Fecal Microbial Diversity of Coyotes and Wild Hogs in Texas Panhandle, USABabafela Awosile - 2023
AbstractThe ecology of infectious diseases involves wildlife, yet the wildlife interface is often neglected and understudied. Pathogens related to infectious diseases are often maintained within wildlife populations and can spread to livestock and humans. In this study, we explored the fecal microbiome of coyotes and wild hogs in the Texas panhandle using polymerase chain reactions and 16S sequencing methods. The fecal microbiota of coyotes was dominated by members of the phyla Bacteroidetes, Firmicutes, and Proteobacteria. At the genus taxonomic level, Odoribacter, Allobaculum, Coprobacillus, and Alloprevotella were the dominant genera of the core fecal microbiota of coyotes. While for wild hogs, the fecal microbiota was dominated by bacterial members of the phyla Bacteroidetes, Spirochaetes, Firmicutes, and Proteobacteria. Five genera, Treponema, Prevotella, Alloprevotella, Vampirovibrio, and Sphaerochaeta, constitute the most abundant genera of the core microbiota of wild hogs in this study. Functional profile of the microbiota of coyotes and wild hogs identified 13 and 17 human-related diseases that were statistically associated with the fecal microbiota, respectively (p < 0.05). Our study is a unique investigation of the microbiota using free-living wildlife in the Texas Panhandle and contributes to awareness of the role played by gastrointestinal microbiota of wild canids and hogs in infectious disease reservoir and transmission risk. This report will contribute to the lacking information on coyote and wild hog microbial communities by providing insights into their composition and ecology which may likely be different from those of captive species or domesticated animals. This study will contribute to baseline knowledge for future studies on wildlife gut microbiomes.
Foodborne Pathogens in Leafy Vegetables Grown and Consumed Locally in Yaounde, Cameroon: A Public Health ConcernMary Nkongho Tanyitiku - 2023
AbstractThis study sought to understand the health risks of foodborne pathogens in fresh leafy vegetables that are grown and consumed locally in Yaounde, Cameroon. Through a survey, 200 respondents were recruited to relate possible food-related illnesses to leafy vegetable consumption. Additionally, a total of 168 vegetable samples consisting of six leafy vegetables and 15 irrigated water samples from five water sources were collected from farms and local markets for microbiological analysis. Using a high-fidelity DNA polymerase, five potential bacterial pathogens, namely, Shiga-toxin producing Escherichia coli (STEC), Campylobacter spp., Salmonella spp., Listeria monocytogenes and Yersinia enterocolitica were also examined. The mean counts of total viable count and total coliforms followed decreasing trends from vegetables obtained on the farms to the local markets, and these ranged from 4.98-8.74 log cfu/g and 1.77-7.42 log cfu/g respectively. All pathogens detected were of significant concern to public health showing high occurrence in some vegetables: STEC (20%) and Yersinia enterolitica (13%) in cabbage, Campylobacter spp. (21%) in lettuce, Listeria monocytogenes (15%) in African nightshade, and Salmonella spp. (15%) in amaranth. Importantly, 42% of respondents highlighted that they frequently got sick from eating leafy vegetables from the study area. These microbiological and qualitative results along with certain vegetable farming and vending practices (such as the use of untreated sewage water for crop irrigation, the sales of physically dirty, muddy, and unpackaged vegetables) indicated that foodborne diseases could be occurring among leafy vegetable-consuming populations in Cameroon.
Segmentation strategy of de novo designed four-helical bundles expands protein oligomerization modalities for cell regulationEstera Merljak - 2023
AbstractProtein–protein interactions govern most biological processes. New protein assemblies can be introduced through the fusion of selected proteins with di/oligomerization domains, which interact specifically with their partners but not with other cellular proteins. While four-helical bundle proteins (4HB) have typically been assembled from two segments, each comprising two helices, here we show that they can be efficiently segmented in various ways, expanding the number of combinations generated from a single 4HB. We implement a segmentation strategy of 4HB to design two-, three-, or four-chain combinations for the recruitment of multiple protein components. Different segmentations provide new insight into the role of individual helices for 4HB assembly. We evaluate 4HB segmentations for potential use in mammalian cells for the reconstitution of a protein reporter, transcriptional activation, and inducible 4HB assembly. Furthermore, the implementation of trimerization is demonstrated as a modular chimeric antigen receptor for the recognition of multiple cancer antigens.
Pilot study of a comprehensive resource estimation method from environmental DNA using universal D-loop amplification primersKazutoshi Yoshitake - 2023
AbstractMany studies have investigated the ability of environmental DNA (eDNA) to identify the species. However, when individual species are to be identified, accurate estimation of their abundance using traditional eDNA analyses is still difficult. We previously developed a novel analytical method called HaCeD-Seq (haplotype count from eDNA by sequencing), which focuses on the mitochondrial D-loop sequence for eels and tuna. In this study, universal D-loop primers were designed to enable the comprehensive detection of multiple fish species by a single sequence. To sequence the full-length D-loop with high accuracy, we performed nanopore sequencing with unique molecular identifiers (UMI). In addition, to determine the D-loop reference sequence, whole genome sequencing was performed with thin coverage, and complete mitochondrial genomes were determined. We developed a UMI-based Nanopore D-loop sequencing analysis pipeline and released it as open-source software. We detected 5 out of 15 species (33%) and 10 haplotypes out of 35 individuals (29%) among the detected species. This study demonstrates the possibility of comprehensively obtaining information related to population size from eDNA. In the future, this method can be used to improve the accuracy of fish resource estimation, which is currently highly dependent on fishing catches.
Essential Oils Reduce Grey Mould Rot of Apples and Modify the Fruit Microbiome during Postharvest StorageGiada Schiavon - 2022
AbstractBotrytis cinerea is the causal agent of grey mould rot of apples. The efficacy of biofumigation with thyme (Thymus vulgaris), savoury (Satureja montana), and basil (Ocimum basilicum) essential oils (EOs) at 1%, 0.5%, and 0.1% concentrations were tested against B. cinerea. In vitro, the results showed 100% growth inhibition at 1% concentration for all oils. Subsequent biofumigation experiments on apples of cultivar ‘Opal’ with 1% EOs showed that, after 60 d storage, thyme and savoury EOs significantly reduced grey mould rot incidence (average incidence 2% for both treatments) compared to the control (7%). Analyses of quality indicated slightly higher fruit firmness for 1% thyme at 30 d and slightly higher titratable acidity for 1% thyme and savoury at 60 d. Sampling of the atmosphere inside the cabinets was performed to characterize and quantify the volatile components of EOs released through biofumigation. Though thymol and p-cymene were the main components of thyme EO, the antimicrobial activity was mainly due to the presence of thymol and, to a lower extent, of carvacrol. In savoury EO, carvacrol and p-cymene were the main components, whereas in basil EO, linalool and estragole were mainly present. Metabarcoding analyses showed that the epiphytic microbiome had higher richness and evenness compared to their endophytic counterpart. By the end of shelf-life, treatments with thyme EO reduced B. cinerea abundance compared to the inoculated control for both endophytes (from 36.5% to 1.5%) and epiphytes (from 7.0% to 0.7%), while favouring a significant increase in Penicillium species both in endophytes (from 0.2% to 21.5%) and epiphytes (from 0.5% to 18.6%). Results indicate that thyme EO (1%) and savoury EO (1%) are equally effective in hampering grey mould rot development in vivo.
Simple and reliable in situ CRISPR-Cas9 nuclease visualization tool is ensuring efficient editing in Streptomyces speciesAlen Pšeničnik - 2022
AbstractCRISPR-Cas9 technology has emerged as a promising tool for genetic engineering of Streptomyces strains. However, in practice, numerous technical hurdles have yet to be overcome when developing robust editing procedures. Here, we developed an extension of the CRISPR-Cas toolbox, a simple and reliable cas9 monitoring tool with transcriptional fusion of cas9 nuclease to a beta glucuronidase (gusA) visual reporter gene. The Cas9-SD-GusA tool enables in situ identification of cells expressing Cas9 nuclease following the introduction of the plasmid carrying the CRISPR-Cas9 machinery. Remarkably, when the Cas9-SD-GusA system was applied under optimal conditions, 100% of the colonies displaying GusA activity carried the target genotype. In contrast, it was shown that the cas9 sequence had undergone major recombination events in the colonies that did not exhibit GusA activity, giving rise to “escaper colonies” carrying unedited genotype. Our approach allows a simple detection of “escaper” phenotype and serves as an efficient CRISPR-Cas9 optimisation tool.
Genomic Characterisation of Canis Familiaris Papillomavirus Type 24, a Novel Papillomavirus Associated with Extensive Pigmented Plaque Formation in a Pug DogJohn S. Munday - 2022
AbstractNumerous large dark plaques developed over the ventrum, legs and head of a 9-year-old pug dog over a 4-year-period. Histology confirmed a diagnosis of viral pigmented plaque and a short section of a novel papillomavirus (PV) type was amplified using consensus PCR primers. Taking advantage of the circular nature of PV DNA, ‘outward facing’ PCR primers allowed amplification of the full sequence. As this is the 24th PV known to infect dogs, the novel PV was designated canine papillomavirus (CPV) type 24. The CPV24 genome contained putative coding regions for 5 early proteins and 2 late ones. The CPV24 open reading frame L1 showed the highest (78.2%) similarity to CPV4 and phylogenetic analysis showed that CPV24 clustered with CPV4 and CPV16 suggesting CPV24 is the third species 2 Chipapillomavirus type identified in dogs. This is the third report of extensive pigmented plaques covering a high proportion of the skin. Both previous cases were caused CPV4 and, considering the high genetic similarity between CPV4 and CP24, infection by these CPV types may predispose to more severe clinical disease. In addition, as plaques caused by CPV16 appear more likely to progress to neoplasia, the detection of a species 2 Chipapillomavirus within a pigmented plaque may indicate the potential for more severe disease.
Essential Oils Reduce Grey Mould Rot of Apples and Modify the Fruit Microbiome during Postharvest StorageGiada Schiavon - 2023
AbstractBotrytis cinerea is the causal agent of grey mould rot of apples. The efficacy of biofumigation
with thyme (Thymus vulgaris), savoury (Satureja montana), and basil (Ocimum basilicum) essential oils
(EOs) at 1%, 0.5%, and 0.1% concentrations were tested against B. cinerea. In vitro, the results showed
100% growth inhibition at 1% concentration for all oils. Subsequent biofumigation experiments
on apples of cultivar ‘Opal’ with 1% EOs showed that, after 60 d storage, thyme and savoury EOs
significantly reduced grey mould rot incidence (average incidence 2% for both treatments) compared
to the control (7%). Analyses of quality indicated slightly higher fruit firmness for 1% thyme at 30 d
and slightly higher titratable acidity for 1% thyme and savoury at 60 d. Sampling of the atmosphere
inside the cabinets was performed to characterize and quantify the volatile components of EOs
released through biofumigation. Though thymol and p-cymene were the main components of thyme
EO, the antimicrobial activity was mainly due to the presence of thymol and, to a lower extent, of
carvacrol. In savoury EO, carvacrol and p-cymene were the main components, whereas in basil
EO, linalool and estragole were mainly present. Metabarcoding analyses showed that the epiphytic
microbiome had higher richness and evenness compared to their endophytic counterpart. By the end
of shelf-life, treatments with thyme EO reduced B. cinerea abundance compared to the inoculated
control for both endophytes (from 36.5% to 1.5%) and epiphytes (from 7.0% to 0.7%), while favouring
a significant increase in Penicillium species both in endophytes (from 0.2% to 21.5%) and epiphytes
(from 0.5% to 18.6%). Results indicate that thyme EO (1%) and savoury EO (1%) are equally effective
in hampering grey mould rot development in vivo.
Fine-scale spatial variation shape fecal microbiome diversity and composition in black- tailed prairie dogs (Cynomys ludovicianus)Sufia A. Neha - 2022
AbstractBackground
Host associated gut microbiota are important in understanding the coevolution of host-microbe, it’s causes
and consequences that may help wildlife population to adapt to its rapid climatic changes. Mammalian
gut microbiota composition and diversity may be affected by a variety of factors including geographic
variation, seasonal variation in diet, habitat disturbance, environmental conditions, age, and sex. However,
there have been few studies that have examined how ecological and environmental factors influence gut
microbiota composition in animals' natural environments. In this study, we explore how host habitat,
geographical location and environmental factors affect the fecal microbiota of Cynomys ludovicianus at a
small spatial scale. We collected fecal samples from five geographically distinct locations in Texas
Panhandle occupying habitat classified as urban and rural areas using high throughput 16S rRNA gene
amplicon sequencing.
Results
The results showed that microbiota of fecal samples was largely dominated by phylum Bacteroidetes.
Fecal microbiome diversity and composition differed significantly across sampling sites and habitats.
Prairie dogs inhabiting urban areas showed reduced fecal diversity due to more homogenous environment
and anthropogenic disturbance. Urban prairie dog colonies displayed greater phylogenetic variation than
those in rural habitats. Differentially abundant analysis revealed that bacterial species pathogenic to
humans and animals were highly abundant in urban areas which indicates that host health and fitness
might be negatively affected. Random forest model identified Alistipes shahii as the important species
driving the changes in fecal microbiome composition. Despite the effects of habitat and geographic
location of host, we found a strong correlation with environmental factors- average maximum temperature
was the best predictor of prairie dog fecal microbial diversity.
Conclusions
Our findings suggest that reduction in alpha diversity in conjunction with greater dispersion in beta
diversity could be indicative of declining host health in urban areas which could help determine in future
conservation efforts. Moreover, several bacterial species pathogenic to humans and other animals were
highly abundant in prairie dog colonies near urban areas, which may in turn adversely affect host
phenotype and fitness.
A Non-Destructive High-Speed Procedure to Obtain DNA Barcodes from Soft-Bodied Insect Samples with a Focus on the Dipteran Section of SchizophoraFrederik Stein - 2022
AbstractWhile the need for biodiversity research is growing, paradoxically, global taxonomical expertise is decreasing as a result of the neglected funding for young academics in taxonomy. Non-destructive approaches for DNA barcoding are necessary for a more efficient use of this dwindling expertise to fill gaps, and identify incorrect entries in sequence databases like BOLD or GenBank. They are efficient because morphological re-examination of species vouchers is still possible post-DNA barcoding. Non-destructive approaches for Diptera with a comprehensive species representation or the consideration of diagnostic fragile morphological characters are missing. Additionally, most non-destructive approaches combine a time intensive and non-destructive digestion step with common DNA extraction methods, such as commercial kits or CTAB DNA isolation. We circumvented those approaches and combined a modified non-destructive TE buffer high-speed DNA extraction, with a PCR inhibitor-resistant PCR reaction system, to a non-destructive DNA barcoding procedure for fresh and frozen samples of the Schizophora (Diptera). This method avoids morphological impairment and the application of harmful chemicals, is cost and time effective, restricts the need for laboratory equipment to a minimum, and prevents cross-contamination risk during DNA isolation. Moreover, the study indicates that the presented non-destructive DNA barcoding procedure is transferable to other soft-bodied insects. We suggest that PCR inhibitor-resistant master mixes enable the development of new—and the modification of existing—non-destructive approaches with the avoidance of further DNA template cleaning.
A Non-Destructive High-Speed Procedure to Obtain DNA Barcodes from Soft-Bodied Insect Samples with a Focus on the Dipteran Section of SchizophoraFrederick Stein - 2022
AbstractWhile the need for biodiversity research is growing, paradoxically, global taxonomical expertise is decreasing as a result of the neglected funding for young academics in taxonomy. Non-destructive approaches for DNA barcoding are necessary for a more efficient use of this dwindling expertise to fill gaps, and identify incorrect entries in sequence databases like BOLD or GenBank. They are efficient because morphological re-examination of species vouchers is still possible post-DNA barcoding. Non-destructive approaches for Diptera with a comprehensive species representation or the consideration of diagnostic fragile morphological characters are missing. Additionally, most non-destructive approaches combine a time intensive and non-destructive digestion step with common DNA extraction methods, such as commercial kits or CTAB DNA isolation. We circumvented those approaches and combined a modified non-destructive TE buffer high-speed DNA extraction, with a PCR inhibitor-resistant PCR reaction system, to a non-destructive DNA barcoding procedure for fresh and frozen samples of the Schizophora (Diptera). This method avoids morphological impairment and the application of harmful chemicals, is cost and time effective, restricts the need for laboratory equipment to a minimum, and prevents cross-contamination risk during DNA isolation. Moreover, the study indicates that the presented non-destructive DNA barcoding procedure is transferable to other soft-bodied insects. We suggest that PCR inhibitor-resistant master mixes enable the development of new—and the modification of existing—non-destructive approaches with the avoidance of further DNA template cleaning.
A diverse group of underappreciated zygnematophytes deserves in-depth explorationAnna Busch - 2022
AbstractThe conjugating green algae (Zygnematophyceae) are the closest relatives of land plants and hence are of great evolutionary interest. Besides the popular placoderm desmids and the filamentous species, there is an underappreciated diversity of unicellular zygnematophytes with a much “simpler” morphology and smooth cell walls – traditionally referred to as “saccoderm desmids”. These saccoderm desmids have a broad geographic distribution and are ecologically diverse. Many species inhabit terrestrial habitats such as dead wood, rock surfaces and glacial ice. Furthermore, several of the saccoderm genera have turned out to be highly polyphyletic and are typically poorly captured by environmental sequencing approaches. One of these genera is Mesotaenium Nägeli, with ~70 described species and infraspecific taxa united only by a relatively simple (plate- or ribbon-like) chloroplast structure. Here, we shed some light on these inconspicuous yet important members of the algal flora and present an updated rbcL gene phylogeny of the conjugating green algae, including several new lineages of Mesotaenium-like zygnematophytes. We depict the subtle morphological differences among these lineages and discuss our updated phylogeny in the light of ecology and cell biology. In addition, we review published knowledge on photoprotective strategies of zygnematophytes, the latest insights into their evolutionary innovations, and address some technical challenges in exploring this elusive group of microalgae. Some new observations of saccoderm desmids in undersampled habitats and of their microbial associates (e.g., parasites)
point to interesting avenues for future research.
Opposite effects of stress on effortful motivation in high and low anxiety are mediated by CRHR1 in the VTAIoannis Zalachoras - 2022
AbstractIndividuals frequently differ in their behavioral and cognitive responses to stress. However, whether motivation
is differently affected by acute stress in different individuals remains to be established. By exploiting natural variation
in trait anxiety in outbred Wistar rats, we show that acute stress facilitates effort-related motivation in low anxious
animals, while dampening effort in high anxious ones. This model allowed us to address the mechanisms underlying
acute stress–induced differences in motivated behavior. We show that CRHR1 expression levels in dopamine neurons
of the ventral tegmental area (VTA)—a neuronal type implicated in the regulation of motivation—depend on
animals’ anxiety, and these differences in CRHR1 expression levels explain the divergent effects of stress on both
effortful behavior and the functioning of mesolimbic DA neurons. These findings highlight CRHR1 in VTA DA
neurons—whose levels vary with individuals’ anxiety—as a switching mechanism determining whether acute
stress facilitates or dampens motivation.
Bacillus velezensis Identification and Recombinant Expression, Purification, and Characterization of Its Alpha-AmylaseXiaodong Zhang - 2021
AbstractAmylases account for about 30% of the global market of industrial enzymes, and the current amylases cannot fully meet industrial needs. This study aimed to identify a high α-amylase producing bacterium WangLB, to clone its α-amylase coding gene, and to characterize the α-amylase. Results showed that WangLB belonged to Bacillus velezensis whose α-amylase gene was 1980 bp coding 659 amino acids designated as BvAmylase. BvAmylase was a hydrophilic stable protein with a signal peptide and a theoretical pI of 5.49. The relative molecular weight of BvAmylase was 72.35 kDa, and was verified by SDS-PAGE. Its modeled structure displayed that it was a monomer composed of three domains. Its optimum temperature and pH were 70 °C and pH 6.0, respectively. It also showed high activity in a wide range of temperatures (40–75 °C) and a relatively narrow pH (5.0–7.0). It was a Ca2+-independent enzyme, whose α-amylase activity was increased by Co2+, Tween 20, and Triton X-100, and severely decreased by SDS. The Km and the Vmax of BvAmylase were 3.43 ± 0.53 and 434.19 ± 28.57 U/mg. In conclusion, the α-amylase producing bacterium WangLB was identified, and one of its α-amylases was characterized, which will be a candidate enzyme for industrial applications.
