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1. Genotypic and Phenotypic Characterization of Incompatibility Group FIB Positive Salmonella enterica Serovar Typhimurium Isolates from Food Animal Sources

   https://doi.org/10.3390/genes11111307  

   Aljahdali, Nesreen H. ; Khajanchi, Bijay K. ; Weston, Kennedi ; Deck, Joanna ; Cox, Justin ; Singh, Ruby ; Gilbert, Jeffrey ; Sanad, Yasser M. ; Han, Jing ; Nayak, Rajesh ; et al ( November 2020 , Genes)

   null (Ed.)

   Salmonella enterica is one of the most common bacterial foodborne pathogens in the United States, causing illnesses that range from self-limiting gastroenteritis to more severe, life threatening invasive disease. Many Salmonella strains contain plasmids that carry virulence, antimicrobial resistance, and/or transfer genes which allow them to adapt to diverse environments, and these can include incompatibility group (Inc) FIB plasmids. This study was undertaken to evaluate the genomic and phenotypic characteristics of IncFIB-positive Salmonella enterica serovar Typhimurium isolates from food animal sources, to identify their plasmid content, assess antimicrobial resistance and virulence properties, and compare their genotypic isolates with more recently isolated S. Typhimurium isolates from food animal sources. Methods: We identified 71 S. Typhimurium isolates that carried IncFIB plasmids. These isolates were subjected to whole genome sequencing and evaluated for bacteriocin production, antimicrobial susceptibility, the ability to transfer resistance plasmids, and a subset was evaluated for their ability to invade and persist in intestinal human epithelial cells. Results: Approximately 30% of isolates (n = 21) displayed bacteriocin inhibition of Escherichia coli strain J53. Bioinformatic analyses using PlasmidFinder software confirmed that all isolates contained IncFIB plasmids along with multiple other plasmid replicon types. Comparative analyses showed that all strains carried multiple antimicrobial resistance genes and virulence factors including iron acquisition genes, such as iucABCD (75%), iutA (94%), sitABCD (76%) and sitAB (100%). In 17 cases (71%), IncFIB plasmids, along with other plasmid replicon types, were able to conjugally transfer antimicrobial resistance and virulence genes to the susceptible recipient strain. For ten strains, persistence cell counts (27%) were noted to be significantly higher than invasion bacterial cell counts. When the genome sequences of the study isolates collected from 1998–2003 were compared to those published from subsequent years (2005–2018), overlapping genotypes were found, indicating the perseverance of IncFIB positive strains in food animal populations. This study confirms that IncFIB plasmids can play a potential role in disseminating antimicrobial resistance and virulence genes amongst bacteria from several food animal species. 

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2. Madagascar’s extraordinary biodiversity: Evolution, distribution, and use

   https://doi.org/10.1126/science.abf0869  

   Antonelli, Alexandre ; Smith, Rhian J. ; Perrigo, Allison L. ; Crottini, Angelica ; Hackel, Jan ; Testo, Weston ; Farooq, Harith ; Torres Jiménez, Maria F. ; Andela, Niels ; Andermann, Tobias ; et al ( December 2022 , Science)

   BACKGROUND The Republic of Madagascar is home to a unique assemblage of taxa and a diverse set of ecosystems. These high levels of diversity have arisen over millions of years through complex processes of speciation and extinction. Understanding this extraordinary diversity is crucial for highlighting its global importance and guiding urgent conservation efforts. However, despite the detailed knowledge that exists on some taxonomic groups, there are large knowledge gaps that remain to be filled. ADVANCES Our comprehensive analysis of major taxonomic groups in Madagascar summarizes information on the origin and evolution of terrestrial and freshwater biota, current species richness and endemism, and the utilization of this biodiversity by humans. The depth and breadth of Madagascar’s biodiversity—the product of millions of years of evolution in relative isolation —is still being uncovered. We report a recent acceleration in the scientific description of species but many remain relatively unknown, particularly fungi and most invertebrates. DIGITIZATION Digitization efforts are already increasing the resolution of species richness patterns and we highlight the crucial role of field- and collections-based research for advancing biodiversity knowledge in Madagascar. Phylogenetic diversity patterns mirror that of species richness and endemism in most of the analyzed groups. Among the new data presented, our update on plant numbers estimates 11,516 described vascular plant species native to Madagascar, of which 82% are endemic, in addition to 1215 bryophyte species, of which 28% are endemic. Humid forests are highlighted as centers of diversity because of their role as refugia and centers of recent and rapid radiations, but the distinct endemism of other areas such as the grassland-woodland mosaic of the Central Highlands and the spiny forest of the southwest is also important despite lower species richness. Endemism in Malagasy fungi remains poorly known given the lack of data on the total diversity and global distribution of species. However, our analysis has shown that ~75% of the fungal species detected by environmental sequencing have not been reported as occurring outside of Madagascar. Among the 1314 species of native terrestrial and freshwater vertebrates, levels of endemism are extremely high (90% overall)—all native nonflying terrestrial mammals and native amphibians are found nowhere else on Earth; further, 56% of the island’s birds, 81% of freshwater fishes, 95% of mammals, and 98% of reptile species are endemic. Little is known about endemism in insects, but data from the few well-studied groups on the island suggest that it is similarly high. The uses of Malagasy species are many, with much potential for the uncovering of useful traits for food, medicine, and climate mitigation. OUTLOOK Considerable work remains to be done to fully characterize Madagascar’s biodiversity and evolutionary history. The multitudes of known and potential uses of Malagasy species reported here, in conjunction with the inherent value of this unique and biodiverse region, reinforce the importance of conserving this unique biota in the face of major threats such as habitat loss and overexploitation. The gathering and analysis of data on Madagascar’s remarkable biota must continue and accelerate if we are to safeguard this unique and highly threatened subset of Earth’s biodiversity. Emergence and composition of Madagascar’s extraordinary biodiversity. Madagascar’s biota is the result of over 160 million years of evolution, mostly in geographic isolation, combined with sporadic long distance immigration events and local extinctions. (Left) We show the age of the oldest endemic Malagasy clade for major groups (from bottom to top): arthropods, bony fishes, reptiles, flatworms, birds, amphibians, flowering plants, mammals, non-flowering vascular plants, and mollusks). Humans arrived recently, some 10,000 to 2000 years (top right) and have directly or indirectly caused multiple extinctions (including hippopotamus, elephant birds, giant tortoises, and giant lemurs) and introduced many new species (such as dogs, zebu, rats, African bushpigs, goats, sheep, rice). Endemism is extremely high and unevenly distributed across the island (the heat map depicts Malagasy palm diversity, a group characteristic of the diverse humid forest). Human use of biodiversity is widespread, including 1916 plant species with reported uses. The scientific description of Malagasy biodiversity has accelerated greatly in recent years (bottom right), yet the diversity and evolution of many groups remain practically unknown, and many discoveries await. 

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3. Using a Human Liver Tissue Equivalent (hLTE) Platform to Define the Functional Impact of Liver-Directed AAV Gene Therapy

   Ramamurthy, R ; Zheng, WT ; George, S ; Wan, M ; Zhou, Y ; Lu, B ; Bishop, C ; Atala, A ; Porada, C ; Almeida-Porada, G ( December 2021 , ASH)

   2938 Using a Human Liver Tissue Equivalent (hLTE) Platform to Define the Functional Impact of Liver-Directed AAV Gene Therapy 63rd ASH Annual Meeting and Exposition, December 11-14, 2021, Georgia World Congress Center, Atlanta, GA Program: Oral and Poster Abstracts Session: 801. Gene Therapies: Poster II Hematology Disease Topics & Pathways: Bleeding and Clotting, Biological, Translational Research, Hemophilia, Genetic Disorders, Clinically Relevant, Diseases, Gene Therapy, Therapies Sunday, December 12, 2021, 6:00 PM-8:00 PM Ritu M Ramamurthy1*, Wen Ting Zheng2*, Sunil George, PhD1*, Meimei Wan1*, Yu Zhou, PhD1*, Baisong Lu, PhD1*, Colin E Bishop, PhD1*, Anthony Atala, M.D.1*, Christopher D Porada, PhD1* and M. Graca Almeida-Porada, MD3 1Fetal Research and Therapy Program, Wake Forest Institute for Regenerative Medicine, Winston Salem, NC 2Massachusetts Institute of Technology, Cambridge, MA 3Fetal Research and Therapy Program, Wake Forest Institute For Regenerative Medicine, Winston-Salem, NC Clinical trials employing AAV vectors for hemophilia A have been hindered by unanticipated immunological and/or inflammatory responses in some of the patients. Also, these trials have often yielded lower levels of transgene expression than were expected based upon preclinical studies, highlighting the poor correlation between the transduction efficiency observed in traditional 2D cultures of primary cells in vitro, and that observed in those same cell types in vivo. It has been also recognized that there are marked species-specific differences in AAV-vector tropism, raising the critical question of the accuracy with which various animal models will likely predict tropism/vector transduction efficiency, and eventual treatment success in humans. Human liver tissue equivalents (hLTEs) are comprised of major cell types in the liver in physiologically relevant frequencies and possess the ability to recapitulate the biology and function of native human liver. Here, we hypothesize that hLTEs can be used as a better model to predict the efficacy and safety of AAV gene therapy in humans. We fabricated hLTEs using 75% hepatocytes, 10% stellate cells, 10% Kupffer cells, and 5% liver sinusoid-derived endothelial cells in 96-well Elplasia plates with 79 microwells per well. hLTEs were transduced at an MOI of 105vg/cell, on the day of fabrication, with the clinically relevant serotypes AAV5 (hLTE-5) or AAV3b (hLTE-3b), both encoding a GFP reporter. After 4 days of self-aggregation, live/dead assay was performed to confirm viability. Non-transduced hLTEs served as negative controls (hLTE(-)), and hLTEs exposed to 20 mM acetaminophen were used as positive controls for liver inflammation/damage. Incucyte® Live-Cell Imaging system was used to track the aggregation and GFP expression of hLTEs. Over the course of the next 5 days, media was collected to determine hepatic functionality, RNA was isolated to assess dysregulation of genes involved in inflammation and fibrosis, DNA was isolated to determine whether AAV vectors integrate into the genome of human hepatocytes and, if so, to define the frequency at which this occurs and the genomic loci of integration, and hLTEs were fixed and processed at appropriate times for histological analyses and transmission electron microscopy (TEM). TEM analysis revealed that all groups exhibited microvilli and bile-canaliculus-like structures, demonstrating the formation of a rudimentary biliary system and, more importantly, proving that hLTEs resemble native liver structure. Incucyte® imaging showed that AAV5 and AAV3b transduction impaired formation of hLTEs (57.57 ± 2.42 and 24.57 ± 4.01 spheroids/well, respectively) in comparison with hLTE(-) (74.86 ± 3.8 spheroids/well). Quantification of GFP expression demonstrated that AAV5 yielded the most efficient transduction of hLTEs (fold change in GFP expression compared to control: 2.73 ± 0.09 and 1.19 ± 0.03 for hLTE-5 and hLTE-3b, respectively). Chromogenic assays showed decreased urea production in cell culture supernatants of AAV transduced groups compared to the non-transduced hLTEs on days 6 and 10 of culture, demonstrating decreased hepatocyte functionality. However, ALT and AST levels were similar in all groups. On day 10, hLTEs were either used for RNA isolation or fixed in 4% PFA and processed for histology. Masson’s Trichrome and Alcian Blue/Sirius Red staining was performed to detect fibrosis, which was then quantified using ImageJ. These analyses showed no significant increase in fibrosis in either hLTE-5 or hLTE-3b compared to hLTE(-). Nevertheless, RT2 PCR Array for Human Fibrosis detected dysregulation of several genes involved in fibrosis/inflammation in both hLTE-5 and hLTE-3b (16/84 and 26/84, respectively). In conclusion, data collected thus far show successful recapitulation of native liver biology and demonstrate that AAV5 transduces hLTEs more efficiently than AAV3b. However, impaired self-aggregation and decreased hepatocyte functionality was observed in both AAV-transduced groups. Studies to address the incidence and location(s) of AAV integration are ongoing. We have thus shown that the hLTE system can provide critical new knowledge regarding the efficacy and safety of AAV gene therapy in the human liver. Disclosures: No relevant conflicts of interest to declare. 

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4. Preferred conformations of lipooligosaccharides and oligosaccharides of Moraxella catarrhalis

   https://doi.org/10.1093/glycob/cwz086  

   Gao, Ya ; Lee, Jumin ; Widmalm, Göran ; Im, Wonpil ( October 2019 , Glycobiology)

   Abstract Moraxella catarrhalis (M. catarrhalis) is a pathogenic gram-negative bacterium that causes otitis media and sinusitis in children. Three major serotypes A, B and C are identified to account for approximately 95% of the clinical isolates. Understanding the conformational properties of different serotypes of M. catarrhalis provides insights into antigenic determinants. In this work, all-atom molecular dynamics simulations were conducted for M. catarrhalis lipooligosaccharide (LOS) bilayer systems and oligosaccharides (OS) in water solution to investigate the conformational similarities and differences of three serotypes. For up to 10 neutral monosaccharides in the core part, the conformational ensembles described by the pair-wise root mean square deviation distributions are similar among the three serotypes of either the LOS or OS. At the central β-($1\to4$)-linkage, anti-$\psi$ conformation in conjunction with the gauche-gauche (g−) conformation of the central trisubstituted glucosyl residue is observed as the dominant conformation to sustain the structural characteristics of M. catarrhalis three types, which is further supported by calculated transglycosidic ${}^3{J}_{C,H}\Big({\psi}_H\Big)$ of serotype A in comparison to experimental data. Interestingly, the conformational variability of three serotypes is more restricted for the OS in water solution than that in the LOS bilayer systems. The LOS–LOS interactions in the bilayer systems are responsible for the increased conformational diversity despite of tight packing. Solvent-accessible surface area analysis suggests that a trisaccharide attached to the β-($1\to 6$)-linked sugar in all three serotypes of LOS could be the common epitope and have the possibility to interact with antibodies. 

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5. Neo‐sex chromosome evolution and phenotypic differentiation across an elevational gradient in horned larks ( Eremophila alpestris )

   https://doi.org/10.1111/mec.16357  

   Shakya, Subir B. ; Wang‐Claypool, Cynthia Y. ; Cicero, Carla ; Bowie, Rauri C. K. ; Mason, Nicholas A. ( January 2022 , Molecular Ecology)

   Genetic structure and phenotypic variation among populations are affected by both geographic distance and environmental variation across species' distributions. Understanding the relative contributions of isolation by distance (IBD) and isolation by environment (IBE) is important for elucidating population dynamics across habitats and ecological gradients. In this study, we compared phenotypic and genetic variation among Horned Lark (Eremophila alpestris) populations from 10 sites encompassing an elevational gradient from low‐elevation desert scrub in Death Valley (285 a.s.l.) to high‐elevation meadows in the White Mountains of the Sierra Nevada of California (greater than 3000 m a.s.l.). Using a ddRAD data set of 28,474 SNPs aligned to a high‐quality reference genome, we compared genetic structure with elevational, environmental, and spatial distance to quantify how different aspects of the landscape drive genomic and phenotypic differentiation in Horned Larks. We found larger‐bodied birds were associated with sites that had less seasonality and higher annual precipitation, and longer spurs occurred in soils with more clay and silt content, less sand, and finer fragments. Larks have large neo‐sex chromosomes, and we found that associations with elevation and environmental variation were much stronger among neo‐sex chromosomes compared to autosomes. Furthermore, we found that putative chromosomal translocations, fusions, and inversions were associated with elevation and may underlie local adaptation across an elevational gradient in Horned Larks. Our results suggest that genetic variation in Horned Larks is affected more by IBD than IBE, but specific phenotypes and genomic regions—particually on neo‐sex chromosomes—bear stronger associations with the environment.

    

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