Contribution to the knowledge of Gongolaria barbata (Sargassaceae, Fucales) from the Mediterranean: insights into infraspecific diversityAbstract
Gongolaria barbata (Sargassaceae, Fucales) is a widespread species for which several infraspecific taxa have been described,
indicating its polymorphism. This study contributes to the understanding of the molecular, nomenclatural, morphological and
ecological aspects of G. barbata in the Mediterranean and sheds light on the infraspecific diversity and its implications for
the taxonomy of this species. Molecular analyses were performed using sequencing of the mitochondrial cox1 gene on both
haptophytic and pleustophytic forms from different sites in the Adriatic and Tyrrhenian Seas. Vegetative and reproductive
morphology was studied on thalli samples from the Adriatic. Our results showed that there are different morphotypes
within G. barbata populations related to specific environmental conditions, suggesting infraspecific variation. In contrast,
molecular analyses showed no differences between samples, regardless of whether individuals are growing “attached” to a
substrate or “unattached”. We also discussed the taxonomic status and nomenclatural issues related to certain infraspecific
taxa previously proposed for G. barbata. In particular, the confusion surrounding Cystoseira aurantia is clarified.
In Vitro Regeneration from Leaf Explants of Helianthus verticillatus, a Critically Endangered SunflowerAbstract
Helianthus verticillatus (Asteraceae), a whorled sunflower, is a perennial species restricted to a few locations in the southeastern United States and is now considered endangered. Therefore, restoring and protecting H. verticillatus as a species is a priority. This study introduces a highly efficient in vitro adventitious plant regeneration system from leaf explants, utilizing five diverse specimens of H. verticillatus, each representing distinct genotypes with phenotypic variations in leaf and stem morphology. Key factors influencing in vitro morphogenesis, including genetic constitution, explant source, and plant growth regulators (PGRs), were identified. The study revealed a remarkably strong genotype-dependent impact on the regeneration efficiency of the investigated H. verticillatus genotypes, ranging from a lack of regeneration to highly effective regeneration. The selection of two genotypes with varying regeneration abilities provides valuable models for genetic analyses, offering insights into factors influencing the regeneration potential of this endangered species. Optimum adventitious shoot regeneration results were achieved using Murashige and Skoog basal media (MS) supplemented with 8.8 µM N6-benzyladenine (BA) and 1.08 µM α-naphthalene acetic acid (NAA). This combination yielded the highest adventitious shoot production. Subsequent successful rooting on ½ MS medium without PGRs further solidified the efficiency of the developed protocol. Regenerated plantlets, demonstrating robust shoots and roots, were successfully acclimatized to greenhouse conditions with a 95% survival rate. The protocol developed in this study is the first such report for this endangered species and is expected to contribute to future genetic manipulation and modification studies.
Smooth muscle–derived adventitial progenitor cells direct atherosclerotic plaque composition complexity in a Klf4-dependent mannerAbstract
We previously established that vascular smooth muscle–derived adventitial progenitor cells (AdvSca1-SM) preferentially differentiate into myofibroblasts and contribute to fibrosis in response to acute vascular injury. However, the role of these progenitor cells in chronic atherosclerosis has not been defined. Using an AdvSca1-SM cell lineage tracing model, scRNA-Seq, flow cytometry, and histological approaches, we confirmed that AdvSca1-SM–derived cells localized throughout the vessel wall and atherosclerotic plaques, where they primarily differentiated into fibroblasts, smooth muscle cells (SMC), or remained in a stem-like state. Krüppel-like factor 4 (Klf4) knockout specifically in AdvSca1-SM cells induced transition to a more collagen-enriched fibroblast phenotype compared with WT mice. Additionally, Klf4 deletion drastically modified the phenotypes of non–AdvSca1-SM–derived cells, resulting in more contractile SMC and atheroprotective macrophages. Functionally, overall plaque burden was not altered with Klf4 deletion, but multiple indices of plaque composition complexity, including necrotic core area, macrophage accumulation, and fibrous cap thickness, were reduced. Collectively, these data support that modulation of AdvSca1-SM cells through KLF4 depletion confers increased protection from the development of potentially unstable atherosclerotic plaques.
Interplay of gut microbiota and host epithelial mitochondrial dysfunction is necessary for the development of spontaneous intestinal inflammation in miceAbstract
Intestinal epithelial cell (IEC) mitochondrial dysfunction involvement in inflammatory bowel diseases (IBD), including Crohn’s disease affecting the small intestine, is emerging in recent studies. As the interface between the self and the gut microbiota, IECs serve as hubs of bidirectional cross-talk between host and luminal microbiota. However, the role of mitochondrial-microbiota interaction in the ileum is largely unexplored. Prohibitin 1 (PHB1), a chaperone protein of the inner mitochondrial membrane required for optimal electron transport chain function, is decreased during IBD. We previously demonstrated that mice deficient in PHB1 specifically in IECs (Phb1i∆IEC) exhibited mitochondrial impairment, Paneth cell defects, gut microbiota dysbiosis, and spontaneous inflammation in the ileum (ileitis). Mice deficient in PHB1 in Paneth cells (epithelial secretory cells of the small intestine; Phb1∆PC) also exhibited mitochondrial impairment, Paneth cell defects, and spontaneous ileitis. Here, we determined whether this phenotype is driven by Phb1 deficiency-associated ileal microbiota alterations or direct effects of loss of PHB1 in host IECs.
Depletion of gut microbiota by broad-spectrum antibiotic treatment in Phb1∆PC or Phb1i∆IEC mice revealed a necessary role of microbiota to cause ileitis. Using germ-free mice colonized with ileal microbiota from Phb1-deficient mice, we show that this microbiota could not independently induce ileitis without host mitochondrial dysfunction. The luminal microbiota phenotype of Phb1i∆IEC mice included a loss of the short-chain fatty acid butyrate. Supplementation of butyrate in Phb1-deficient mice ameliorated Paneth cell abnormalities and ileitis. Phb1-deficient ileal enteroid models suggest deleterious epithelial-intrinsic responses to ileal microbiota that were protected by butyrate.
These results suggest a mutual and essential reinforcing interplay of gut microbiota and host IEC, including Paneth cell, mitochondrial health in influencing ileitis. Restoration of butyrate is a potential therapeutic option in Crohn’s disease patients harboring epithelial cell mitochondrial dysfunction.
Optimization of the 16S rRNA sequencing analysis pipeline for studying in vitro communities of gut commensalsAbstract
While microbial communities inhabit a wide variety of complex natural environments, in vitro culturing enables highly controlled conditions and high-throughput interrogation for generating mechanistic insights. In vitro assemblies of gut commensals have recently been introduced as models for the intestinal microbiota, which plays fundamental roles in host health. However, a protocol for 16S rRNA sequencing and analysis of in vitro samples that optimizes financial cost, time/effort, and accuracy/reproducibility has yet to be established. Here, we systematically identify protocol elements that have significant impact, introduce bias, and/or can be simplified. Our results indicate that community diversity and composition are generally unaffected by substantial protocol streamlining. Additionally, we demonstrate that a strictly aerobic halophile is an effective spike-in for estimating absolute abundances in communities of anaerobic gut commensals. This time- and money-saving protocol should accelerate discovery by increasing 16S rRNA data reliability and comparability and through the incorporation of absolute abundance estimates.
Exploring the diversity of the deep sea—four new species of the amphipod genus Oedicerina described using morphological and molecular methodsAbstract
Collections of the amphipod genus Oedicerina were obtained during six expeditions devoted to the study of deep-sea environments of the Pacific Ocean. The material revealed four species new to science. Two species (Oedicerina henrici sp. nov. and sp. nov.) were found at abyssal depths of the central eastern Pacific in the Clarion-Clipperton Zone; one species (sp. nov.) (Oedicerina claudei sp. nov.) was recovered in the Sea of Okhotsk (north-west Pacific), and one (Oedicerina lesci sp. nov.) in the abyss adjacent to the Kuril-Kamchatka Trench (KKT). The four new species differ from each other and known species by the shapes of the rostrum, coxae 1 and 4, basis of pereopod 7, armatures of pereonite 7, pleonites and urosomites. An identification key for all known species is provided. The study of the cytochrome c oxidase subunit I gene of the four new species and Oedicerina ingolfi collected in the North Atlantic confirmed their genetic distinction. However, small intraspecific variation within each of the studied species was observed. In the case of the new species occurring across the KKT, the same haplotype was found on both sides of the trench, providing evidence that the trench does not constitute an insurmountable barrier for population connectivity. None of the species have so far been found on both sides of the Pacific.
A DNA microarray for the authentication of giant tiger prawn (Penaeus monodon) and whiteleg shrimp (Penaeus (Litopenaeus) vannamei): a proof-of-principleAbstract
This proof-of-principle study describes the development of a rapid and easy-to-use DNA microarray assay for the authentication of giant tiger prawns and whiteleg shrimp. Following DNA extraction and conventional end-point PCR of a 16S rDNA segment, the PCR products are hybridised to species-specific oligonucleotide probes on DNA microarrays located at the bottom of centrifuge tubes (ArrayTubes) and the resulting signal patterns are compared to those of reference specimens. A total of 21 species-specific probes were designed and signal patterns were recorded for 47 crustacean specimens belonging to 16 species of seven families. A hierarchical clustering of the signal patterns demonstrated the specificity of the DNA microarray for the two target species. The DNA microarray can easily be expanded to other important crustaceans. As the complete assay can be performed within half a day and does not require taxonomic expertise, it represents a rapid and simple alternative to tedious DNA barcoding and could be used by crustacean trading companies as well as food control authorities for authentication of crustacean commodities.
Characterization of Emerging Swine Viral Diseases through Oxford Nanopore Sequencing Using Senecavirus A as a ModelAbstract
Emerging viral infectious diseases present a major threat to the global swine industry.
Since 2015, Senecavirus A (SVA) has been identified as a cause of vesicular disease in different
countries and is considered an emerging disease. Despite the growing concern about SVA, there is
a lack of preventive and diagnostic strategies, which is also a problem for all emerging infectious
diseases. Using SVA as a model, we demonstrated that Oxford Nanopore MinION sequencing could
be used as a robust tool for the investigation and surveillance of emerging viral diseases. Our results
identified that MinION sequencing allowed for rapid, unbiased pathogen detection at the species and
strain level for clinical cases. SVA whole genome sequences were generated using both direct RNA
sequencing and PCR-cDNA sequencing methods, with an optimized consensus accuracy of 94% and
99%, respectively. The advantages of direct RNA sequencing lie in its shorter turnaround time, higher
analytical sensitivity and its quantitative relationship between input RNA and output sequencing
reads, while PCR-cDNA sequencing excelled at creating highly accurate sequences. This study
developed whole genome sequencing methods to facilitate the control of SVA and provide a reference
for the timely detection and prevention of other emerging infectious diseases.
Experimental infection reveals transmission of tilapia lake virus (TiLV) from tilapia broodstock to their reproductive organs and fertilized eggsAbstract
Early developmental stages of tilapia, including fertilized eggs were tested positive for TiLV in our previous study (Dong et al., 2017a). We, therefore, hypothesized that infected broodstock is able to pass the virus to their reproductive organs and then to the fertilized eggs. In order to prove this hypothesis, Nile tilapia (Oreochromis niloticus) broodstock were experimentally infected with TiLV by intramuscular injection and non-infected broodstock were used as control group. At day 6 post infection, eggs and semen from each breeding pair were aseptically collected for in vitro fertilization. Fertilized eggs at 3, 12 and 64 h post-fertilization were subjected to detection of TiLV by PCR, ISH, and cell culture. In parallel, blood, serum, liver and reproductive organs from each broodstock were subjected to TiLV analysis. The results revealed that all collected tissues (liver, blood, ovary and testis) from infected broodstock tested positive for TiLV by PCR, ISH, and cell culture. ISH revealed strong positive signals in hepatocytes surrounding blood vessels in the liver, connective tissue and membrane surrounding the oocytes in the ovary and the connective tissue close to blood vessels in the testis. These findings suggested that TiLV causes systemic infection in tilapia broodstock with the virus being able to spread into the reproductive organs, most likely through the blood circulatory system. Subsequently, the fertilized eggs produced by infected broodstock tested positive for TiLV by PCR and ISH revealed location of the virus inside the fertilized eggs. The results of this study suggested that TiLV can be transmitted vertically. We thus recommend for hatchery and multiplication center to use TiLV-tested negative broodstock for the production of TiLV-free tilapia seeds.
A novel 8.7-kb mitochondrial genome deletion accurately detects endometriosis in the plasma of symptomatic womenAbstract
Aim: To evaluate an 8.7-kb mitochondrial DNA (mtDNA) deletion as a potential biomarker of endometriosis. Materials & methods: We tested the diagnostic accuracy of the 8.7-kb deletion real-time PCR assay using 182 prospectively collected blood samples from females presenting with symptoms of endometriosis in a case–control format. Results: The assay differentiated between endometriosis and controls (area under curve: 0.74–0.89) with a statistically significant difference (p < 0.05) in 8.7-kb deletion levels measured for all disease subtypes and stages. No correlation was seen between 8.7-kb deletion levels and participant or specimen age, hormone status or menstrual phase. Conclusion: The diagnostic accuracy of the 8.7-kb deletion for endometriosis suggests potential utility in the clinic to improve patient management.
Endometriosis occurs when endometrial tissue grows outside the uterus and affects approximately 5–10% of women of reproductive age, with infertility, a symptom of this condition, reported in 30–50% of patients [1–8]. Symptoms vary in severity, and include pelvic pain, painful menstrual cramps, discomfort during intercourse and chronic tiredness . This painful condition can significantly impact a patient’s quality of life, resulting in days off work and loss of productivity . Endometriosis is associated with substantial costs and has a comparable economic burden to that of other chronic diseases such as diabetes, Crohn’s disease and rheumatoid arthritis .
Laparoscopic surgery followed by histopathological confirmation is needed to make a definitive diagnosis [5,12], but this is invasive and patients are, understandably, reluctant to go through with the procedure causing further delays in diagnosis and treatment . There is, however, an increasingly important role for a clinical or presumptive diagnosis of disease, which can support the initiation of treatment and management strategies to control symptoms . Ballard et al.  have reported the value of a diagnosis of endometriosis to the patient beyond the availability of treatment options including legitimizing access to social support, excusing absences from work and social events due to symptoms and providing a language with which to communicate about their disease. These can be considered a benefit of both presumptive and surgical diagnoses.
Increasingly, molecular biomarkers are being used in many areas of medicine to detect and manage diseases [16–22]. However, to date, an endometriosis-specific biomarker has not been found that can be used successfully in clinical practice [23–25]. Nonstandard procedures for sample collection and data analysis have hampered researchers’ ability to find such a biomarker. However, recent efforts to harmonize sample collection and storage, analysis methods and the reporting of data, encouraged by the publication of the World Endometriosis Research Foundation EPHect Protocols , have contributed toward the development of disease-specific assays.
In this paper, we describe the investigation of an 8.7-kb mitochondrial DNA (mtDNA) deletion as a potential biomarker for diagnosing endometriosis, including an initial assessment of diagnostic accuracy followed by an evaluation of disease specificity by comparing the biomarker’s frequency in plasma from women with: endometriosis and symptomatic controls, and endometrial cancer, ovarian cancer and breast cancer.
British Red Squirrels Remain the Only Known Wild Rodent Host for Leprosy BacilliAbstract
Eurasian red squirrels (Sciurus vulgaris) in the British Isles are the most recently discovered animal reservoir for the leprosy bacteria Mycobacterium leprae and Mycobacterium lepromatosis. Initial data suggest that prevalence of leprosy infection is variable and often low in different squirrel populations. Nothing is known about the presence of leprosy bacilli in other wild squirrel species despite two others (Siberian chipmunk [Tamias sibiricus], and Thirteen-lined ground squirrel [Ictidomys tridecemlineatus]) having been reported to be susceptible to experimental infection with M. leprae. Rats, a food-source in some countries where human leprosy occurs, have been suggested as potential reservoirs for leprosy bacilli, but no evidence supporting this hypothesis is currently available. We screened 301 squirrel samples covering four species [96 Eurasian red squirrels, 67 Eastern gray squirrels (Sciurus carolinensis), 35 Siberian chipmunks, and 103 Pallas's squirrels (Callosciurus erythraeus)] from Europe and 72 Mexican white-throated woodrats (Neotoma albigula) for the presence of M. leprae and M. lepromatosis using validated PCR protocols. No DNA from leprosy bacilli was detected in any of the samples tested. Given our sample-size, the pathogen should have been detected if the prevalence and/or bacillary load in the populations investigated were similar to those found for British red squirrels.
Temporal escalation of Pyrethroid Resistance in the major malaria vector Anopheles coluzzii from Sahelo-Sudanian Region of northern NigeriaAbstract
Despite the highest global burden of malaria, information on bionomics and insecticide resistance status of malaria vectors is grossly lacking in the densely populated Sahelo-Sudanian region of Nigeria. To support evidence-based vector control we characterised transmission and resistance profiles of Anopheles coluzzii populations from three sites in northern Nigeria. High sporozoite infection (~19.51%) was found in the An. coluzzii populations. A high pyrethroid resistance was observed with only 1% mortality against deltamethrin, a high LD50 (96.57 µg/ml), and a high LT50 (170.27 min, resistance ratio of ~51 compared with the fully susceptible Ngoussou colony). Moderate carbamate resistance was observed. Synergist bioassays significantly recovered deltamethrin susceptibility implicating CYP450s (mortality = 85%, χ2 = 134.04, p < 0.0001) and esterases (mortality = 56%, χ2 = 47.31, p < 0.0001). Reduced bed net efficacy was also observed, with mortalities on exposure to the roof of PermaNet3.0 (PBO + deltamethrin) more than 22 times compared to the side panel (deltamethrin). TaqMan genotyping revealed a high frequency of 1014F kdr mutation (82%) with significant difference in genotype distribution associated with permethrin resistance [OR = 4.69 (CI:1.53–14.35, χ2 = 8.22 p = 0.004]. Sequencing of exons 18–21 of the VGSC led to detection of two additional nonsynonymous mutations, Ile10148Asn and Ser1156Gly. These findings highlight the threats posed by the highly resistant An. coluzzii to malaria control in Nigeria.
Pyrethroid exposure alters internal and cuticle surface bacterial communities in Anopheles albimanusAbstract
A deeper understanding of the mechanisms underlying insecticide resistance is needed to mitigate its threat to malaria vector control. Following previously identified associations between mosquito microbiota and insecticide resistance, we demonstrate for the first time, the effects of pyrethroid exposure on the microbiota of F1 progeny of field-collected Anopheles albimanus. Larval and adult mosquitoes were exposed to the pyrethroids alphacypermethrin (only adults), permethrin, and deltamethrin. While there were no significant differences in bacterial composition between insecticide-resistant and insecticide-susceptible mosquitoes, bacterial composition between insecticide-exposed and non-exposed mosquitoes was significantly different for alphacypermethrin and permethrin exposure. Along with other bacterial taxa not identified to species, Pantoea agglomerans (a known insecticide-degrading bacterial species) and Pseudomonas fragi were more abundant in insecticide-exposed compared to non-exposed adults, demonstrating that insecticide exposure can alter mosquito bacterial communities. We also show for the first time that the cuticle surfaces of both larval and adult An. albimanus harbor more diverse bacterial communities than their internal microbial niches. Together, these findings demonstrate how insecticide pressure could be selecting for certain bacteria within mosquitoes, especially insecticide-metabolizing bacteria, thus potentially contributing to insecticide resistance.
High Plasmodium infection and multiple insecticide resistance in a major malaria vector Anopheles coluzzii from Sahel of Niger RepublicAbstract
Background: Information on insecticide resistance and the mechanisms driving it in the major malaria vectors is
grossly lacking in Niger Republic, thus hindering control eforts. To facilitate evidence-based malaria control, the role
of Anopheles coluzzii population from southern Niger, in malaria transmission, its insecticides resistance profle and the
molecular mechanisms driving the resistance were characterized.
Methods: Blood fed female Anopheles gambiae sensu lato resting indoor were collected at Tessaoua, Niger. Source
of blood was established using PCR and infection with Plasmodium determined using TaqMan assay. Resistance
profle was established with the major public health insecticides, and resistance intensity determined with deltamethrin. Synergist assays were conducted with piperonyl butoxide and diethyl maleate. Presence of L1014F and L1014S
knockdown resistance (kdr) mutations in the voltage-gated sodium channel (VGSC) was investigated using TaqMan
genotyping, and strength of selection pressure acting on the Anopheles populations determined by assessing the
genetic diversity of a fragment spanning exon-20 of the VGSC from alive and dead females.
Results: High human blood index (96%) and high Plasmodium falciparum infection (~13%) was observed in the An.
coluzzii population. Also, a single mosquito was found infected with Plasmodium vivax. High pyrethroid and organochloride resistance was observed with mortalities of less than 20% for deltamethrin, permethrin, α-cypermethrin,
and DDT. A high LD50 (156.65 min) was obtained for deltamethrin, with a resistance ratio of~47.18 compared to
the susceptible Ngoussou colony. Moderate carbamate resistance was observed, and a full susceptibility to organophosphates recorded. Synergist bioassays with piperonyl butoxide and diethyl maleate signifcantly recovered
deltamethrin and DDT susceptibility, respectively implicating CYP450 s (mortality=82%, χ2=84.51, p<0.0001) and
glutathione S-transferases (mortality=58%, χ2=33.96, p<0.001) in resistance. A high frequency of 1014F kdr mutation (82%) was established, with signifcant diference in genotype distribution associated with permethrin resistance
[odds ratio=7.71 (95% CI 2.43–14.53, χ2=13.67, p=0.001]. Sequencing of intron-1 of the voltage-gated sodium
channel (VGSC) revealed a low genetic diversity.
Conclusion: High pyrethroid resistance highlight the challenges to the efectiveness of the pyrethroids-based ITNs
and indoor residual spraying (IRS) against An. coluzzii in Niger. The pyrethroids-synergists LLINs and organophosphatebased IRS maybe the alternatives for malaria control in southern Niger
Community ecology across bacteria, archaea and microbial eukaryotes in the sediment and seawater of coastal Puerto Nuevo, Baja CaliforniaAbstract
Microbial communities control numerous biogeochemical processes critical for ecosystem function and health. Most analyses of coastal microbial communities focus on the characterization of bacteria present in either sediment or seawater, with fewer studies characterizing both sediment and seawater together at a given site, and even fewer studies including information about non-bacterial microbial communities. As a result, knowledge about the ecological patterns of microbial biodiversity across domains and habitats in coastal communities is limited–despite the fact that archaea, bacteria, and microbial eukaryotes are present and known to interact in coastal habitats. To better understand microbial biodiversity patterns in coastal ecosystems, we characterized sediment and seawater microbial communities for three sites along the coastline of Puerto Nuevo, Baja California, Mexico using both 16S and 18S rRNA gene amplicon sequencing. We found that sediment hosted approximately 500-fold more operational taxonomic units (OTUs) for bacteria, archaea, and microbial eukaryotes than seawater (p < 0.001). Distinct phyla were found in sediment versus seawater samples. Of the top ten most abundant classes, Cytophagia (bacterial) and Chromadorea (eukaryal) were specific to the sediment environment, whereas Cyanobacteria and Bacteroidia (bacterial) and Chlorophyceae (eukaryal) were specific to the seawater environment. A total of 47 unique genera were observed to comprise the core taxa community across environment types and sites. No archaeal taxa were observed as part of either the abundant or core taxa. No significant differences were observed for sediment community composition across domains or between sites. For seawater, the bacterial and archaeal community composition was statistically different for the Major Outlet site (p < 0.05), the site closest to a residential area, and the eukaryal community composition was statistically different between all sites (p < 0.05). Our findings highlight the distinct patterns and spatial heterogeneity in microbial communities of a coastal region in Baja California, Mexico.