n=9) or home-based stretching (STRETCH, n=8) 3 days per week. Outcomes were assessed at baseline and post-intervention. Muscle biopsies were analyzed by RT-PCR for mRNA expression. Body composition was assessed through dual-energy X-ray absorptiometry, and physical function through muscular strength, timed up and go, stair climb, and 400m walk. Results: MuRF-1 mRNA expression was significantly greater in EXE compared to STRETCH post-intervention (P=.005). Change in MuRF-1 mRNA expression significantly correlated with improvements in strength and physical function (P < .05), while change in IGF-1 expression correlated with change in lean mass (P =.015). Conclusion: Twelve weeks of resistance training increased mRNA expression of MuRF-1 in skeletal muscle of PCS on ADT. Elevations in resting mRNA expression of IGF-1, myogenin and PGC-1α4, and reduction in mRNA expression of myostatin that are typically expected following resistance training were not observed.
ENTRY AND EARLY INFECTION OF NON-SEGMENTED NEGATIVE SENSE RNA VIRUSESJean Mawuena Branttie - 2021
AbstractParamyxoviruses, pneumoviruses, and other non-segmented negative
sense (NNS) RNA viruses have historically been of public health concern.
Although their genomes are typically small (up to 19kbs) they are able to inflict
large-scale detrimental pathologies on host cells. Human metapneumovirus
(HMPV) is a widespread pathogen and is a NNS RNA virus. HMPV results
respiratory tract infections and is particularly dangerous for preterm infants, the
elderly, and immunocompromised individuals. Other viruses within the NNS
RNA virus order include the deadly Ebola, Hendra, and Nipah viruses (EBOV,
HeV, and NiV), as well as the re-emerging measles virus (MeV). Despite their
public impact, there are currently very limited available FDA-approved
therapeutics and antivirals against NNS RNA viruses.
During the infectious cycle, viral surface glycoproteins play critical roles
in establishing infection. For most NNS RNA viruses, the attachment protein is
important for the tethering of a viral membrane to host cells, while the fusion
protein is responsible for the membrane merger of the virus and host. The fusion
protein of paramyxo-and pneumovirus proteins are class I proteins that are
folded into trimers, must be proteolytically cleaved to be functional, and are held
in a metastable prefusion conformation until the signal for fusion occurs. Upon
being signaled, the fusion protein undergoes dramatic essentially irreversible
conformational changes for membrane mixing. Because of its important role in
starting infection, F has garnered interest as a potentially powerful target against
infection. For paramyxoviruses, the ectodomain regions of F have been wellstudied; however, the hydrophobic nature of the transmembrane domain (TMD)
of the protein has resulted in difficulties in crystallization. To address this,
several biochemical assays have been utilized to address the function of the
TMDs of paramyxo-and pneumovirus fusion proteins. Although initially thought
to be solely a membrane anchor, the transmembrane domains of several
viruses have been shown to be important for the functionality of fusion proteins.
For some paramyxoviruses, replacement of the proteinaceous TMD resulted in
the premature triggering. Further studies showed that the TMDs of
paramyxoviruses and several other viral F proteins exist in isolation as trimers,
and these trimeric associations in turn drive trimeric associations of the full
protein. Studies of the HeV F TMD in isolation identified a leucine/isoleucine
(L/I) zipper as an important motif for TMD-TMD trimerization. Mutations to this
L/I zipper motif in the context of the full protein resulted in reduced surface
expression, and a loss of functionality. The L/I zipper was found to be present
in 140 paramyxo- and pneumovirus fusion protein TMDs. This work examines
whether the importance of the wh iimporether L/Izipper in the context of another paramyxvovirus. Weused the model system, PIV5 F to dissect the role of the TMD L/I zipper
fusogenic activity. We found that the (L/I) zipper plays important roles in in expression and
functionality of the PIV5 F protein, but not surface expression of the protein.
Following membrane merging, a series of events occur that facilitate the
release of viral contents into the host cell. The NNS RNA carried by the virus
into the cell is used as a template for viral replication and transcription; two
important steps in generation of viral progeny. In the life cycle of NNS viruses,
viral proteins assume multi-functional roles to optimize their replication and
spread. One of the key players during the course of infection is the matrix
protein (M). The matrix protein has been identified as a master regulator of viral
infection with most studies focusing on its roles in late-stage infection, during
assembly and budding of viral progeny. The matrix proteins of many enveloped
viruses have been shown to associate in high order oligomers to form a gridlike array underneath the plasma membrane, where they can induce
membrane curvature to allow for the budding of viral particles. Not surprisingly,
the absence of M in some NNS RNA viruses results in a significant viral titer
decrease. Interestingly, some recent studies show that the matrix protein has
other critical roles in viral infection such as immune modulation and host cell
translation antagonism. One of these newly uncovered roles for viral matrix
proteins involves the regulation of viral RNA synthesis. Studies with EBOV and
MeV demonstrate that the matrix protein is involved in early infection events,
as inhibits viral replication. To study the roles of the HMPV M protein in early
infection, we performed a spatiotemporal analysis of M in HMPV-infected cells.
We noted the presence of HMPV M within the nucleus during early infection.
Our knockdown studies of HMPV M indicate that HMPV M is a positive
regulator of viral replication and transcription, as in its absence, the rates of
mRNA and viral genomic RNA synthesis are dramatically reduced. Additionally,
within the NNS RNA virus order, HMPV M is the only matrix protein found to
bind calcium. We created alanine mutants to the calcium coordinating residues
of HMPV M and found that these residues were important in properly folding
the protein. Together, these findings contribute to our understanding of the
mechanisms of NNS RNA viral infection
Polycomb-group recruitment to a Drosophila target gene is the default state that is inhibited by a transcriptional activatorElnaz Ghotbi - 2021
AbstractPolycomb-group (PcG) proteins are epigenetic regulators that maintain the transcriptional repression of target
genes following their initial repression by transcription factors. PcG target genes are repressed in some cells, but
active in others. Therefore, a mechanism must exist by which PcG proteins distinguish between the repressed and
active states and only assemble repressive chromatin environments at target genes that are repressed. Here, we
present experimental evidence that the repressed state of a Drosophila PcG target gene, giant (gt), is not identified by the presence of a repressor. Rather, de novo establishment of PcG-mediated silencing at gt is the default
state that is prevented by the presence of an activator or coactivator, which may inhibit the catalytic activity of
Polycomb-repressive complex 2 (PRC2).
Mutations that increase expression of the EmrAB-TolC efflux pump confer increased resistance to nitroxoline in Escherichia coliFabiola Puértolas-Balint - 2020
AbstractObjectives
To determine the mechanism of resistance to the antibiotic nitroxoline in Escherichia coli.
Methods
Spontaneous nitroxoline-resistant mutants were selected at different concentrations of nitroxoline. WGS and strain reconstruction were used to define the genetic basis for the resistance. The mechanistic basis of resistance was determined by quantitative PCR (qPCR) and by overexpression of target genes. Fitness costs of the resistance mutations and cross-resistance to other antibiotics were also determined.
Results
Mutations in the transcriptional repressor emrR conferred low-level resistance to nitroxoline [nitroxoline MIC (MICNOX)=16 mg/L] by increasing the expression of the emrA and emrB genes of the EmrAB-TolC efflux pump. These resistant mutants showed no fitness reduction and displayed cross-resistance to nalidixic acid. Second-step mutants with higher-level resistance (MICNOX=32–64 mg/L) had mutations in the emrR gene, together with either a 50 kb amplification, a mutation in the gene marA, or an IS upstream of the lon gene. The latter mutations resulted in higher-level nitroxoline resistance due to increased expression of the tolC gene, which was confirmed by overexpressing tolC from an inducible plasmid in a low-level resistance mutant. Furthermore, the emrR mutations conferred a small increase in resistance to nitrofurantoin only when combined with an nfsAB double-knockout mutation. However, nitrofurantoin-resistant nfsAB mutants showed no cross-resistance to nitroxoline.
Conclusions
Mutations in different genes causing increased expression of the EmrAB-TolC pump lead to an increased resistance to nitroxoline. The structurally similar antibiotics nitroxoline and nitrofurantoin appear to have different modes of action and resistance mechanisms.
Sphingomyelin-Based Nanosystems (SNs) for the Development of Anticancer miRNA TherapeuticsSurasa Nagachinta - 2020
AbstractGene replacement therapy with oncosuppressor microRNAs (miRNAs) is a promising alternative to interfere with cancer progression. However, miRNAs are highly inefficient in a biological environment, hampering a successful translation to clinics. Nanotechnology can tackle this drawback by providing delivery systems able to efficiently deliver them to cancer cells. Thus, the objective of this work was to develop biocompatible nanosystems based on sphingomyelin (SM) for the intracellular delivery of miRNAs to colorectal cancer cells. We pursued two different approaches to select the most appropriate composition for miRNA delivery. On the one hand, we prepared sphingomyelin-based nanosystems (SNs) that incorporate the cationic lipid stearylamine (ST) to support the association of miRNA by the establishment of electrostatic interactions (SNs–ST). On the other hand, the cationic surfactant (DOTAP) was used to preform lipidic complexes with miRNA (Lpx), which were further encapsulated into SNs (SNs-Lpx). Restitution of miRNA145 levels after transfection with SNs-Lpx was related to the strongest anticancer effect in terms of tumor proliferation, colony forming, and migration capacity assays. Altogether, our results suggest that SNs have the potential for miRNA delivery to develop innovative anticancer therapies.
THROUGH THE LOOKING GLASS: Real-Time Imaging in Brachypodium Roots and Osmotic Stress AnalysisZaemma Khan - 2019
AbstractTo elucidate dynamic developmental processes in plants, live tissues and organs must be visualised frequently and for extended periods. The development of roots is studied at a cellular resolution not only to comprehend the basic processes fundamental to maintenance and pattern formation but also study stress tolerance adaptation in plants. Despite technological advancements, maintaining continuous access to samples and simultaneously preserving their morphological structures and physiological conditions without causing damage presents hindrances in the measurement, visualisation and analyses of growing organs including plant roots. We propose a preliminary system which integrates the optical real-time visualisation through light microscopy with a liquid culture which enables us to image at the tissue and cellular level horizontally growing Brachypodium roots every few minutes and up to 24 h. We describe a simple setup which can be used to track the growth of the root as it grows including the root tip growth and osmotic stress dynamics. We demonstrate the system’s capability to scale down the PEG-mediated osmotic stress analysis and collected data on gene expression under osmotic stress.
α-Lipoic Acid Reduces Iron-induced Toxicity and Oxidative Stress in a Model of Iron OverloadGiuseppina Camiolo - 2019
AbstractIron toxicity is associated with organ injury and has been reported in various clinical
conditions, such as hemochromatosis, thalassemia major, and myelodysplastic syndromes. Therefore, iron chelation therapy represents a pivotal therapy for these patients during their lifetime. The aim of the present study was to assess the iron chelating properties of α-lipoic acid (ALA) and how such an effect impacts on iron overload mediated toxicity. Human mesenchymal stem cells (HS-5) and animals (zebrafish, n = 10 for each group) were treated for 24 h with ferric ammonium citrate (FAC, 120 µg/mL) in the presence or absence of ALA (20 µg/mL). Oxidative stress was evaluated by reduced glutathione content, reactive oxygen species formation, mitochondrial dysfunction, and gene expression of heme oxygenase-1b and mitochondrial superoxide dismutase; organ injury, iron accumulation, and autophagy were measured by microscopical, cytofluorimetric analyses, and inductively coupled plasma-optical mission Spectrometer (ICP-OES). Our results showed that FAC results in a significant increase of tissue iron accumulation, oxidative stress, and autophagy and such detrimental effects were reversed by ALA treatment. In conclusion, ALA possesses excellent iron chelating properties that may be exploited in a clinical setting for organ preservation, as well as exhibiting a good safety profile and low cost for the national health system.
Single-Cell Heterogeneity Analysis and CRISPR Screen Identify Key β-Cell-Specific Disease GenesZhou Fang - 2019
AbstractIdentification of human disease signature genes typically requires samples from many donors to achieve statistical significance. Here, we show that single-cell heterogeneity analysis may overcome this hurdle by significantly improving the test sensitivity. We analyzed the transcriptome of 39,905 single islets cells from 9 donors and observed distinct β cell heterogeneity trajectories associated with obesity or type 2 diabetes (T2D). We therefore developed RePACT, a sensitive single-cell analysis algorithm to identify both common and specific signature genes for obesity and T2D. We mapped both β-cell-specific genes and disease signature genes to the insulin regulatory network identified from a genome-wide CRISPR screen. Our integrative analysis discovered the previously unrecognized roles of the cohesin loading complex and the NuA4/Tip60 histone acetyltransferase complex in regulating insulin transcription and release. Our study demonstrated the power of combining single-cell heterogeneity analysis and functional genomics to dissect the etiology of complex diseases.
Rhodnius prolixus: Identification of missing components of the IMD immune signaling pathway and functional characterization of its role in eliminating bacteriaNicolas Salcedo-Porras - 2019
AbstractThe innate immune system in insects is regulated by specific signalling pathways. Most immune related pathways were identified and characterized in holometabolous insects such as Drosophila melanogaster, and it was assumed they would be highly conserved in all insects. The hemimetabolous insect, Rhodnius prolixus, has served as a model to study basic insect physiology, but also is a major vector of the human parasite, Trypanosoma cruzi, that causes 10,000 deaths annually. The publication of the R. prolixus genome revealed that one of the main immune pathways, the Immune-deficiency pathway (IMD), was incomplete and probably non-functional, an observation shared with other hemimetabolous insects including the pea aphid (Acyrthosiphon pisum) and the bedbug (Cimex lectularius). It was proposed that the IMD pathway is inactive in R. prolixus as an adaptation to prevent eliminating beneficial symbiont gut bacteria. We used bioinformatic analyses based on reciprocal BLAST and HMM-profile searches to find orthologs for most of the “missing” elements of the IMD pathway and provide data that these are regulated in response to infection with Gram-negative bacteria. We used RNAi strategies to demonstrate the role of the IMD pathway in regulating the expression of specific antimicrobial peptides (AMPs) in the fat body of R. prolixus. The data indicate that the IMD pathway is present and active in R. prolixus, which opens up new avenues of research on R. prolixus-T. cruzi interactions.
TCO, a Putative Transcriptional Regulator in Arabidopsis, Is a Target of the Protein Kinase CK2Laina M. Weinman - 2019
AbstractAs multicellular organisms grow, spatial and temporal patterns of gene expression are strictly regulated to ensure that developmental programs are invoked at appropriate stages. In this work, we describe a putative transcriptional regulator in Arabidopsis, TACO LEAF (TCO), whose overexpression results in the ectopic activation of reproductive genes during vegetative growth. Isolated as an activation-tagged allele, tco-1D displays gene misexpression and phenotypic abnormalities, such as curled leaves and early flowering, characteristic of chromatin regulatory mutants. A role for TCO in this mode of transcriptional regulation is further supported by the subnuclear accumulation patterns of TCO protein and genetic interactions between tco-1D and chromatin modifier mutants. The endogenous expression pattern of TCO and gene misregulation in tco loss-of-function mutants indicate that this factor is involved in seed development. We also demonstrate that specific serine residues of TCO protein are targeted by the ubiquitous kinase CK2. Collectively, these results identify TCO as a novel regulator of gene expression whose activity is likely influenced by phosphorylation, as is the case with many chromatin regulators.
Octopus maya white body show sex-specific transcriptomic profiles during the reproductive phase, with high differentiation in signaling pathwaysOscar E. Juarez - 2019
AbstractWhite bodies (WB), multilobulated soft tissue that wraps the optic tracts and optic lobes, have been considered the hematopoietic organ of the cephalopods. Its glandular appearance and its lobular morphology suggest that different parts of the WB may perform different functions, but a detailed functional analysis of the octopus WB is lacking. The aim of this study is to describe the transcriptomic profile of WB to better understand its functions, with emphasis on the difference between sexes during reproductive events. Then, validation via qPCR was performed using different tissues to find out tissue-specific transcripts. High differentiation in signaling pathways was observed in the comparison of female and male transcriptomic profiles. For instance, the expression of genes involved in the androgen receptorsignaling pathway were detected only in males, whereas estrogen receptor showed higher expression in females. Highly expressed genes in males enriched oxidation-reduction and apoptotic processes, which are related to the immune response. On the other hand, expression of genes involved in replicative senescence and the response to cortisol were only detected in females. Moreover, the transcripts with higher expression in females enriched a wide variety of signaling pathways mediated by molecules like neuropeptides, integrins, MAPKs and receptors like TNF and Toll-like. In addition, these putative neuropeptide transcripts, showed higher expression in females’ WB and were not detected in other analyzed tissues. These results suggest that the differentiation in signaling pathways in white bodies of O. maya influences the physiological dimorphism between females and males during the reproductive phase.
Specific sequences of infectious challenge lead to secondary hemophagocytic lymphohistiocytosis-like disease in miceAndrew Wang - 2019
AbstractSecondary hemophagocytic lymphohistiocytosis (sHLH) is a highly mortal complication associated with sepsis. In adults, it is often seen in the setting of infections, especially viral infections, but the mechanisms that underlie pathogenesis are unknown. sHLH is characterized by a hyperinflammatory state and the presence hemophagocytosis. We found that sequential challenging of mice with a nonlethal dose of viral toll-like receptor (TLR) agonist followed by a nonlethal dose of TLR4 agonist, but not other permutations, produced a highly lethal state that recapitulates many aspects of human HLH. We found that this hyperinflammatory response could be recapitulated in vitro in bone marrow-derived macrophages. RNA sequencing analyses revealed dramatic up-regulation of the red-pulp macrophage lineage-defining transcription factor SpiC and its associated transcriptional program, which was also present in bone marrow macrophages sorted from patients with sHLH. Transcriptional profiling also revealed a unique metabolic transcriptional profile in these macrophages, and immunometabolic phenotyping revealed impaired mitochondrial function and oxidative metabolism and a reliance on glycolytic metabolism. Subsequently, we show that therapeutic administration of the glycolysis inhibitor 2-deoxyglucose was sufficient to rescue animals from HLH. Together, these data identify a potential mechanism for the pathogenesis of sHLH and a potentially useful therapeutic strategy for its treatment.
Impact of malaria and hepatitis B co-infection on clinical and cytokine profiles among pregnant womenNsoh Godwin Anabire - 2019
AbstractBackground
The overlap of malaria and chronic hepatitis B (CHB) is common in endemic regions, however, it is not known if this co-infection could adversely influence clinical and immunological responses. This study investigated these interactions in pregnant women reporting to antenatal clinics in Ghana.
Methods
Clinical parameters (hemoglobin, liver function biomarker, peripheral malaria parasitemia, and hepatitis B viremia) and cytokine profiles were assayed and compared across four categories of pregnant women: un-infected, mono-infected with Plasmodium falciparum (Malaria group), mono-infected with chronic hepatitis B virus (CHB group) and co-infected (Malaria+CHB group).
Results
Women with Malaria+CHB maintained appreciably normal hemoglobin levels (mean±SEM = 10.3±0.3 g/dL). That notwithstanding, Liver function test showed significantly elevated levels of alanine aminotransferase, aspartate aminotransferase and total bilirubin [P<0.001 for all comparisons]. Similarly, the Malaria+CHB group had significantly elevated pro-inflammatory cytokines, including tumour necrosis factor alpha (TNF-α), interleukin (IL)-1β, and IL-6 [P<0.05 for all comparisons]. In women with Malaria+CHB, correlation analysis showed significant negative association of the pro-inflammatory cytokines responses with malaria parasitemia [IL-1β (P<0.001; r = -0.645), IL-6 (P = 0.046; r = -0.394) and IL-12 (P = 0.011; r = -0.49)]. On the other hand, the pro-inflammatory cytokine levels positively correlated with HBV viremia [TNF-α (P = 0.004; r = 0.549), IL-1β (P<0.001; r = 0.920), IL-6 (P<0.001; r = 0.777), IFN-γ (P = 0.002; r = 0.579), IL-2 (P = 0.008; r = 0.512) and IL-12 (P<0.001; r = 0.655)]. Also, for women in the Malaria+CHB group, parasitemia was observed to diminish HBV viremia [P = 0.003, r = -0.489].
Conclusion
Put together the findings suggests that Malaria+CHB could exacerbate inflammatory cytokine responses and increase susceptibility to liver injury among pregnant women in endemic settings.
Selection for novel metabolic capabilities in Salmonella entericaOmar Warsi - 2019
AbstractBacteria are known to display extensive metabolic diversity and many studies have shown that they can use an extensive repertoire of small molecules as carbon‐ and energy sources. However, it is less clear to what extent a bacterium can expand its existing metabolic capabilities by acquiring mutations that, for example, rewire its metabolic pathways. To investigate this capability and potential for evolution of novel phenotypes, we sampled large populations of mutagenized Salmonella enterica to select very rare mutants that can grow on minimal media containing 124 low molecular weight compounds as sole carbon sources. We found mutants growing on 18 of these novel carbon sources, and identified the causal mutations that allowed growth for four of them. Mutations that relieve physiological constraints or increase expression of existing pathways were found to be important contributors to the novel phenotypes. For the remaining 14 novel phenotypes, whole genome sequencing of independent mutants and genetic analysis suggested that these novel metabolic phenotypes result from a combination of multiple mutations. This work, by virtue of identifying the genetic and mechanistic basis for new metabolic capabilities, sheds light on the properties of adaptive landscapes underlying the evolution of novel phenotypes.
Bacterial diet and weak cadmium stress affect the survivability of Caenorhabditis elegans and its resistance to severe stressRamona Dolling - 2019
AbstractStress may have negative or positive effects in dependence of its intensity (hormesis). We studied this phenomenon in Caenorhabditis elegans by applying weak or severe abiotic (cadmium, CdCl2) and/or biotic stress (different bacterial diets) during cultivation/breeding of the worms and determining their developmental speed or survival and performing transcriptome profiling and RT-qPCR analyses to explore the genetic basis of the detected phenotypic differences. To specify weak or severe stress, developmental speed was measured at different cadmium concentrations, and survival assays were carried out on different bacterial species as feed for the worms. These studies showed that 0.1 μmol/L or 10 mmol/L of CdCl2 were weak or severe abiotic stressors, and that E. coli HT115 or Chitinophaga arvensicola feeding can be considered as weak or severe biotic stress. Extensive phenotypic studies on wild type (WT) and different signaling mutants (e.g., kgb-1Δ and pmk-1Δ) and genetic studies on WT revealed, inter alia, the following results. WT worms bred on E. coli OP50, which is a known cause of high lipid levels in the worms, showed high resistance to severe abiotic stress and elevated gene expression for protein biosynthesis. WT worms bred under weak biotic stress (E. coli HT115 feeding which causes lower lipid levels) showed an elevated resistance to severe biotic stress, elevated gene expression for the innate immune response and signaling but reduced gene expression for protein biosynthesis. WT worms bred under weak biotic and abiotic stress (E. coli HT115 feeding plus 0.1 μmol/L of CdCl2) showed high resistance to severe biotic stress, elevated expression of DAF-16 target genes (e.g., genes for small heat shock proteins) but further reduced gene expression for protein biosynthesis. WT worms bred under weak biotic but higher abiotic stress (E. coli HT115 feeding plus 10 μmol/L of CdCl2) showed re-intensified gene expression for the innate immune response, signaling, and protein biosynthesis, which, however, did not caused a higher resistance to severe biotic stress. E. coli OP50 feeding as well as weak abiotic and biotic stress during incubations also improved the age-specific survival probability of adult WT worms. Thus, this study showed that a bacterial diet resulting in higher levels of energy resources in the worms (E. coli OP50 feeding) or weak abiotic and biotic stress promote the resistance to severe abiotic or biotic stress and the age-specific survival probability of WT.
Progesterone decreases gut permeability through upregulating occludin expression in primary human gut tissues and Caco-2 cellsZejun Zhou - 2019
AbstractProgesterone plays a protective role in preventing inflammation and preterm delivery during pregnancy. However, the mechanism involved is unknown. Microbial product translocation from a permeable mucosa is demonstrated as a driver of inflammation. To study the mechanism of the protective role of progesterone during pregnancy, we investigated the effect of physiologic concentrations of progesterone on tight junction protein occludin expression and human gut permeability in vitro and systemic microbial translocation in pregnant women in vivo. Plasma bacterial lipopolysaccharide (LPS), a representative marker of in vivo systemic microbial translocation was measured. We found that plasma LPS levels were significantly decreased during 24 to 28 weeks of gestation compared to 8 to 12 weeks of gestation. Moreover, plasma LPS levels were negatively correlated with plasma progesterone levels but positively correlated with plasma tumor necrosis factor-alpha (TNF-α) levels at 8 to 12 weeks of gestation but not at 24 to 28 weeks of gestation. Progesterone treatment increased intestinal trans-epithelial electrical resistance (TEER) in primary human colon tissues and Caco-2 cells in vitro through upregulating tight junction protein occludin expression. Furthermore, progesterone exhibited an inhibitory effect on nuclear factor kappa B (NF-κB) activation following LPS stimulation in Caco-2 cells. These results reveal a novel mechanism that progesterone may play an important role in decreasing mucosal permeability, systemic microbial translocation, and inflammation during pregnancy.
Chronic liver injury drives non‐traditional intrahepatic fibrin(ogen) crosslinking via tissue transglutaminaseL. G. Poole - 2019
AbstractBackground
Intravascular fibrin clots and extravascular fibrin deposits are often implicated in the progression of liver fibrosis. However, evidence supporting a pathological role of fibrin in hepatic fibrosis is indirect and based largely on studies using anticoagulant drugs that inhibit activation of the coagulation protease thrombin, which has other downstream targets that promote fibrosis. Therefore, the goal of this study was to determine the precise role of fibrin deposits in experimental hepatic fibrosis.
Methods
Liver fibrosis was induced in mice expressing mutant fibrinogen insensitive to thrombin‐mediated proteolysis (i.e. locked in the monomeric form), termed FibAEK mice, and factor XIII A2 subunit‐deficient (FXIII−/−) mice. Female wild‐type mice, FXIII−/− mice and homozygous FibAEK mice were challenged with carbon tetrachloride (CCl4) twice weekly for 4 weeks or 6 weeks (1 mL kg−1, intraperitoneal).
Results
Hepatic injury and fibrosis induced by CCl4 challenge were unaffected by FXIII deficiency or inhibition of thrombin‐catalyzed fibrin polymer formation (in FibAEK mice). Surprisingly, hepatic deposition of crosslinked fibrin(ogen) was not reduced in CCl4‐challenged FXIII−/− mice or FibAEK mice as compared with wild‐type mice. Rather, deposition of crosslinked hepatic fibrin(ogen) following CCl4 challenge was dramatically reduced in tissue transglutaminase‐2 (TGM2)‐deficient (TGM2−/−) mice. However, the reduction in crosslinked fibrin(ogen) in TGM2−/− mice did not affect CCl4‐induced liver fibrosis.
Conclusions
These results indicate that neither traditional fibrin clots, formed by the thrombin–activated FXIII pathway nor atypical TGM2‐crosslinked fibrin(ogen) contribute to experimental CCl4‐induced liver fibrosis. Collectively, the results indicate that liver fibrosis occurs independently of intrahepatic fibrin(ogen) deposition.
MiRNA-27a sensitizes breast cancer cells to treatment with Selective Estrogen Receptor ModulatorsBojan Ljepoja - 2019
AbstractBackground
MicroRNA-27a (miR-27a) is a small non-coding RNA, shown to play a role in multiple cancers, including the regulation of ERα expression in breast cancer. Most ERα positive tumors are treated with Selective Estrogen Receptor Modulators (SERMs) and thus the role of miR-27a expression in response to SERM treatment is of interest.
Methods
Tamoxifen resistant cells were generated by molecular evolution with six cycles of tamoxifen treatment. MCF7 and T47D luminal A breast cancer cell lines were either treated with miR-27a mimics, or ER-signaling was modulated ectopically. The changes were analyzed with RT-qPCR, western blotting and transcriptional activity ERE-reporter assays. Moreover, the response to SERM treatments (tamoxifen, endoxifen and toremifen) was investigated by cell viability and apoptosis measurements. An in silico analysis of survival data from the METABRIC study was performed in order to assess the prognostic value of miR-27a for response to SERM treatment.
Results
Tamoxifen-resistant cells showed decreased expression of ERα and miR-27a. The overexpression of miR-27a increased the levels of ERα, while modulation of ERα decreased miR-27a expression. High miR-27a expression increased the sensitivity of MCF7 and T47D cells to SERM treatments and re-sensitized the cells to tamoxifen. Patient survival of luminal A breast cancer patients that underwent endocrine therapies was better in groups with high miR-27a expression.
Conclusion
MiR-27a sensitizes luminal A breast cancer cells to SERM treatments based on a positive feedback loop with ERα. An increased overall-survival of ER-positive breast cancer patients that underwent endocrine treatments and displayed high miR-27a levels was found.
Presence of Circulating miR-145, miR-155, and miR-382 in Exosomes Isolated from Serum of Breast Cancer Patients and Healthy DonorsVianey Gonzalez-Villasana - 2019
AbstractmiR-145, miR-155, and miR-382 have been proposed as noninvasive biomarkers to distinguish breast cancer patients from healthy individuals. However, it is unknown if these three miRNAs are secreted by exosomes. Thus, we hypothesized that miR-145, miR-155, and miR-382 in breast cancer patients are present in exosomes. We isolated exosomes from serum of breast cancer patients and healthy donors, then we characterized them according to their shape, size, and exosome markers by scanning electron microscopy, atomic force microscopy, nanoparticle tracking analysis (NTA), and Western blot and determined the exosome concentration in all samples by NTA. Later, exosomal small RNA extraction was done to determine the expression levels of miR-145, miR-155, and miR-382 by qRT-PCR. We observed a round shape of exosomes with a mean size of 119.84 nm in breast cancer patients and 115.4 nm in healthy donors. All exosomes present the proteins CD63, Alix, Tsg, CD9, and CD81 commonly used as markers. Moreover, we found a significantly high concentration of exosomes in breast cancer patients with stages I, III, and IV compared to healthy donors. We detected miR-145, miR-155, and miR-382 in the exosomes isolated from serum of breast cancer patients and healthy donors. Our results show that the exosomes isolated from the serum of breast cancer patients and healthy donors contains miR-145, miR-155, and miR-382 but not in a selective manner in breast cancer patients. Moreover, our data support the association between exosome concentration and the presence of breast cancer, opening the possibility to study how miRNAs packaged into exosomes play a role in BC progression.
Quantitative PCR Measurement of miR-371a-3p and miR-372-p Is Influenced by HemolysisMette Pernille Myklebust - 2019
AbstractCell-free microRNAs have been reported as biomarkers for several diseases. For testicular germ cell tumors (GCT), circulating microRNAs 371a-3p and 372-3p in serum and plasma have been proposed as biomarkers for diagnostic and disease monitoring purposes. The most widely used method for quantification of specific microRNAs in serum and plasma is reverse transcriptase real-time quantitative PCR (RT-qPCR) by the comparative Ct-method. In this method one or several reference genes or reference microRNAs are needed in order to normalize and calculate the relative microRNA levels across samples. One of the pitfalls in analysis of microRNAs from serum and plasma is the release of microRNAs from blood cells during hemolysis. This is an important issue because varying degrees of hemolysis are not uncommon in routine blood sampling. Thus, hemolysis must be taken into consideration when working with circulating microRNAs from blood. miR-93-5p, miR-30b-5p, and miR-20a-5p have been reported as reference microRNA in analysis of the miR-371a-373 cluster. We here show how these three microRNAs are influenced by hemolysis. We also propose a new reference microRNA, miR-191-5p, which is relatively stable in serum samples with mild hemolysis. In addition, we show how hemolysis can have effect on the reported microRNA levels in patient samples when these reference microRNAs are used in samples with varying levels of hemolysis.
On resolving ambiguities in microbial community analysis of partial nitritation anammox reactorsLaura Orschler - 2019
AbstractPCR-based methods have caused a surge for integration of eco-physiological approaches into research on partial nitritation anammox (PNA). However, a lack of rigorous standards for molecular analyses resulted in widespread data misinterpretation and consequently lack of consensus. Data consistency and accuracy strongly depend on the primer selection and data interpretation. An in-silico evaluation of 16S rRNA gene eubacterial primers used in PNA studies from the last ten years unraveled the difficulty of comparing ecological data from different studies due to a variation in the coverage of these primers. Our 16S amplicon sequencing approach, which includes parallel sequencing of six 16S rRNA hypervariable regions, showed that there is no perfect hypervariable region for PNA microbial communities. Using qPCR analysis, we emphasize the significance of primer choice for quantification and caution with data interpretation. We also provide a framework for PCR based analyses that will improve and assist to objectively interpret and compare such results.
Metformin induces the AP-1 transcription factor network in normal dermal fibroblastsZoe E. Gillespie - 2019
AbstractMetformin is a widely-used treatment for type 2 diabetes and is reported to extend health and lifespan as a caloric restriction (CR) mimetic. Although the benefits of metformin are well documented, the impact of this compound on the function and organization of the genome in normal tissues is unclear. To explore this impact, primary human fibroblasts were treated in culture with metformin resulting in a significant decrease in cell proliferation without evidence of cell death. Furthermore, metformin induced repositioning of chromosomes 10 and 18 within the nuclear volume indicating altered genome organization. Transcriptome analyses from RNA sequencing datasets revealed that alteration in growth profiles and chromosome positioning occurred concomitantly with changes in gene expression profiles. We further identified that different concentrations of metformin induced different transcript profiles; however, significant enrichment in the activator protein 1 (AP-1) transcription factor network was common between the different treatments. Comparative analyses revealed that metformin induced divergent changes in the transcriptome than that of rapamycin, another proposed mimetic of CR. Promoter analysis and chromatin immunoprecipitation assays of genes that changed expression in response to metformin revealed enrichment of the transcriptional regulator forkhead box O3a (FOXO3a) in normal human fibroblasts, but not of the predicted serum response factor (SRF). Therefore, we have demonstrated that metformin has significant impacts on genome organization and function in normal human fibroblasts, different from those of rapamycin, with FOXO3a likely playing a role in this response.
Cytoprotective effects of Avenathramide C against oxidative and inflammatory stress in normal human dermal fibroblastsChenxuan Wang - 2019
AbstractNatural polyphenols are promising anti-aging compounds not only for their antioxidant activity, but also their ability to activate specific cellular pathways mediating the aging process. Avenanthramide C (Avn C), found exclusively in oats, is a natural antioxidant associated with free radical scavenging; however, it is how this compound elicits other protective effects. We investigated the intracellular antioxidant activity of Avn C and other cytoprotective potential in normal human skin fibroblasts exposed to extracellular stress. Avn C reduced H2O2-induced oxidative stress by reducing intracellular free radical levels and antioxidant gene transcripts. Avn C also resulted in decreased levels of gene transcripts encoding pro-inflammatory cytokines in response to H2O2 or tumor necrosis factor-α (TNF-α). This reduction in cytokine gene transcription occurred concomitantly with reduced phosphorylated nuclear factor-κB (NF-κB) p65, and decreased NF-κB DNA binding. Avn C further induced heme oxygense-1 (HO-1) expression through increased Nrf2 DNA binding activity, demonstrating a second mechanism by which Avn C attenuates cellular stress. Collectively, our findings indicate that Avn C protects normal human skin fibroblasts against oxidative stress and inflammatory response through NF-κB inhibition and Nrf2/HO-1 activation
Three Types of Functional Regulatory T Cells Control T Cell Responses at the Human Maternal-Fetal InterfaceMaria Salvany-Celades - 2019
AbstractDuring pregnancy, maternal regulatory T cells (Tregs) are important in establishing immune tolerance to invading fetal extravillous trophoblasts (EVTs). CD25HIFOXP3+ Tregs are found at high levels in decidual tissues and have been shown to suppress fetus-specific and nonspecific responses. However, limited data are available on additional decidual Treg types and the mechanisms by which they are induced. This study investigated three distinct decidual CD4+ Treg types in healthy pregnancies with a regulatory phenotype and the ability to suppress T cell responses: CD25HIFOXP3+, PD1HIIL-10+, and TIGIT+FOXP3dim. Moreover, co-culture of HLA-G+ EVTs or decidual macrophages with blood CD4+ T cells directly increased the proportions of CD25HIFOXP3+ Tregs compared to T cells cultured alone. EVTs also increased PD1HI Tregs that could be inhibited by HLA-C and CD3 antibodies, suggesting an antigen-specific induction. The presence of distinct Treg types may allow for the modulation of a variety of inflammatory responses in the placenta.
Surmounting Cytarabine-resistance in acute myeloblastic leukemia cells and specimens with a synergistic combination of hydroxyurea and azidothymidineMay Levin - 2019
AbstractAcute myeloid leukemia (AML) patients display dismal prognosis due to high prevalence of refractory and relapsed disease resulting from chemoresistance. Treatment protocols, primarily based on the anchor drug Cytarabine, remained chiefly unchanged in the past 50 years with no standardized salvage regimens. Herein we aimed at exploring potential pre-clinical treatment strategies to surmount Cytarabine resistance in human AML cells. We established Cytarabine-resistant sublines derived from human leukemia K562 and Kasumi cells, and characterized the expression of Cytarabine-related genes using real-time PCR and Western blot analyses to uncover the mechanisms underlying their Cytarabine resistance. This was followed by growth inhibition assays and isobologram analyses testing the sublines’ sensitivity to the clinically approved drugs hydroxyurea (HU) and azidothymidine (AZT), compared to their parental cells. All Cytarabine-resistant sublines lost deoxycytidine kinase (dCK) expression, rendering them refractory to Cytarabine. Loss of dCK function involved dCK gene deletions and/or a novel frameshift mutation leading to dCK transcript degradation via nonsense-mediated decay. Cytarabine-resistant sublines displayed hypersensitivity to HU and AZT compared to parental cells; HU and AZT combinations exhibited a marked synergistic growth inhibition effect on leukemic cells, which was intensified upon acquisition of Cytarabine-resistance. In contrast, HU and AZT combination showed an antagonistic effect in non-malignant cells. Finally, HU and AZT synergism was demonstrated on peripheral blood specimens from AML patients. These findings identify a promising HU and AZT combination for the possible future treatment of relapsed and refractory AML, while sparing normal tissues from untoward toxicity.
CYLD Regulates Centriolar Satellites Proteostasis by Counteracting the E3 Ligase MIB1Tiphaine Douanne - 2019
AbstractThe tumor suppressor CYLD is a deubiquitinatingenzyme that removes non-degradative ubiquitin link-ages bound to a variety of signal transduction adap-tors. CYLD participates in the formation of primarycilia, a microtubule-based structure that protrudesfrom the cell body to act as a ‘‘sensing antenna.’’Yet, how exactly CYLD regulates ciliogenesis is notfully understood. Here, we conducted an unbiasedproteomic screen of CYLD binding partners andidentified components of the centriolar satellites.These small granular structures, tethered to the scaf-fold protein pericentriolar matrix protein 1 (PCM1),gravitate toward the centrosome and orchestrateciliogenesis. CYLD knockdown promotes PCM1degradation and the subsequent dismantling of thecentriolar satellites. We found that CYLD marshalsthe centriolar satellites by deubiquitinating andpreventing the E3 ligase Mindbomb 1 (MIB1) frommarking PCM1 for proteasomal degradation. Theseresults link CYLD to the regulation of centriolar satel-lites proteostasis and provide insight into howreversible ubiquitination finely tunes ciliogenesis.
Disruption of Intestinal Homeostasis and Intestinal Microbiota During Experimental Autoimmune UveitisCathleen Janowitz - 2019
AbstractPurpose: We determine the changes in intestinal microbiota and/or disruptions in intestinal homeostasis during uveitis.
Methods: Experimental autoimmune uveitis (EAU) was induced in B10.RIII mice with coadministration of interphotoreceptor retinoid-binding protein peptide (IRBP) and killed mycobacterial antigen (MTB) as an adjuvant. Using 16S rRNA gene sequencing, we looked at intestinal microbial differences during the course of uveitis, as well as intestinal morphologic changes, changes in intestinal permeability by FITC-dextran leakage, antimicrobial peptide expression in the gastrointstinal tract, and T lymphocyte prevalence before and at peak intraocular inflammation.
Results: We demonstrate that increased intestinal permeability and antimicrobial peptide expression in the intestinal tract coincide in timing with increased effector T cells in the mesenteric lymph nodes, during the early stages of uveitis, before peak inflammation. Morphologic changes in the intestine were most prominent during this phase, but also occurred with adjuvant MTB alone, whereas increased intestinal permeability was found only in IRBP-immunized mice that develop uveitis. We also demonstrate that the intestinal microbiota were altered during the course of uveitis, and that some of these changes are specific to uveitic animals, whereas others are influenced by adjuvant MTB alone. Intestinal permeability peaked at 2 weeks, coincident with an increase in intestinal bacterial strain differences, peak lipocalin production, and peak uveitis.
Conclusions: An intestinal dysbiosis accompanies a disruption in intestinal homeostasis in autoimmune uveitis, although adjuvant MTB alone promotes intestinal disruption as well. This may indicate a novel axis for future therapeutic targeting experimentally or clinically.
Tart Cherry Prevents Bone Loss through Inhibition of RANKL in TNF-Overexpressing MiceNicholas Moon - 2019
AbstractCurrent drugs for the treatment of rheumatoid arthritis-associated bone loss come with concerns about their continued use. Thus, it is necessary to identify natural products with similar effects, but with fewer or no side effects. We determined whether tart cherry (TC) could be used as a supplement to prevent inflammation-mediated bone loss in tumor necrosis factor (TNF)-overexpressing transgenic (TG) mice. TG mice were assigned to a 0%, 5%, or 10% TC diet, with a group receiving infliximab as a positive control. Age-matched wild-type (WT) littermates fed a 0% TC diet were used as a normal control. Mice were monitored by measurement of body weight. Bone health was evaluated via serum biomarkers, microcomputed tomography (µCT), molecular assessments, and mechanical testing. TC prevented TNF-mediated weight loss, while it did not suppress elevated levels of interleukin (IL)-1β and IL-6. TC also protected bone structure from inflammation-induced bone loss with a reduced ratio of receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) to a degree comparable to infliximab. Furthermore, unlike with infliximab, TC exhibited a moderate improvement in TNF-mediated decline in bone stiffness. Thus, TC could be used as a prophylactic regimen against future fragility fractures in the context of highly chronic inflammation.
Insulin-like Growth Factor (IGF)-1 treatment stabilizes the microvascularcytoskeleton under ischemic conditionsShameena Bake - 2019
AbstractOur previous studies showed that Insulin-like Growth Factor (IGF)-1 reduced blood brain barrier permeabilityand decreased infarct volume caused by middle cerebral artery occlusion (MCAo) in middle aged female rats.Similarly, cultures of primary brain microvessel endothelial cells from middle-aged female rats and exposed tostroke-like conditions (oxygen glucose deprivation; OGD) confirmed that IGF-1 reduced dye transfer across thiscell monolayer. Surprisingly, IGF-1 did not attenuate endothelial cell death caused by OGD. To reconcile thesefindings, the present study tested the hypothesis that, at the earliest phase of ischemia, IGF-1 promotes barrierfunction by increasing anchorage and stabilizing cell geometry of surviving endothelial cells. Cultures of humanbrain microvessel endothelial cells were subject to oxygen-glucose deprivation (OGD) in the presence of IGF-1,IGF-1 + JB-1 (IGFR inhibitor) or vehicle. OGD disrupted the cell monolayer and reduced cell-cell interactions,which was preserved in IGF-1-treated cultures and reversed by concurrent treatment with JB-1. IGF-1-mediatedpreservation of the endothelial monolayer was reversed with LY294002 treatment, but not by Rapamycin, in-dicating that IGF-1 s actions on cell-cell contacts are likely mediated via the PI3K pathway. In vivo, microvesselmorphology was evaluated in middle-aged female rats that were subjected to ischemia by MCAo, and treated ICVwith IGFeI, IGF-1 + JB-1, or artificial CSF (aCSF; vehicle) after reperfusion. Compared to vehicle controls, IGF-1treated animals displayed larger microvessel diameters in the peri-infarct area and increased staining density forvinculin, an anchorage protein. Both these measures were reversed by concurrent IGF-1 + JB-1 treatment.Moreover these effects were restricted to 24 h after ischemia-reperfusion and no treatment effects were seen at5d post stroke. Collectively, these data suggest that in the earliest hours during ischemia, IGF-1 promotes re-ceptor-mediated anchorage of endothelial cells, and its actions may be accurately characterized as vasculo-protective.
High diagnostic yield and novel variants in very late-onset spasticityMomen Almomen - 2019
AbstractHereditary spastic paraplegias (HSPs) are a diverse group of genetic conditions with variable severity
and onset age. From a neurogenetic clinic, we identified 14 patients with very late-onset HSP, with
symptoms starting after the age of 35. In this cohort, sequencing of known genetic causes was performed using clinically available HSP sequencing panels. We identified 4 patients with mutations in
SPG7 and 3 patients with SPAST mutations, representing 50% of the cohort and indicating a very high
diagnostic yield. In the SPG7 group, we identified novel variants in two patients. We have also identified two novel mutations in the SPAST group. We present sequencing data from cDNA and RT-qPCR to
support the pathogenicity of these variants, and provide observations regarding the poor genotypephenotype correlation in these conditions that should be the subject of future study
Three-Dimensional Printed Polylactic Acid Scaffolds Promote Bonelike Matrix Deposition in VitroRayan Fairag - 2019
AbstractLarge bone defects represent a significant challenge for
clinicians and surgeons. Tissue engineering for bone regeneration represents
an innovative solution for this dilemma and may yield attractive alternate bone
substitutes. Three-dimensional (3D) printing with inexpensive desktop printers
shows promise in generating high-resolution structures mimicking native
tissues using biocompatible, biodegradable, and cost-effective thermoplastics,
which are already FDA-approved for food use, drug delivery, and many medical
devices. Microporous 3D-printed polylactic acid scaffolds, with different pore
sizes (500, 750, and 1000 μm), were designed and manufactured using an
inexpensive desktop 3D printer, and the mechanical properties were assessed.
The scaffolds were compared for cell growth, activity, and bone-like tissue
formation using primary human osteoblasts. Osteoblasts showed high
proliferation, metabolic activity, and osteogenic matrix protein production, in
which 750 μm pore-size scaffolds showed superiority. Further experimentation
using human mesenchymal stem cells on 750 μm pore scaffolds showed their ability in supporting osteogenic differentiation.
These findings suggest that even in the absence of any surface modifications, low-cost 750 μm pore-size 3D-printed scaffolds
may be suitable as a bone substitute for repair of large bone defects.
Biallelic CCM3 mutations cause a clonogenic survival advantage and endothelial cell stiffeningKonrad Schwefel - 2019
AbstractCCM3, originally described as PDCD10, regulates blood‐brain barrier integrity and vascular maturation in vivo. CCM3 loss‐of‐function variants predispose to cerebral cavernous malformations (CCM). Using CRISPR/Cas9 genome editing, we here present a model which mimics complete CCM3 inactivation in cavernous endothelial cells (ECs) of heterozygous mutation carriers. Notably, we established a viral‐ and plasmid‐free crRNA:tracrRNA:Cas9 ribonucleoprotein approach to introduce homozygous or compound heterozygous loss‐of‐function CCM3 variants into human ECs and studied the molecular and functional effects of long‐term CCM3 inactivation. Induction of apoptosis, sprouting, migration, network and spheroid formation were significantly impaired upon prolonged CCM3 deficiency. Real‐time deformability cytometry demonstrated that loss of CCM3 induces profound changes in cell morphology and mechanics: CCM3‐deficient ECs have an increased cell area and elastic modulus. Small RNA profiling disclosed that CCM3 modulates the expression of miRNAs that are associated with endothelial ageing. In conclusion, the use of CRISPR/Cas9 genome editing provides new insight into the consequences of long‐term CCM3 inactivation in human ECs and supports the hypothesis that clonal expansion of CCM3‐deficient dysfunctional ECs contributes to CCM formation.