title: Genome sequences of Aegilops species of section Sitopsis reveal phylogenetic relationships and provide resources for wheat improvement

abstract:
Aegilops is a close relative of wheat (Triticum spp.), and Aegilops species in the section Sitopsis represent a rich reservoir of genetic diversity for improvement of wheat. To understand their diversity and advance their utilization, we produced whole-genome assemblies of Ae. longissima and Ae. speltoides. Whole-genome comparative analysis, along with the recently sequenced Ae. sharonensis genome, showed that the Ae. longissima and Ae. sharonensis genomes are highly similar and most closely related to the wheat D subgenome. By contrast, the Ae. speltoides genome is more closely related to the B subgenome. Haplotype block analysis supported the idea that Ae. speltoides is the closest ancestor of the wheat B subgenome and highlighted variable and similar genomic regions between the three Aegilops species and wheat. Genome-wide analysis of nucleotide-binding site leucine-rich repeat (NLR) genes revealed species-specific and lineage-specific NLR genes and variants, demonstrating the potential of Aegilops genomes for wheat improvement.

TeaserGenome sequences of Aegilops species provides a key for efficient exploitation of this rich genetic resource in wheat improvement.

category: plant biology


title: Ability of nucleoside-modified mRNA to encode HIV-1 envelope trimer nanoparticles

abstract: The success of nucleoside-modified mRNAs in lipid nanoparticles (mRNA-LNP) as COVID-19 vaccines heralded a new era of vaccine development. For HIV-1, multivalent envelope (Env) trimer protein nanoparticles are superior immunogens compared to trimers alone for priming of broadly neutralizing antibody (bnAb) B cell lineages. The successful expression of complex multivalent nanoparticle immunogens with mRNAs has not been demonstrated. Here we show that mRNAs can encode antigenic Env trimers on ferritin nanoparticles that initiate bnAb precursor B cell expansion and induce serum autologous tier 2 neutralizing activity in bnAb precursor VH + VL knock-in mice. Next generation sequencing demonstrated acquisition of critical mutations, and monoclonal antibodies that neutralized heterologous HIV-1 isolates were isolated. Thus, mRNA- LNP can encode complex immunogens and are of use in design of germline-targeting and sequential boosting immunogens for HIV-1 vaccine development.

category: immunology


title: Transcriptome profiling of Candidatus Liberibacter asiaticus in citrus and psyllids

abstract: Candidatus Liberibacter asiaticus (Las) is an emergent bacterial pathogen that is associated with the devastating citrus Huanglongbing (HLB). Vectored by the Asian citrus psyllid, Las colonizes the phloem tissue of citrus, causing severe damage to infected trees. So far, cultivating pure Las culture in axenic media has not been successful and dual-transcriptome analyses aiming to profile gene expression in both Las and its host(s) have a low coverage of the Las genome due to the low abundance of bacterial RNA in total RNA extracts from infected tissues. Therefore, the lack of a Las transcriptome remains as a significant knowledge gap. Here, we used a bacterial cell enrichment procedure and confidently determined the expression profiles of approximately 84% of the Las genes. Genes that exhibited the highest expression levels in citrus include ion transporters, ferritin, outer membrane porins, and genes involved in phage-related functions, pilus formation, cell wall modification, and stress responses. One hundred and six genes were found to be differentially expressed in citrus vs psyllids. Genes related to transcription/translation and resilience to host defense response were upregulated in citrus; whereas genes involved in energy generation and the flagella system were expressed to higher levels in psyllids. We also determined the relative expression levels of potential Sec-dependent effectors, which are considered as key virulence factors of Las. This work advances our understanding of HLB biology and offers novel insight into the interactions of Las with its plant host and insect vector.

category: microbiology


title: Who is the best surrogate for germ stem cell transplantation in fish?

abstract: Surrogate reproduction technology in fish has potential for aquaculture as well as endangered species preservation and propagation. Species with some unfavourable biological characteristics for culturing such as a late maturation or a large body size are ideal candidates for surrogate reproduction using smaller and faster-maturing host. One of the general prerequisites for the successful surrogacy and the pure donor-derived gamete production is the sterility of the host. Various sterilization methods have been developed and used in fish surrogacy; however, a direct comparison of available methods is missing. Such a knowledge gap hinders choice for the surrogate in various fish species, including those in high commercial demand such as tuna or sturgeons, where is a particular limitation from the point of the live material availability and difficulty to perform a high throughput assessment of different surrogates. Yet, large sturgeons or tuna species are one of the most prominent candidates for surrogacy. Zebrafish was utilized in this study as a model species to answer whether and to which extent different sterilization strategies can affect the surrogacy. Germ cell-depleted recipients (produced using knockdown of dead end gene), triploid recipients, and zebrafish x pearl danio hybrid recipients were tested as they represent the most frequently used types of surrogates. Spermatogonia isolated from vas::EGFP transgenic strain were intraperitoneally transplanted into swim-up 5-day old zebrafish. Transplantation success, survival, gonadal development, and reproductive output of the fish was analyzed. Germ cell-depleted recipients with empty gonads were identified as the most convenient among tested sterilization methods considering surrogacy induction success and reproductive output. The present study stands as significant aid for selecting suitable surrogates in various fish species.

category: cell biology


title: Global prediction of candidate R-loop binding and R-loop regulatory proteins

abstract: In the past decade there has been a growing appreciation for R-loop structures as important regulators of the epigenome, telomere maintenance, DNA repair and replication. Given these numerous functions, dozens, or potentially hundreds, of proteins could serve as direct or indirect regulators of R-loop writing, reading, and erasing. In order to understand common properties shared amongst potential R-loop binding proteins (RLBPs) we mined published proteomic studies and distilled 10 features that were enriched in RLBPs compared to the rest of the proteome. We used these RLBP-specific features along with their amino acid composition to create a random forest classifier which predicts the likelihood of a protein to bind to R-loops. In parallel, we employed a whole-genome CRISPR screen coupled with flow-cytometry using the S9.6 monoclonal antibody to sort guide RNAs associated with induction of high S9.6 staining. Known R-loop regulating pathways such as splicing and DNA damage repair are highly enriched in our datasets, and we validate two new R-loop modulating proteins. Together these resources provide a reference to pursue analyses of novel R-loop regulatory proteins.


title: Blocking cell fusion inhibits age-induced polyploidy and maintains epithelial organization in Drosophila

abstract: A characteristic of normal aging and age-related diseases is the remodeling of a tissues cellular organization through polyploid cell growth. Polyploidy arises from an increase in nuclear ploidy or the number of nuclei per cell. However, it is not known whether age-induced polyploidy is an adaption to stressors or a precursor to degeneration. Here, we find that the adult fruit flys abdominal epithelium becomes polyploid with age through generation of large multinucleated cells that make up more than 40% of the tissue area. The syncytia arise by cell fusion, not endomitosis. Epithelial multinucleation is also a characteristic of macular degeneration, including Ctnna1tvrm5, a mouse model for pattern dystrophy. Similarly, we find that the knockdown of alpha-catenin enhances multinucleation in the fly epithelium. We further show that age-induced polyploidy can be suppressed by inhibiting cell fusion revealing a means to maintain tissue organization in older animals.


title: A Drosophila RNAi screen reveals conserved glioblastoma-related adhesion genes that regulate collective cell migration

abstract: Migrating cell collectives are key to embryonic development but also contribute to invasion and metastasis of a variety of cancers. Cell collectives can invade deep into tissues, leading to tumor progression and resistance to therapies. Collective cell invasion is also observed in the lethal brain tumor glioblastoma, which infiltrates the surrounding brain parenchyma leading to tumor growth and poor patient outcomes. Drosophila border cells, which migrate as a small cell cluster in the developing ovary, are a well-studied and genetically accessible model used to identify general mechanisms that control collective cell migration within native tissue environments. Most cell collectives remain cohesive through a variety of cell-cell adhesion proteins during their migration through tissues and organs. In this study, we first identified cell adhesion, cell junction, and associated regulatory genes that are expressed in human brain tumors. We performed RNAi knockdown of the Drosophila orthologs in border cells to evaluate if migration and/or cohesion of the cluster was impaired. From this screen, we identified eight adhesion genes that disrupted border cell collective migration upon RNAi knockdown. Bioinformatics analyses further demonstrated that subsets of the orthologous genes were elevated in the margin and invasive edge of human glioblastoma patient tumors. These data together show that conserved cell adhesion and adhesion regulatory proteins with potential roles in tumor invasion also modulate collective cell migration. This dual screening approach for adhesion genes linked to glioblastoma and border cell migration thus may reveal conserved mechanisms that drive collective tumor cell invasion.

category: developmental biology


title: Integrative annotation of genomic features reveal functional elements and epigenetic landscapes in the developing zebrafish

abstract: Zebrafish, a popular model for embryonic development and for modelling human diseases, has so far lacked a systematic functional annotation programme akin to those in other animal models. To address this, we formed the international DANIO-CODE consortium and created the first central repository to store and process zebrafish developmental functional genomic data. Our Data Coordination Center (https://danio-code.zfin.org) combines a total of 1,802 sets of unpublished and reanalysed published genomics data, which we used to improve existing annotations and show its utility in experimental design. We identified over 140,000 cis-regulatory elements in development, including novel classes with distinct features dependent on their activity in time and space. We delineated the distinction between regulatory elements active during zygotic genome activation and those active during organogenesis, identifying new aspects of how they relate to each other. Finally, we matched regulatory elements and epigenomic landscapes between zebrafish and mouse and predict functional relationships between them beyond sequence similarity, extending the utility of zebrafish developmental genomics to mammals.


title: Integrated annotation and analysis of genomic features reveal new types of functional elements and large-scale epigenetic phenomena in the developing zebrafish


title: The miR-430 locus with extreme promoter density is a transcription body organizer, which facilitates long range regulation in zygotic genome activation

abstract: In anamniote embryos the major wave of zygotic genome activation (ZGA) starts during the mid-blastula transition. This major wave of ZGA is facilitated by several mechanisms, including dilution of repressive maternal factors and accumulation of activating transcription factors during the fast cell division cycles preceding the mid-blastula transition. However, a set of genes escape global genome repression and are activated substantially earlier, during what is called, the minor wave of genome activation. While the mechanisms underlying the major wave of genome activation have been studied extensively, the minor wave of genome activation is little understood. In zebrafish the earliest expressed RNA polymerase II (Pol II) transcribed genes are activated in a pair of large transcription bodies depleted of chromatin, abundant in elongating Pol II and nascent RNAs (Hadzhiev et al., 2019; Hilbert et al., 2021). This transcription body includes the miR-430 gene cluster required for maternal mRNA clearance. Here we explored the genomic, chromatin organisation and cis-regulatory mechanisms of the minor wave of genome activation occurring in the transcription body. By long read genome sequencing we identified a remarkable cluster of miR-430 genes with over 300 promoters and spanning 0.6 Mb, which represent the highest promoter density of the genome. We demonstrate that the miR-430 gene cluster is required for the formation of the transcription body and acts as a transcription organiser for minor wave activation of a set of zinc finger genes scattered on the same chromosome arm, which share promoter features with the miR-430 cluster. These promoter features are shared among minor wave genes overall and include the TATA-box and sharp transcription start site profile. Single copy miR-430 promoter transgene reporter experiments indicate the importance of promoter-autonomous mechanisms regulating escape from global repression of the early embryo. These results together suggest that formation of the transcription body in the early embryo is the result of high promoter density coupled to a minor wave-specific core promoter code for transcribing key minor wave ZGA genes, which are required for the overhaul of the transcriptome during early embryonic development.


title: Intraguild predation does not necessarily have negative effects on pest biological control: insights from a multiple consumer-multiple resource food web population model

abstract: We develop a food web population dynamical based on an experimental pest biocontrol setup consisting of thrips and aphids (pests) being consumed by two agents Macrolophus pygmaeus and Orius laevigatus, and with O. laevigatus being an intraguild predator of M. pygmaeus. By means of numerical simulations, we show that pest biocontrol disruption can be avoided depending on initial population densities of pests and agents, despite the intraguild predation (IGP) of O. laevigatus upon M. pygmaeus. This possible avoidance of pest biocontrol disruption is in accordance with the referred experimental setup and moreover, the proposed model corroborates the importance of initial densities of pests and agents in the determination of the failure or success of pest biocontrol found in this and other biocontrol experimental setups.

category: ecology


title: Preferential associations of soil fungal taxa under mixed compositions of eastern American tree species

abstract: Soil fungi are vital to forest ecosystem functions, in part through their role mediating tree responses to environmental factors, as well as directly through effects on resource cycling. While the distribution of these key taxa may vary with a suite of abiotic and biotic factors, the relative role of host species identity on soil fungal community composition and function remains unresolved. In this study, we used a combination of amplicon sequencing and enzymatic assays to assess soil fungal composition and associated function under three tree species, Quercus rubra, Betula nigra, and Acer rubrum, planted individually and in all combinations in a greenhouse, with added fungal inoculum collected below mature field trees. Across treatments, fungal communities were dominated by the phylum Ascomycota, followed by Basidiomycota and Mortierellomycota. Nonetheless, fungal communities differed between each of the solo planted treatments, suggesting at least some taxa may associate preferentially with these tree species. Additionally, fungal community composition under mixed sapling treatments broadly differed from solo saplings. The data also suggests that there were larger enzymatic activities in the solo treatments as compared with all mixed treatments. This difference may be due to the greater relative abundance of saprobic taxa found in the solo treatments. This study provides evidence of the importance of tree identity on soil microbial communities and functional changes to forest soils.


title: On the relationships between rarity, uniqueness, distinctiveness, originality and functional/phylogenetic diversity

abstract: Rarity reflects the low abundance of a species while distinctiveness reflects its quality of being easy to recognize because it has unique functional characteristics and/or an isolated phylogenetic position. As such, the assemblage-level rarity of a species functional and phylogenetic characteristics (that we name  effective originality) results from both the rarity and the distinctiveness of this species. The functional and phylogenetic diversity of an assemblage then results from a compromise between the abundances and the effective originalities of the species it contains. Although the distinctiveness of a species itself depends on the abundance of the other species in the assemblage, distinctiveness indices that are available in the ecological literature scarcely consider abundance data. We develop a unifying framework that demonstrates the direct connections between measures of diversity, rarity, distinctiveness and effective originality. While developing our framework, we discovered a family of distinctiveness indices that permit a full control of the influence one wants to give to the strict uniqueness of a species (=its smallest functional or phylogenetic distance to another species in the assemblage). Illustrating our framework with bat phylogenetic diversity along a disturbance gradient in Mexico, we show how each component of rarity, distinctiveness and originality can be controlled to obtain efficient indicators for conservation. Overall our framework is aimed to improve conservation actions directed towards highly diverse areas and/or towards species whose loss would considerably decrease biodiversity by offering flexible quantitative tools where the influence of abundant versus rare, and ordinary versus original, species is understood and controlled.


title: Does the evolution of division of labour require accelerating returns from individual specialisation?

abstract: Recent theory has overturned the assumption that accelerating returns from individual specialisation are required to favour the evolution of division of labour. Yanni et al. (2020) showed that topologically constrained groups, where cells cooperate with only direct neighbours such as for filaments or branching growths, can evolve a reproductive division of labour even with diminishing returns from individual specialisation. We developed a conceptual framework and specific models to investigate the factors that can favour the initial evolution of reproductive division of labour. We found that selection for division of labour in topologically constrained groups: (1) is not a single mechanism to favour division of labour - depending upon details of the group structure, division of labour can be favoured for different reasons; (2) always involves an efficiency benefit at the level of group fitness; and (3) requires a mechanism of coordination to determine which individuals perform which tasks. Given that such coordination is unlikely to evolve before division of labour, this limits the extent to which topological constraints could have favoured the initial evolution of division of labour. We conclude by suggesting experimental designs that could determine why division of labour is favoured in the natural world.

category: evolutionary biology


title: A gene-by-gene mosaic of dosage compensation strategies on the human X chromosome

abstract: The human X chromosome exists in active (Xa) and inactive (Xi) states. To quantify Xi gene expression and explore its implications, we conducted RNA sequencing in two cell types from individuals with one Xa and zero to three Xis. Linear models fit the data well, indicating equal contributions from each Xi and quantifying significant Xi-driven expression changes for 124 of 406 expressed X-chromosome genes. As revealed by allele-specific expression analyses, these Xi-driven changes reflect both i) Xi transcription and ii) Xi modulation - both positive and negative - of Xa transcript levels. Incorporating population and evolutionary measures of constraint on expression levels, we identified 13 X-chromosome genes most likely to drive the phenotypic consequences of natural variation in Xi copy number. These analytics and the underlying datasets offer a foundation for understanding Turner, Klinefelter, and other sex chromosome aneuploidy syndromes, and sex-chromosome effects on health and disease in euploid males and females.

category: genetics


title: Quantitative analysis of gene expression on the inactive human X chromosome


title: A broadly neutralizing biparatopic Nanobody protects mice from lethal challenge with SARS-CoV-2 variants of concern

abstract: The ongoing COVID-19 pandemic and the frequent emergence of new SARS-CoV-2 variants of concern (VOCs), requires continued development of fast and effective therapeutics. Recently, we identified high-affinity neutralizing nanobodies (Nb) specific for the receptor-binding domain (RBD) of SARS-CoV-2, which are now being used as biparatopic Nbs (bipNbs) to investigate their potential as future drug candidates. Following detailed in vitro characterization, we chose NM1267 as the most promising candidate showing high affinity binding to several recently described SARS-CoV-2 VOCs and strong neutralizing capacity against a patient isolate of B.1.351 (Beta). To assess if bipNb NM1267 confers protection against SARS-CoV-2 infection in vivo, human ACE2 transgenic mice were treated by intranasal route before infection with a lethal dose of SARS-CoV-2. NM1267-treated mice showed significantly reduced disease progression, increased survival rates and secreted less infectious virus via their nostrils. Histopathological analyses and in situ hybridization further revealed a drastically reduced viral load and inflammatory response in lungs of NM1267-treated mice. These data suggest, that bipNb NM1267 is a broadly active and easily applicable drug candidate against a variety of emerging SARS-CoV-2 VOCs.

category: biochemistry


title: A Systematic Comparison of Differential Analysis Methods for CyTOF Data

abstract:
Cytometry techniques are widely used to discover cellular characteristics at single-cell resolution. Many data analysis methods for cytometry data focus solely on identifying subpopulations via clustering and testing for differential cell abundance. For differential expression analysis of markers between conditions, only few tools exist. These tools either reduce the data distribution to medians, discarding valuable information, or have underlying assumptions that may not hold for all expression patterns.

Here, we systematically evaluated existing and novel approaches for differential expression analysis on real and simulated CyTOF data. We found that methods using median marker expressions compute fast and reliable results when the data is not strongly zero-inflated. Methods using all data detect changes in strongly zero-inflated markers, but partially suffer from overprediction or cannot handle big datasets. We present a new method, CyEMD, based on calculating the Earth Movers Distance between expression distributions that can handle strong zero-inflation without being too sensitive.

Additionally, we developed CYANUS, a user-friendly R Shiny App allowing the user to analyze cytometry data with state-of-the-art tools, including well-performing methods from our comparison. A public web interface is available at https://exbio.wzw.tum.de/cyanus/.

category: bioinformatics


title: Noncanonical Notch signals have opposing roles during cardiac development

abstract: The Notch pathway is an ancient intercellular signaling system with crucial roles in numerous cell-fate decision processes across species. While the canonical pathway is activated by ligand-induced cleavage and nuclear localization of membrane-bound Notch, Notch can also exert its activity in a ligand/transcription-independent fashion, which is conserved in Drosophila, Xenopus, and mammals. However, the noncanonical role remains poorly understood in in vivo processes. Here we show that increased levels of the Notch intracellular domain (NICD) in the early mesoderm inhibit heart development, potentially through impaired induction of the second heart field (SHF), independently of the transcriptional effector RBP-J. Similarly, inhibiting Notch cleavage, shown to increase noncanonical Notch activity, suppressed SHF induction in embryonic stem cell (ESC)-derived mesodermal cells. In contrast, NICD overexpression in late cardiac progenitor cells lacking RBP-J resulted in an increase in heart size. Our study suggests that noncanonical Notch signaling has stagespecific roles during cardiac development.


title: Activity budget and gut microbiome structure across reproductive states in wild, female capuchin monkeys

abstract: Energy demands associated with pregnancy and lactation are significant forces in mammalian evolution. To mitigate increased energy costs associated with reproduction, female mammals have evolved behavioural and physiological responses. Some species alter activity to conserve energy during pregnancy and lactation, while others experience changes in metabolism and fat deposition. Restructuring of gut microbiota with shifting reproductive states may also help females increase energy harvest from foods, especially during pregnancy. Here, we combine life history data with >13,000 behavioural scans and >300 fecal samples collected longitudinally across multiple years from 33 white-faced capuchin monkey females to examine the relationships among behaviour, gut microbiota composition, and reproductive state. We used 16S-based amplicon sequencing and the DADA2 pipeline to analyze microbial diversity and putative functions. Reproductive state explained some variation in activity, but overall resting and foraging behaviours were relatively stable across the reproductive cycle. We found evidence for increases in biotin synthesis pathways among microbes in lactating females, and that relatoe abundance of major phyla among the states was small but significant. Otherwise, gut microbiota structure and estimated functions were not substantially different among reproductive states. These data contribute to a broader understanding of plasticity in response to physiological shifts associated with mammalian reproduction.


title: piRNAs regulate a Hedgehog germline-to-soma pro-aging signal

abstract: The reproductive system regulates the aging of the soma through competing anti- and pro-aging signals. Germline removal extends somatic lifespan through conserved pathways including Insulin, mTOR, and steroid signaling, while germline hyperactivity cuts lifespan short through mechanisms that remain elusive. Here, we show that mating-induced germline hyperactivity leads to the dramatic downregulation of piRNAs, which in turn releases silencing of their targets, including the Hedgehog-like ligand encoding genes wrt-1 and wrt-10, ultimately causing somatic collapse and early death. Germline-produced Hedgehog signals require PTR-6 and PTR-16 receptors for mating-induced body shrinking and lifespan shortening. Our results reveal an unconventional role of the piRNA pathway in transcriptional regulation of Hedgehog signaling, as well as a new role of Hedgehog signaling in the regulation of longevity and somatic maintenance. Our data suggest that Hedgehog signaling is controlled by the tunable piRNA pathway to encode the previously unknown germline-to-soma pro-aging signal. Mating-induced downregulation of piRNAs in the germline and subsequent signaling to the soma via the Hedgehog pathway enables the animal to tune its somatic resource allocation in response to germline needs to optimize reproductive timing and survival.


title: OTUs clustering should be avoided for defining oral microbiome

abstract:
This in silico investigation aimed to: 1) evaluate a set of primer pairs with high coverage, including those most commonly used in the literature, to find the different oral species with 16S rRNA gene amplicon similarity/identity (ASI) values [≥]97%; and 2) identify oral species that may be erroneously clustered in the same operational taxonomic unit (OTU) and ascertain whether they belong to distinct genera or other higher taxonomic ranks.

Thirty-nine primer pairs were employed to obtain amplicon sequence variants (ASVs) from the complete genomes of 186 bacterial and 135 archaeal species. For each primer, ASVs without mismatches were aligned using BLASTN and their similarity values were obtained. Finally, we selected ASVs from different species with an ASI value [≥]97% that were covered 100% by the query sequences. For each primer, the percentage of species-level coverage with no ASI[≥]97% (SC-NASI[≥]97%) was calculated.

Based on the SC-NASI[≥]97% values, the best primer pairs were OP_F053-KP_R020 for bacteria (65.05%), KP_F018-KP_R002 for archaea (51.11%), and OP_F114-KP_R031 for bacteria and archaea together (52.02%). Eighty percent of the oral-bacteria and oralarchaea species shared an ASI[≥]97% with at least one other taxa, including Campylobacter, Rothia, Streptococcus, and Tannerella, which played conflicting roles in the oral microbiota. Moreover, around a quarter and a third of these two-by-two similarity relationships were between species from different bacteria and archaea genera, respectively. Furthermore, even taxa from distinct families, orders, and classes could be grouped in the same cluster.

Consequently, irrespective of the primer pair used, OTUs constructed with a 97% similarity provide an inaccurate description of oral-bacterial and oral-archaeal species, greatly affecting microbial diversity parameters. As a result, clustering by OTUs impacts the credibility of the associations between some oral species and certain health and disease conditions. This limits significantly the comparability of the microbial diversity findings reported in oral microbiome literature.


title: Construction of competing endogenous RNA interaction networks as prognostic markers in metastatic melanoma

abstract: Malignant melanoma (MM) is a highly aggressive, metastatic cancer originating from melanocytes. These tumors have an extremely poor prognosis. MM accounts for 4% of skin cancers with an 80% mortality rate. The median survival of patients with metastatic melanoma is approximately six months, with a five-year survival rate of less than 10%. In recent years, the incidence of melanoma has gradually increased and has become one of the deadliest cancers. Competitive endogenous RNA (ceRNA) models the mechanism by which long chain non-coding RNAs (lncRNAs) play a regulatory role in the disease. LncRNAs can act as a sponge, competitively attracting small RNAs (micoRNAs; miRNAs) and interfering with miRNA function. This can affect the expression of target gene messenger RNAs (mRNAs), ultimately promoting tumorigenesis and tumor progression. Bioinformatics analysis may identify potentially prognostic and therapeutically-relevant differentially expressed genes in MM, as well as lncRNAs, miRNAs and mRNAs that are interconnected through the ceRNA network. This may provide further insight into gene regulation and the prognosis of metastatic melanoma. Weighted co-expression networks were used to identify lncRNA and mRNA modules associated with the metastatic phenotype, as well as the co-expression genes contained in the modules. We used 17 lncRNAs, six miRNAs, and 11 mRNAs to construct a ceRNA interaction network with a regulatory role in patients with metastatic melanoma. The prognostic risk model was used as a sorter to classify the survival prognosis of melanoma patients. Four groups of ceRNA interaction triplets were obtained, and miR-3662 may be used in the treatment of metastatic melanoma patients. Experiments confirmed the regulating relationship and phenotype of this assumption. This study provides new targets to regulate the metastatic process, predict metastatic potential and determine that miR-3662 may be used in the treatment of melanoma.

category: cancer biology


title: Interaction between β-lactoglobulin and EGCG under high-pressure by molecular dynamics simulation

abstract: The binding between {beta}-lactoglobulin and epigallocatechin gallate (EGCG) under the pressure of 600 MPa was explored using molecular docking and molecular dynamics (MD) simulation. EGCG bound mainly in two regions with site 1 in internal cavity of the {beta}-barrel and site 2 on the surface of protein. 150 ns MD was performed starting from the structure with the lowest binding energy at the two sites in molecular docking, respectively, it was found that the system fluctuated greatly when small molecule bound to site 2 at 0.1 MPa. The protein fluctuation and solvent accessible surface area became smaller under high-pressure. The binding of small molecules made the protein structure more stable with increasing of -helix and {beta}-sheet, while high-pressure destroyed -helix of protein. The binding free energy of small molecules at site 1was higher than that at site 2 under 0.1 MPa, with higher van der Waals and hydrophobic interaction at site 1 while more hydrogen bonds at site 2. The binding free energy of both sites decreased under high-pressure, especially at site 1, causing binding free energy at site 1 was lower than that at site 2 under high-pressure.

category: molecular biology


title: Complex feline disease mapping using a dense genotyping array

abstract: The current feline genotyping array of 63k single nucleotide polymorphisms has proven its utility within breeds, and its use has led to the identification of variants associated with Mendelian traits in purebred cats. However, compared to single gene disorders, association studies of complex diseases, especially with the inclusion of random bred cats with relatively low linkage disequilibrium, require a denser genotyping array and an increased sample size to provide statistically significant associations. Here, we undertook a multi-breed study of 1,122 cats, most of which were admitted and phenotyped for nine common complex feline diseases at the Cornell University Hospital for Animals. Using a proprietary 340k single nucleotide polymorphism mapping array, we identified significant genome-wide associations with hyperthyroidism, diabetes mellitus, and eosinophilic keratoconjunctivitis. These results provide genomic locations for variant discovery and candidate gene screening for these important complex feline diseases, which are relevant not only to feline health, but also to the development of disease models for comparative studies.


title: Genetic diversity of local and introduced cassava germplasm in Burundi using DArTseq molecular analyses

abstract: In Burundi, most of small-scale farmers still grow traditional cassava landraces that are adapted to local conditions and have been selected for consumer preferred attributes. They tend to be susceptible, in varying degrees, to devastating cassava viral diseases such as Cassava Brown Steak Disease (CBSD) and Cassava Mosaic Disease (CMD) with production annual losses of US$1 billion annually. For long term resistance to the disease, several breeding strategies have been proposed. A sound basis for a breeding program is to understand the genetic diversity of both landraces and elite introduced breeding cultivars. This will also assist in efforts to conserve landraces ahead of the broad distribution of improved varieties which have the possibility of replacing landraces. Our study aimed at determining the genetic diversity and relationships of local landraces and introduced elite germplasm using morphological and simple nucleotide polymorphism (SNP) markers as well as identifying a core set of germplasm from the local varieties to be used in the cassava breeding program. A total of 118 cultivars were characterized for morphological trait variation based on leaf, stem and root traits, and genetic variation using SNP markers. Results of morphological characterization based on Wards Method revealed three main clusters and five accessions sharing similar characteristics. Molecular characterization identified over 18,000 SNPs and six main clusters and three pairs of duplicates which should be pooled together as one cultivar to avoid redundancy. Results of population genetic analysis showed low genetic distance between populations and between local landraces and elite germplasm. Accessions that shared similar morphological traits were divergent at the molecular level indicating that clustering using morphological traits was inconsistent. Despite the variabilities found within the collection, it was observed that cassava germplasm in Burundi have a narrow genetic base.


title: In vivo study on the healing of bone defect treated with non-thermal atmospheric pressure gas discharge plasma

abstract: Medical treatment using non-thermal atmospheric pressure gas discharge plasma is rapidly gaining recognition. Gas discharge plasma is thought to generate highly reactive species in an ambient atmosphere, which could be exposed to biological targets (e.g., cells and tissues). If plasma-generated reactive species could stimulate bone regeneration, gas discharge plasma could provide a new treatment opportunity in regenerative medicine. We investigated the impact of plasma on bone regeneration using a large bone defect in model rabbits and simple atmospheric pressure plasma (helium microplasma jet). We tracked the recovery progress of the large bone defects by X-ray imaging over eight weeks. The X-ray results showed a clear difference in the filling of the large bone defect among groups with different plasma treatment times, whereas filling was not substantial in the untreated control group. According to the results of micro-computed tomography analysis at eight weeks, the most successful bone regeneration was achieved using a plasma treatment time of 10 min, wherein the new bone volume was 1.51 times larger than that in the control group. Overall, these results suggest that non-thermal atmospheric pressure gas discharge plasma is promising for fracture treatment.

category: pathology


title: Investigating the active substance and mechanism of Jing-Fu-Kang granules via mass spectrometry technology and network pharmacology method

abstract: Jing-Fu-Kang granules (JFKG) is a famous Chinese patent medicine for the treatment of cervical spondylosis around the China, whereas the active substance and mechanism are not completely investigated clearly. In the current study, a rapid separation and identification method using UPLC-QE-Orbitrap-MS was established, 97 chemical constituents from JFKG were identified, and 16 prototype components from plasma samples after administration of JFKG were observed within 16 min. The structures of typical compounds were preliminarily speculated by comparing the retention time and fragmentation pattern. Furthermore, multiple databases were used to integrate the compound targets of JFKG, and the disease targets related to cervical spondylosis. After the intersection of the two sets of targets, a protein-protein interaction (PPI) network and a TCM-component-target-pathway-disease network were established, then using the DAVID database to perform gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes analysis on the common targets to find related pathways. Finally, a total of 531 common targets and 136 pathways were found to participate in the mechanism. Our findings will help to further confirm the mechanism of JFKG for relieving cervical spondylosis, which will improve the scientific rationality of JFKG in clinical use, and can also assist in guiding doctors.

category: pharmacology and toxicology


title: Rapid screening methods of potato cultivars for low glycemic traits

abstract: Potatoes are a dietary staple consumed by a significant portion of the world, providing valuable carbohydrates and vitamins with minimal fat. Most commercially produced potatoes have a high content of highly branched amylopectin starch, which generally results in a high glycemic index (GI). Consumption of foods with high levels of amylopectin elicit a rapid spike in blood glucose levels, which is undesirable for individuals who are pre-diabetic, diabetic, or obese. Some cultivars of potatoes with lower amylopectin levels have previously been identified and are commercially available in niche markets in some countries but are relatively unavailable in the United States and Latin America. Among Native communities in North America and in the high Andes countries of South America, some foods that include certain potato cultivars, may have been used to help people mitigate what is now defined as the effects of blood sugar and obesity. These cultivars are not widely available on a global market. This study utilizes three independent analyses of potato starch: microscopic examination of granule structure, water absorption, and spectrophotometry of iodine complexes to identify potato cultivars with low amylopectin that are thought to have low GI potential. Differences among cultivars tested were detected by all three types of analyses. Of the 60 potato cultivars evaluated the most promising are Huckleberry Gold, Muru, Multa, Green Mountain, and an October Blue x Colorado Rose cross. Further work is necessary to document the ability of these low amylopectin cultivars to reduce blood glucose spike levels in human subjects.


title: Molecular Changes in Prader-Willi Syndrome Neurons Reveals Clues About Increased Autism Susceptibility.

abstract:
BackgroundPrader-Willi syndrome (PWS) is a neurodevelopmental disorder characterized by hormonal dysregulation, obesity, intellectual disability, and behavioral problems. Most PWS cases are caused by paternal interstitial deletions of 15q11.2-q13.1, while a smaller number of cases are caused by chromosome 15 maternal uniparental disomy (PW-UPD). Children with PW-UPD are at higher risk for developing autism spectrum disorder (ASD) than the neurotypical population. In this study, we used expression analysis of PW-UPD neurons to try to identify the molecular cause for increased autism risk.

MethodsDental pulp stem cells (DPSC) from neurotypical control and PWS subjects were differentiated to neurons for mRNA sequencing. Significantly differentially expressed transcripts among all groups were identified. Downstream protein analysis including immunocytochemistry and immunoblots were performed to confirm the transcript level data and pathway enrichment findings.

ResultsWe identified 9 transcripts outside of the PWS critical region (15q11.2-q13.1) that may contribute to core PWS phenotypes. Moreover, we discovered a global reduction in mitochondrial transcripts in the PW-UPD +ASD group. We also found decreased mitochondrial abundance along with mitochondrial aggregates in the cell body and neural projections of +ASD neurons.

LimitationsDPSC derived neuronal cultures used here were immature (3 weeks old), while important for studying the development of the disorder, it will be critical to confirm these mitochondrial defects in more mature neurons or postmortem brain tissue. Our PW-UPD -ASD group included only females, but the sample size in downstream image analysis was increased to include males for the analysis of mitochondrial phenotypes. The ASD diagnostic tool we use is the Social Communication Questionnaire (SCQ), which has been used extensively, but is not the gold standard for ASD diagnosis.

ConclusionsThe 9 transcripts we identified common to all PWS subtypes may reveal PWS specific defects during neurodevelopment. Importantly, we found a global reduction in mitochondrial transcripts in PW-UPD +ASD neurons versus control and other PWS subtypes. We then confirmed mitochondrial defects in neurons from individuals with PWS at the cellular level. Quantification of this phenotype supports our hypothesis that the increased incidence of ASD in PW-UPD subjects may arise from mitochondrial defects in developing neurons.

category: neuroscience


title: Integrating genomics and multivariate evolutionary quantitative genetics: A case study of multivariate constraints on sexual selection in Drosophila serrata

abstract: In evolutionary quantitative genetics, the genetic variance-covariance matrix, G, and the vector of directional selection gradients, {beta}, are key parameters for predicting multivariate selection responses and genetic constraints. Historically, investigations of G and {beta} have not overlapped with those dissecting the genetic basis of quantitative traits. Thus, it remains unknown whether these parameters reflect pleiotropic effects at individual loci. Here, we integrate multivariate GWAS with G and {beta} estimation in a well-studied system of multivariate constraint; sexual selection on male cuticular hydrocarbons (CHCs) in Drosophila serrata. In a panel of wild-derived resequenced lines, we augment genome-based REML, (GREML) to estimate G alongside multivariate SNP effects, detecting 532 significant associations from 1,652,276 SNPs. Constraint was evident, with {beta} lying in a direction of G with low evolvability. Interestingly, minor frequency alleles typically increased male CHC-attractiveness suggesting opposing natural selection on {beta}. SNP effects were significantly misaligned with the major eigenvector of G, gmax, but well aligned to the second and third eigenvectors g2 and g3. We discuss potential factors leading to these varied results including multivariate stabilising selection and mutational bias. Our framework may be useful as researchers increasingly access genomic methods to study multivariate selection responses in wild populations.


title: LoFTK: a framework for fully automated calculation of predicted Loss-of-Function variants

abstract:
MotivationLoss-of-Function (LoF) variants in human genes are important due to their impact on clinical phenotypes and frequent occurrence in the genomes of healthy individuals. Current approaches predict high-confidence LoF variants without identifying the specific genes or the number of copies they affect. Moreover, there is a lack of methods for detecting knockout genes caused by compound heterozygous (CH) LoF variants.

ResultsWe have developed the Loss-of-Function ToolKit (LoFTK), which allows efficient and automated prediction of LoF variants from both genotyped and sequenced genomes. LoFTK enables the identification of genes that are inactive in one or two copies and provides summary statistics for downstream analyses. LoFTK can identify CH LoF variants, which result in LoF genes with two copies lost. Using data from parents and offspring we show that 96% of CH LoF genes predicted by LoFTK in the offspring have the respective alleles donated by each parent.

Availability and implementationLoFTK is an open source software and is freely available to non-commercial users at https://github.com/CirculatoryHealth/LoFTK

Contactj.vansetten@umcutrecht.nl

Supplementary informationSupplementary data are available at Bioinformatics online.


title: Genomic style: yet another deep-learning approach to characterize bacterial genome sequences

abstract:
MotivationBiological sequence classification is the most fundamental task in bioinformatics analysis. For example, in metagenome analysis, binning is a typical type of DNA sequence classification. In order to classify sequences, it is necessary to define sequence features. The k-mer frequency, base composition, and alignment-based metrics are commonly used. In contrast, in the field of image recognition using machine learning, image classification is broadly divided into those based on shape and those based on style. A style matrix was introduced as a method of expressing the style of an image (e.g., color usage and texture).

ResultsWe propose a novel sequence feature, called genomic style, inspired by image classification approaches, for classifying and clustering DNA sequences. As with the style of images, the DNA sequence is considered to have a genomic style unique to the bacterial species, and the style matrix concept is applied to the DNA sequence. Our main aim is to introduce the genomics style as yet another basic sequence feature for metagenome binning problem in replace of the most commonly used sequence feature k-mer frequency. Performance evaluations show that our method using style matrix achieves the superior accuracy than state-of-the-art binning tools based on k-mer frequency.


title: Deterministic shifts in molecular evolution correlate with convergence to annualism in killifishes

abstract: The repeated evolution of novel life histories correlating with ecological variables offer opportunities to test scenarios of convergence and determinism in genetic, developmental, and metabolic features. Here we leverage the diversity of aplocheiloid killifishes, a clade of teleost fishes that contains over 750 species on three continents. Nearly half of these are "annual" or seasonal species that inhabit bodies of water that desiccate and are unfeasible for growth, reproduction, or survival for weeks to months at a time. We present a large-scale phylogenomic reconstruction of aplocheiloid killifish evolution using newly sequenced transcriptomes from all major clades. We show that developmental dormancy (diapause) and annualism have up to seven independent origins in Africa and America. We then measure evolutionary rates of orthologous genes and show that annual life history is correlated with higher dN/dS ratios. Many of these fast-evolving genes in annual species constitute key developmental genes and nuclear-encoded metabolic genes that control oxidative phosphorylation. Lastly, we compare these fast-evolving genes to genes associated with developmental dormancy and metabolic shifts in killifishes and other vertebrates and thereby identify molecular evolutionary signatures of repeated evolutionary transitions to extreme environments.


title: Molecular studies into Copine-4 function in Retinal Ganglion Cells

abstract: The molecular mechanisms underlying morphological diversity in retinal cell types are poorly understood. We have previously reported that several members of the Copine family of Ca-dependent membrane adaptors are expressed in Retinal Ganglion Cells (RGCs) and transcriptionally regulated by Brn3 transcription factors. Several Copines are enriched in the retina and their over-expression leads to morphological changes reminiscent of neurite formation in HEK293 cells. However, the role of Copines in the retina is largely unknown. Here we focus on Cpne4, a Copine whose expression is restricted to RGCs. Over-expression of Cpne4 in RGCs in vivo led to formation of large varicosities on the dendrites but did not otherwise visibly affect dendrite or axon formation. Protein interactions studies using yeast two hybrid analysis from whole retina cDNA revealed two Cpne4 interacting proteins - HCFC1 and Morn2. Mass Spectrometry analysis of retina lysate pulled down using Cpne4 or its vonWillebrand A (vWA) domain identified a further 207 interacting proteins. Gene Ontology (GO) analysis of Cpne4 interactors suggests its involvement in several metabolic and signaling pathways, including processes related to vesicle trafficking, intracellular membrane bound organelles, and plasma membrane associated structures, including neurites. We conclude that, consistent with its domain structure, Cpne4 may be involved in assembly and trafficking of several membrane associated cell compartments.


title: The LbcA lipoprotein and CtpA protease assemble an oligomeric complex to control peptidoglycan hydrolases in Pseudomonas aeruginosa

abstract:
Pseudomonas aeruginosa CtpA is a carboxyl terminal-processing protease that partners with the outer membrane lipoprotein LbcA to degrade at least five cell wall-associated proteins, four of which are cell wall hydrolases. This activity plays an important role in supporting P. aeruginosa virulence in a mouse model of acute pneumonia. However, almost nothing is known about the molecular mechanisms underlying CtpA and LbcA function. Here, we used structural analysis to show that CtpA alone assembles into an inactive hexamer comprising a trimer of dimers, which limits its substrate access and prevents nonspecific degradation. The adaptor protein LbcA is a right-handed open spiral with 11 tetratricopeptide repeats, which might wrap around a substrate to deliver it to CtpA for degradation. By structure-guided mutagenesis and functional assays, we also showed that the interfaces of the CtpA trimer-of-dimers, and an N-terminal helix of LbcA, are important for LbcA-mediated substrate degradation by CtpA both in vitro and in vivo. This work improves our understanding of the molecular mechanism of a CTP within the C-terminal processing peptidase-3 group.

IMPORTANCECarboxyl-terminal processing proteases (CTPs) are found in all three domains of life. In bacteria, some CTPs have been associated with virulence, raising the possibility that they could be theraputic targets. However, relatively little is known about their molecular mechanisms of action. In Pseudomonas aeruginosa, CtpA supports virulence by working in complex with the outer membrane lipoprotein LbcA to degrade cell wall hydrolases. Here, we report structure-function analyses of CtpA and LbcA, which reveals that CtpA assembles into an inactive hexamer comprising a trimer of dimers. LbcA is monomeric, with an N-terminal region important for binding to and activating CtpA, followed by a spiral structure composed of 11 tetratricopetide repeats, which could wrap around a substrate for delivery to CtpA. This work provides the first structure of a CTP-3 group member, revealing a unique mutimeric arrangement and insight into how this important proteolytic system functions.


title: Pseudomonas aeruginosa C-terminal processing protease CtpA assembles into a hexameric structure that requires activation by a spiral-shaped lipoprotein binding partner


title: Implementing solid-phase-enhanced sample preparation for Co-Immunoprecipitation with Mass Spectrometry for C. elegans

abstract:
Studying protein-protein interactions in vivo can reveal key molecular mechanisms of biological processes. Co-Immunoprecipitation followed by Mass Spectrometry (CoIP-MS) allows detection of protein-protein interactions in high-throughput. The nematode Caenorhabditis elegans (C. elegans) is a powerful genetic model organism for in vivo studies. Yet, its rigid cuticle and complex tissues require optimization for protein biochemistry applications to ensure robustness and reproducibility of experimental outcomes. Therefore, we optimized CoIP-MS application to C. elegans protein lysates by combining a native CoIP procedure with an efficient sample preparation method called single-pot, solid-phase-enhanced, sample preparation method (SP3). Our results based on the subunits of the conserved chromatin remodeler FACT demonstrate that our SP3-integrated CoIP-MS procedure for C. elegans samples is highly accurate and robust. Moreover, in a previous study (Baytek et al. 2021), we extended our technique to studying the chromodomain factor MRG-1 (MRG15 in human), which resulted in unprecedented findings.

Method SummaryCombination of cryo-fracture with single-pot, solid-phase-enhanced, sample preparation (SP3) to perform Co-Immuno-Precipitation followed by Mass Spectrometry (CoIP-MS) provides robust assessments of protein-protein interaction using C. elegans whole animals.


title: Condensation of the influenza viral fusion peptide alters membrane structure and water permeability

abstract: To infect, enveloped viruses employ spike protein, spearheaded by its amphipathic fusion peptide (FP), that upon activation extends out from the viral surface to embed into the target cellular membrane. Here we report that isolated FP of influenza virus are membrane active, by themselves generating pores in giant unilamellar vesicles (GUV), and thus potentially explain both influenza virus hemolytic activity and the liposome poration seen in cryo-electron tomography. Consistent with FP segregation on GUV, molecular dynamics (MD) simulations of asymmetric bilayers with different numbers of FP in one leaflet show FP clustering. At the center of this FP condensate, a profound change in the membrane structure results in thinning, higher water permeability, and curvature. Ultimately, a hybrid bilayer domain can form with one lipid leaflet and one FP leaflet. Membrane elastic theory predicts a reduced barrier to water pore formation when even a dimer of FP thins the membrane as above, and the FP tilt to initiate the required leaflet bending.

category: biophysics


title: Combined single cell proteomics and transcriptomics reveals discrete human tendon cells populations persist in vitro and on fibrous scaffolds

abstract:
Chronic tendinopathy represents a growing burden to healthcare services in an active and ageing global population. The ability to identify, isolate and interrogate, in vitro, key pathogenic and reparative tendon cell populations is essential to developing precision therapies and implantable materials.

Human hamstring tendon cells were cultured for 8 days on either tissue culture plastic or aligned electrospun fibres made of polydioxanone (absorbable polymer). Combined single cell surface proteomics and unbiased single cell transcriptomics (CITE-Seq) revealed six discrete cell clusters, four of which shared key gene expression determinants with ex vivo human cell clusters. These were PTX3_PAPPA, POST_SCX, DCN_LUM and ITGA7_NES cell clusters. Surface proteomics found that PTX3_PAPPA cells were CD10+CD26+CD54+. ITGA7_NES cells were CD146+, and POSTN_SCX cells were CD90+CD95+CD10+.

Three clusters preferentially survived and proliferated on the aligned electrospun fibres; DCN_LUM, POSTN_SCX, and PTX3_PAPPA. They maintained high expression of tendon matrix associated genes, including COL1A1, COL1A2, COL3A1, ELN, FBLN1, and up-regulated genesets enriched for TNF-{gamma} signalling via NF{kappa}B, IFN-{gamma} signalling and IL-6/ STAT3 signalling. When cells were pre-selected based on surface protein markers, a similar up-regulation of pro-inflammatory signalling pathways was observed, particularly in PTX3 gene expressing CD10+CD26+CD54+ cells, with increased expression of genes associated with TNF- signalling and IFN-{gamma} signalling.

Discrete human tendon cell sub populations persist in vitro culture and can be recognised by specific gene and surface protein signatures. Aligned PDO fibres promote the survival of three clusters, including pro-inflammatory PTX3 expressing CD10+CD26+CD54+ cells found in chronic tendon disease.


title: The developing epicardium regulates cardiac chamber morphogenesis by promoting cardiomyocyte growth

abstract: The epicardium, the outermost layer of the heart, is an important regulator of cardiac regeneration. However, a detailed understanding of the crosstalk between the epicardium and myocardium during development requires further investigation. Here, we generated three models of epicardial impairment in zebrafish by mutating the transcription factor genes tcf21 and wt1a, and by ablating tcf21+ epicardial cells. Notably, all three epicardial-impairment models exhibit smaller ventricles. We identified the initial cause of this phenotype as defective cardiomyocyte growth, resulting in reduced cell surface and volume. This failure of cardiomyocytes to grow is followed by decreased proliferation and increased abluminal extrusion. By temporally manipulating its ablation, we show that the epicardium is required to support ventricular growth during early cardiac morphogenesis. By transcriptomic profiling of sorted epicardial cells, we identified reduced expression of FGF and VEGF ligand genes in tcf21-/- hearts, and pharmacological inhibition of these signaling pathways partially recapitulated the ventricular growth defects. Thus, the analysis of these epicardial-impairment models further elucidates the distinct roles of the epicardium during cardiac morphogenesis and the signaling pathways underlying epicardial-myocardial crosstalk.


title: Evaluating the impact of in silico predictors on clinical variant classification

abstract:
BackgroundIn silico evidence is important to consider when interpreting genetic variants. According to the ACMG/AMP, in silico evidence is applied at the supporting strength level using the PP3 and BP4 criteria, for pathogenic and benign evidence, respectively. While PP3 has been determined to be one of the most commonly applied criteria, less is known about the effect of these two criteria on variant classification outcomes.

MethodsIn this study, a total of 727 missense variants curated by Clinical Genome Resource (ClinGen) Variant Curation Expert Panels (VCEPs) were analyzed to determine how often PP3 and BP4 were applied and how often they influenced final variant classifications. The current categorical system of variant classification was compared with a point-based system being developed by the ClinGen Sequence Variant Interpretation Working Group. In addition, the performance of four in silico tools (REVEL, VEST, FATHMM, and MPC) was assessed by using a gold set of 237 variants (classified as benign or pathogenic independent of PP3 or BP4) to calculate pathogenicity likelihood ratios.

ResultsCollectively, the PP3 and BP4 criteria were applied by ClinGen VCEPs to 55% of missense variants in this data set. Removing in silico criteria from variants where they were originally applied caused variants to change classification from pathogenic to likely pathogenic (14%), likely pathogenic to variant of uncertain significance (VUS) (24%), or likely benign to VUS (64%). The proportion of downgrades with the categorical classification system was similar to that of the point-based system, though the latter resolved borderline classifications. REVEL and VEST performed at a level consistent with moderate strength towards either benign or pathogenic evidence, while FATHMM performed at the supporting level.

ConclusionsOverall, this study demonstrates that in silico criteria PP3 and BP4 are commonly applied in variant classification and often affect the final classification. Our results suggest that when sufficient thresholds for in silico predictors are established, PP3 and BP4 may be appropriate to use at a moderate strength. However, further calibration with larger datasets is needed to optimize the performance of current in silico tools given the impact they have on clinical variant classification.

category: genomics


title: Benchmarking artificial intelligence methods for end-to-end computational pathology

abstract: Artificial intelligence (AI) can extract subtle visual information from digitized histopathology slides and yield scientific insight on genotype-phenotype interactions as well as clinically actionable recommendations. Classical weakly supervised pipelines use an end-to-end approach with residual neural networks (ResNets), modern convolutional neural networks such as EfficientNet, or non-convolutional architectures such as vision transformers (ViT). In addition, multiple-instance learning (MIL) and clustering-constrained attention MIL (CLAM) are being used for pathology image analysis. However, it is unclear how these different approaches perform relative to each other. Here, we implement and systematically compare all five methods in six clinically relevant end-to-end prediction tasks using data from N=4848 patients with rigorous external validation. We show that histological tumor subtyping of renal cell carcinoma is an easy task which approaches successfully solved with an area under the receiver operating curve (AUROC) of above 0.9 without any significant differences between approaches. In contrast, we report significant performance differences for mutation prediction in colorectal, gastric and bladder cancer. Weakly supervised ResNet-and ViT-based workflows significantly outperformed other methods, in particular MIL and CLAM for mutation prediction. As a reason for this higher performance we identify the ability of ResNet and ViT to assign high prediction scores to highly informative image regions with plausible histopathological image features. We make all source codes publicly available at https://github.com/KatherLab/HIA, allowing easy application of all methods on any end-to-end problem in computational pathology.


title: Bisphosphonate drugs have actions outside the skeleton and inhibit the mevalonate pathway in alveolar macrophages

abstract: Bisphosphonates drugs target the skeleton and are used globally for the treatment of common bone disorders. Nitrogen-containing bisphosphonates act by inhibiting the mevalonate pathway in bone-resorbing osteoclasts but, surprisingly, also appear to reduce the risk of death from pneumonia. We overturn the long-held belief that these drugs act only in the skeleton and show that a fluorescently-labelled bisphosphonate is internalised by alveolar macrophages and peritoneal macrophages in vivo. Furthermore, a single dose of a nitrogen-containing bisphosphonate (zoledronic acid) in mice was sufficient to inhibit the mevalonate pathway in tissue-resident macrophages, causing the build-up of a mevalonate metabolite and preventing protein prenylation. Importantly, one dose of bisphosphonate enhanced the immune response to bacterial endotoxin in the lung and increased the level of cytokines and chemokines in bronchoalveolar fluid. These studies suggest that bisphosphonates, as well as preventing bone loss, may boost immune responses to infection in the lung and provide a mechanistic basis to fully examine the potential of bisphosphonates to help combat respiratory infections that cause pneumonia.


title: Mechanism of hyperproteinemia-induced blood cell homeostasis imbalance in an animal model

abstract:
Hyperproteinemia is a metabolic disorder associated with increased plasma protein concentration (PPC). It is often clinically complicated by malignant diseases or severe infections. Research on the molecular mechanism of High PPC (HPPC) is scant. Here, an animal model of primary hyperproteinemia was constructed in an invertebrate, Bombyx mori, to investigate the effect of HPPC on circulating blood cells. We showed that HPPC affected blood cell homeostasis and enhanced blood cell phagocytosis, leading to increased reactive oxygen species levels, and induced programmed cell death that depended on the endoplasmic reticulum-calcium ion signaling pathway. HPPC induced the proliferation of blood cells, mainly granulocytes, by activating the JAK/STAT signaling pathway. Supplementation with endocrine hormone active substance 20E significantly reduced the impact of HPPC on blood cell homeostasis. Herein, we reported a novel signaling pathway by which HPPC affected blood cell homeostasis, which was different from hyperglycemia, hyperlipidemia, and hypercholesterolemia. In addition, we showed that down-regulation of gene expression of the hematopoietic factor Gcm could be used as a potential early monitoring index for hyperproteinemia.

Highlights* HPPC induced proliferation of circulating blood cells through the JAK/STAT pathway, leading to a significant increase in the proportion of granulocytes.
* Supplementing endocrine hormone 20E improved hematopoietic function and restored the homeostasis of circulating blood cells through modulation of the JAK/STAT signaling pathway.
* HPPC induced PCD in blood cells through the endoplasmic reticulum-calcium ion release signaling pathway.
* Down-regulation of the gene expression of the hematopoietic factor Gcm could be used as a potential early monitoring index for hyperproteinemia.


title: Relevance of Auditory Errors Decreases When Errors Are Introduced Suddenly

abstract:
PurposeWhen the speech motor system encounters errors, it generates adaptive responses to compensate for the errors. We previously showed that adaptive responses to task-irrelevant errors are significantly smaller than responses to task-relevant errors when errors are introduced gradually. The current study aimed to examine responses to task-irrelevant and task-relevant errors when errors are introduced suddenly.

MethodWe used an adaptation paradigm in which participants experienced task-relevant errors (induced by formant-shift perturbations) and task-irrelevant errors (induced by formant-clamp perturbations). For one group of participants (N = 30), we applied the perturbations gradually. The second group of participants (N = 30) received the perturbations suddenly. We designed the perturbations based on participant-specific vowel configurations such that a participants first and second formants of /{varepsilon}/ were perturbed toward their /ae/. To estimate adaptive responses, we measured formant changes (within 0-100 ms of the vowel onset) in response to the formant perturbations.

ResultsWe found that (1) the difference between adaptive responses to formant-shift and formant-clamp perturbations was the smallest for the suddenly introduced perturbations, and (2) responses to formant-shift perturbations positively correlated with responses to formant-clamp perturbations for the suddenly (but not gradually) introduced perturbations.

ConclusionsThese results showed that the speech motor system responds to task-relevant errors and task-irrelevant errors more differently when errors are introduced gradually than suddenly. Overall, the speech motor system evaluates the relevance of errors and uses its evaluation to modulate its adaptive responses to errors.


title: Combined in situ hybridization chain reaction and immunostaining to visualize gene expression in whole-mount Drosophila central nervous systems

abstract: Methods to visualize gene expression in the Drosophila central nervous system are important in fly neurogenetic studies. In this chapter, we describe a detailed protocol that sequentially combines in situ hybridization chain reaction (HCR) and immunostaining to detect mRNA and protein expression in whole-mount Drosophila larval and adult central nervous systems. We demonstrate the application of in situ HCR in comparisons of nervous system gene expression between Drosophila species, and in the validation of single-cell RNA-Seq results in the fly nervous system. Our protocol provides a simple, robust, multiplexable, and relatively affordable means to quantitatively visualize gene expression in the nervous system of flies, facilitating its general use in fly neurogenetic studies.


title: The Normative Modeling Framework for Computational Psychiatry

abstract: Normative modeling is an emerging and innovative framework for mapping individual differences at the level of a single subject or observation in relation to a reference model. It involves charting centiles of variation across a population in terms of mappings between biology and behavior which can then be used to make statistical inferences at the level of the individual. The fields of computational psychiatry and clinical neuroscience have been slow to transition away from patient versus "healthy" control analytic approaches, likely due to a lack of tools designed to properly model biological heterogeneity of mental disorders. Normative modeling provides a solution to address this issue and moves analysis away from case-control comparisons that rely on potentially noisy clinical labels. In this article, we define a standardized protocol to guide users through, from start to finish, normative modeling analysis using the Predictive Clinical Neuroscience toolkit (PCNtoolkit). We describe the input data selection process, provide intuition behind the various modeling choices, and conclude by demonstrating several examples of down-stream analyses the normative model results may facilitate, such as stratification of high-risk individuals, subtyping, and behavioral predictive modeling.


title: Atmospheric particulate matter aggravates CNS demyelination via TLR-4/NF-κB-mediated microglia pathogenic activities

abstract:
Atmospheric Particulate Matter (PM) is one of the leading environmental risk factors for the global burden of disease. Increasing epidemiological studies demonstrated that PM plays a significant role in CNS demyelinating disorders; however, there is no direct testimony of this, and yet the molecular mechanism by which the occurrence remains unclear. Using multiple in vivo and in vitro strategies, in the present study we demonstrate that PM exposure aggravates neuroinflammation, myelin injury, and dysfunction of movement coordination ability via boosting microglial pro-inflammatory activities, in both the pathological demyelination and physiological myelinogenesis animal models. Indeed, pharmacological disturbance combined with RNA-seq and ChIP-seq suggests that TLR-4/NF-{kappa}B signaling mediated a core network of genes that control PM-triggered microglia pathogenicity. In summary, our study defines a novel atmospheric environmental mechanism that mediates PM-aggravated microglia pathogenic activities, and establishes a systematic approach for the investigation of the effects of environmental exposure in neurologic disorders.

SignificanceAn atmospheric trigger, the respirable particulate matter boost microglia pathogenic activities in the context of CNS demyelination by activating TLR-4/NF-{kappa}B signaling axis in animal models of immune- and toxicity-induced demyelination, as well as myelinogenesis during postnatal development.


title: Efficient and Rapid generation of neural stem cells by direct conversion Fibroblasts with A Single microRNA

abstract: Neural stem cells (NSCs) have great potential in the application of neurodegenerative disease therapy, drug screening and disease modeling. NSC can be generated by reprogramming from terminally differentiated cells with transcription factors or small molecules. However, current methods for producing NSCs involve the danger of integrating foreign genes into the genome and the problem of low efficiency. Here, we report an efficient method to generate NSCs from human skin-derived fibroblasts with microRNA (mir-302a) in 2-3 days. The induced NSCs (iNSCs) have more than 90% of purity. Their morphology is similar to regular NSCs, expressing key markers including Nestin, Pax6 and Sox2, and can be expanded for more than 20 passages in vitro. They can also differentiate into functional neuron progeny, astrocytes and oligodendrocytes as well. Those cells can elicit action potential, can be xeno-transplanted into the brain of immune-deficient mice, and can survive and differentiate in vivo without tumor formation. This study shows that a single part of pluripotency-inducing mir-302 cluster can drive fibroblasts reprogramming, providing a general platform for high-efficiency generation of individual-specific human NSCs for studies of neuron system development and regenerative cell therapy.


title: Immune Cell Profiling Reveals MAIT and Effector Memory CD4+ T Cell Recovery Link to Control of Cytomegalovirus Reactivation after Stem Cell Transplant

abstract: Human cytomegalovirus (HCMV) reactivation is a major opportunistic infection after allogeneic haematopoietic stem cell transplantation and has a complex relationship with post-transplant immune reconstitution. Here, we used mass cytometry to comprehensively define global patterns of innate and adaptive immune cell reconstitution at key phases of HCMV reactivation (before detection, initial detection, peak and near resolution) in the first 100 days post-transplant. In addition to identifying patterns of immune reconstitution in those with or without HCMV reactivation, we found mucosal-associated invariant T (MAIT) cell levels at the initial detection of HCMV DNAemia distinguished patients who subsequently developed low-level versus high-level HCMV reactivation. In addition, early recovery of effector-memory CD4+ T cells distinguished low-level and high-level reactivation. Our data describe distinct immune signatures that emerged with HCMV reactivation post-HSCT, and highlight MAIT cell levels at the initial detection of reactivation as a potential prognostic marker to guide clinical decisions regarding pre-emptive therapy.


title: Increasing temporal variance leads to stable species range limits

abstract: What prevents populations of a species from adapting to the novel environments outside the species geographic distribution? Previous models highlighted how gene flow across spatial environmental gradients determines species expansion vs. extinction and the location of species range limits. However, space is only one of two axes of environmental variation -- environments also vary in time, and we know temporal environmental variation has important consequences for population demography and evolution. We used analytical and individual-based evolutionary models to explore how temporal variation in environmental conditions influences the spread of populations across a spatial environmental gradient. We find that temporal variation greatly alters our predictions for range dynamics compared to temporally static environments. When temporal variance is equal across the landscape, the fate of species (expansion vs. extinction) is determined by the interaction between the degree of temporal autocorrelation in environmental fluctuations and the steepness of the spatial environmental gradient. When the magnitude of temporal variance changes across the landscape, stable range limits form where this variance increases maladaptation sufficiently to prevent local persistence. These results illustrate the pivotal influence of temporal variation on the likelihood of populations colonizing novel habitats and the location of species range limits.


title: Phased Mutations and Complex Rearrangements in Human Prostate Cancer Cell Lines through Linked-Read Whole Genome Sequencing

abstract: A limited number of cell lines have fueled the majority of preclinical Prostate cancer (PCa) research. Despite tremendous effort in characterizing their molecular profiles, comprehensive whole genome sequencing with allelic phasing of somatic genome alterations has not been undertaken to date. Here, we utilized whole genome Linked-read sequencing to obtain haplotype information from the seven most commonly used PCa cell lines (PC3, LNCaP, DU145, CWR22Rv1, VCaP, LAPC4, MDA-PCa-2b), four castrate resistant (CR) subclones (LNCaP_Abl, LNCaP_C42b, VCaP-CR, LAPC4-CR), and an immortalized prostate epithelial line RWPE-1. Phasing of mutations allowed derivation of "Gene-level Haplotype" to assess whether a gene harbored heterozygous mutations in one or both alleles, providing a comprehensive catalogue of mono or bi-allelically inactivated genes. Phased structural variant analysis allowed identification of complex rearrangement chains consistent with chromothripsis and chromoplexy, with breakpoints occurred across a single allele, providing further evidence that complex SVs occurred in a concerted event, rather than through accumulation of multiple independent rearrangements. Additionally, comparison of parental and CR subclones revealed previously known and novel genomic alterations associated with the CR clones. This study therefore comprehensively characterized phased genomic alterations in the commonly used PCa cell lines and provided a useful resource for future cancer research.


title: Patient-derived triple negative breast cancer organoids provide robust model systems that recapitulate tumor intrinsic characteristics

abstract:
Triple negative breast cancer (TNBC) is an aggressive form of breast cancer with poor patient outcomes, and an unmet clinical need for targeted therapies and better model systems. Here, we developed and comprehensively characterized a diverse biobank of normal and breast cancer patient-derived organoids (PDOs) with a focus on TNBCs. PDOs recapitulated patient tumor intrinsic properties and a subset of PDOs can be propagated for long-term culture (LT-TNBCs). Single cell profiling of PDOs identified cell types and gene candidates affiliated with different aspects of cancer progression. The LT-TNBC organoids exhibit signatures of aggressive MYC-driven basal-like breast cancers and are largely comprised of luminal progenitor (LP)-like cells. The TNBC LP-like cells are distinct from normal LPs and exhibit hyperactivation of NOTCH and MYC signaling. Overall, our study validates TNBC PDOs as robust models for understanding breast cancer biology and progression, paving the way for personalized medicine and tailored treatment options.

Statement of SignificanceA comprehensive analysis of TNBC patient-derived organoids is presented by genomic, transcriptomic, and in-vivo analyses, providing insights into cellular heterogeneity and mechanisms of tumorigenesis at the single cell level.


title: The Mismatch Between Neuroscience Graduate Training and Professional Skill Sets

abstract: Understanding the skill sets required for career paths is a prerequisite for preparing students for those careers. Neuroscience career paths are rapidly changing as the field expands and increasingly overlaps with computational and data-heavy job sectors. With the steady growth in neuroscience trainees and the diversification of jobs for those trainees, it is important to assess whether or not our training is matching the skill sets required in the workforce. Here, we surveyed hundreds of neuroscience professionals and graduate students to assess their use and valuation of a range of skills, from bench skills to communication and management. We find that professionals with neuroscience degrees can be clustered into three main groups based on their skill sets: academic research, industry research and technical work, and non-research. Further, we find that while graduate students do not use or highly value management and communication skills, almost all neuroscience professionals report strongly needing those skills. Finally, coding and data analysis skills are widely used in academic and industry research and predict higher salaries. Our findings can help trainees assess their own skill sets as well as encourage educational leaders to offer training in management and communication-skills which may help catapult trainees into the next stages of their careers.

category: scientific communication and education


title: Inhibiting retinoic acid mitigates vision loss in a mouse model of retinal degeneration

abstract: In degenerative retinal disorders, rod and cone photoreceptors die, causing vision impairment and blindness. Downstream neurons survive but undergo morphological and physiological remodeling, with some retinal ganglion cells (RGC) exhibiting heightened spontaneous firing. Retinoic acid (RA) has been implicated as the key signaling molecule that induces RGC hyperactivity, obscuring RGC light responses and reducing light avoidance behaviors triggered by residual rods and cones. However, evidence that RA-dependent remodeling corrupts image-forming vision has been lacking. Here we show that disulfiram, an FDA-approved drug that inhibits RA synthesis, and BMS 493, an RA receptor (RAR) inhibitor, reduce RGC hyperactivity and augment image detection in visually impaired mice. Functional imaging of visual cortical neurons shows that disulfiram and BMS 493 sharpen orientation-tuning and strengthen response fidelity to naturalistic scenes. These findings establish a causal link between RA-induced retinal hyperactivity and vision impairment and define molecular targets and candidate drugs for boosting image-forming vision in retinal degeneration.


title: Phox2b mutation mediated by Atoh1 expression impaired respiratory rhythm and ventilatory responses to hypoxia and hypercapnia

abstract: Retrotrapezoid nucleus (RTN) neurons are involved in central chemoreception and respiratory control. Lineage tracing studies demonstrate RTN neurons to be derived from Phox2b and Atoh1 expressing progenitor cells in rhombere 4. Phox2b exon 3 mutations cause congenital central hypoventilation syndrome (CCHS), producing an impaired respiratory response to hypercapnia and hypoxia. Our goal was to investigate the extent to which a conditional mutation of Phox2b within Atoh1-derived cells might affect a) respiratory rhythm; b) ventilatory responses to hypercapnia and hypoxia and c) number of RTN-chemosensitive neurons. Here, we used a transgenic mouse line carrying a conditional Phox2b{Delta}8 mutation activated by cre-recombinase. We crossed them with Atoh1Cre mice. Ventilation was measured by whole body plethysmograph during neonate and adult life. In room air, experimental and control groups showed similar basal ventilation; however, Atoh1Cre/Phox2b{Delta}8 increased breath irregularity. The hypercapnia and hypoxia ventilatory responses were impaired in neonates. In contrast, adult mice recovered ventilatory response to hypercapnia, but not to hypoxia. Anatomically, we observed a reduction of the Phox2b+/TH- expressing neurons within the RTN region. Our data indicates that conditionally expression of Phox2b mutation by Atoh1 affect development of the RTN neurons and are essential for the activation of breathing under hypoxic and hypercapnia condition, providing new evidence for mechanisms related to CCHS neuropathology.


title: Immediate Impact of Yogic Breathing on Pulsatile Cerebrospinal Fluid Dynamics

abstract: Cerebrospinal fluid (CSF), a clear fluid bathing the central nervous system (CNS), undergoes pulsatile movements, and plays a critical role for the removal of waste products from the brain including amyloid beta, a protein associated with Alzheimers disease. Regulation of CSF dynamics is critical for maintaining CNS health, and increased pulsatile CSF dynamics may alter brains waste clearance due to increased mixing and diffusion. As such, understanding the mechanisms driving CSF movement, and interventions that influence its resultant removal of wastes from the brain is of high scientific and clinical impact. Since pulsatile CSF dynamics is sensitive and synchronous to respiratory movements, we are interested in identifying potential integrative therapies such as yogic breathing to regulate and enhance CSF dynamics, which has not been reported before. Here, we investigated the pre-intervention baseline data from our ongoing randomized controlled trial, and examined whether yogic breathing immediately impacts pulsatile CSF dynamics compared to spontaneous breathing. We utilized our previously established non-invasive real-time phase contrast magnetic resonance imaging (RT-PCMRI) approach using a 3T MRI instrument, and computed and rigorously tested differences in CSF velocities (instantaneous, respiratory, cardiac 1st and 2nd harmonics) at the level of foramen magnum during spontaneous versus four yogic breathing patterns. In examinations of 18 healthy participants (eight females, ten males; mean age 34.9 {+/-} 14 (SD) years; age range: 18-61 years), we discovered immediate increase in cranially-directed velocities of instantaneous-CSF 16% - 28% and respiratory-CSF 60% - 118% during yogic versus spontaneous breathing, with most statistically significant changes during deep abdominal breathing (28%, p=0.0008, and 118%, p=0.0001, respectively). Further, cardiac pulsation was the primary source of pulsatile CSF during all breathing conditions except during deep abdominal breathing, when there was a comparable contribution of respiratory and cardiac 1st harmonic power [0.59 {+/-} 0.78], demonstrating respiration can be the primary regulator of CSF depending on individual differences in breath depth and location. Further work is needed to investigate the impact of sustained training yogic breathing on increased pulsatile CSF dynamics and brain waste clearance for CNS health.


title: Hotair and Malat1 Long Noncoding RNAs regulate Bdnf Expression and Oligodendrocyte Precursor Cells Differentiation

abstract: BDNF has remarkable protective roles in the central nervous system to ensure neurons and glial cells survival and proper functions. The regulatory processes behind the BDNF expression have not been revealed completely. Here, it was explored whether Malat1 and Hotair lncRNAs play roles in the regulation of Bdnf expression level, modification of fingolimod downstream pathway, and oligodendrocytes precursor cells maturation. By Hotair and Malat1 downregulation, their regulatory mechanism on Bdnf expression was investigated. Immunostaining and RT-qPCR assays were employed to assess the effects of fingolimod and lncRNAs on OPCs maturation. The results represented that Hotair and Malat1 lncRNAs may regulate Bdnf expression in primary glial cells significantly, and also can coordinate fingolimod stimulatory effect on Bdnf expression. Furthermore, Malat1 may have a role in the last stages of the intrinsic oligodendrocyte myelination. Here it was demonstrated that these lncRNAs have critical roles in the Bdnf level, fingolimod mechanism of action, and OPCs maturation. Understanding the regulatory mechanism of neurotrophins leads to a better comprehension of the neurodegenerative disorders pathogenesis and designing more effective treatments.


title: Regulation of sexually dimorphic abdominal courtship behaviors in Drosophila by the Tlx/tailless-like nuclear receptor, Dissatisfaction

abstract: Sexually dimorphic courtship behaviors in Drosophila melanogaster develop from the activity of the sexual differentiation genes, doublesex (dsx) and fruitless (fru), functioning with other regulatory factors that have received little attention. The dissatisfaction gene (dsf) encodes an orphan nuclear receptor homologous to vertebrate Tlx and Drosophila tailless that is critical for the development of several aspects of female- and male-specific sexual behaviors. Here, we report the pattern of dsf expression in the central nervous system and show that the activity of sexually dimorphic abdominal interneurons that co-express dsf and dsx is necessary and sufficient for vaginal plate opening in virgin females, ovipositor extrusion in mated females, and abdominal curling in males during courtship. We find that dsf activity results in different neuroanatomical outcomes in females and males, promoting and suppressing, respectively, female development and function of these neurons depending upon the sexual state of dsx expression. We posit that dsf and dsx interact to specify sex differences in the neural circuitry for dimorphic abdominal behaviors.


title: TNFa and IL-6 promote ex-vivo proliferation of lineage-committed human regulatory T cells

abstract: Treg therapy is being tested in clinical trials in transplantation and autoimmune diseases, however, the impact of inflammation on Tregs is unclear. In this study, we challenged human Tregs ex-vivo with pro-inflammatory cytokines, TNF and IL-6. These cytokines enhanced Treg proliferation induced by anti-CD3 and anti-CD28 or CD28 superagonist (CD28SA) while maintaining high expression of FOXP3 and HELIOS, demethylated FOXP3 enhancer, and low expression of cytokines IFNg, IL-4 and IL-17. Blocking TNF receptor signaling using etanercept or deletion of TNF receptor 2 using CRISPR/Cas9 blunted Treg proliferation and attenuated FOXP3 and HELIOS expression, revealing the importance of TNFR2 signaling in Treg proliferation and lineage stability. The robust proliferation induced by CD28SA with IL-6 and TNF may be adopted for the expansion of therapeutic Tregs. Metabolomics analysis showed that Tregs expanded with CD28SA plus cytokines had more active glycolysis and oxidative phosphorylation, increased energy production, and higher antioxidant potential. Finally, CD28SA plus cytokine-expanded Tregs had comparable suppressive activity in vitro and in vivo in a humanized mouse model of graft-versus-host-disease when compared to Tregs expanded using the conventional protocol. These results demonstrate that human Tregs positively respond to proinflammatory cytokines with enhanced proliferation without compromising their lineage identity or function.

category: immunology


title: Quantitative Restoration of Immune Defense in Old Animals Determined by Naive Antigen-Specific CD8 T cell Numbers

abstract:
Older humans and animals often exhibit reduced immune responses to infection and vaccination, and this often directly correlates to the numbers and frequency of naive T (Tn) cells. We found such a correlation between reduced numbers of blood CD8+ Tn cells and severe clinical outcomes of West Nile virus (WNV) in both humans naturally exposed to, and mice experimentally infected with, WNV.

To examine possible causality, we sought to increase the number of CD8 Tn cells by treating C57BL/6 mice with IL-7 complexes (IL-7C, anti-IL-7 mAb bound to IL-7), shown previously to efficiently increase peripheral T cell numbers by homeostatic proliferation. T cells underwent robust expansion following IL-7C administration to old mice increasing the number of total T cells (>four-fold) and NS4b:H-2Db-restricted antigen-specific CD8 T cells (two-fold). This improved the numbers of NS4b-specific CD8 T cells detected at the peak of the response against WNV, but not survival of WNV challenge. IL-7C treated old animals also showed no improvement in WNV-specific effector immunity (neutralizing antibody and in vivo T cell cytotoxicity). To test quantitative limits to which CD8 Tn cell restoration could improve protective immunity, we transferred graded doses of Ag-specific precursors into old mice and showed that injection of 5,400 (but not of 1,800 or 600) adult naive WNV-specific CD8 T cells significantly increased survival after WNV. These results set quantitative limits to the level of Tn reconstitution necessary to improve immune defense in older organisms and are discussed in light of targets of immune reconstitution.


title: Prevalence of heterotrophic methylmercury detoxifying bacteria across oceanic regions

abstract:
Microbial reduction of inorganic divalent mercury (Hg2+) and methylmercury (MeHg) demethylation is performed by the mer operon, specifically by merA and merB genes respectively, but little is known about the mercury tolerance capacity of marine microorganisms and its prevalence in the global ocean. Here, we explored the distribution of these genes in 290 marine heterotrophic bacteria (Alteromonas and Marinobacter spp.) isolated from different oceanographic regions and depths, and assessed their tolerance to diverse concentrations of Hg2+ and MeHg. About 25% of the isolates presented merA and only 8.9% presented both merAB genes, including the strain ISS312 that exhibited the highest tolerance capacity and a degradation efficiency of 98.2% in 24 h. Fragment recruitment analyses of ISS312 genome against microbial metagenomes indicated an extensive distribution across the global bathypelagic ocean. Our findings highlighted that mercury resistance genes are widely distributed in a non-highly polluted environment such as the pelagic marine environment, and that degradation of the neurotoxic MeHg can be performed through the ocean water column by some heterotrophic bacteria at high efficiency with important implications in the biogeochemical cycle of mercury and potentially for the environment and human health.

TeaserActive mercury resistance genes detected in marine cultured bacteria are widely distributed in the ocean including the bathypelagic zone.

category: microbiology


title: Genetic diversity of wild and cultivated Coffea canephora in northeastern DR Congo and the implications for conservation.

abstract:
PremiseMany cultivated coffee varieties descend from Coffea canephora, commonly known as Robusta coffee. The Congo Basin has a century long history of Robusta coffee cultivation and breeding, and is hypothesized to be the region of origin of many of the cultivated Robusta varieties. Since little is known about the genetic composition of C. canephora in this region, we assessed the genetic diversity of wild and cultivated C. canephora shrubs in the Democratic Republic of the Congo.

MethodsUsing 18 microsatellite markers, we studied the genetic composition of wild and backyard-grown C. canephora shrubs in the Tshopo and Ituri provinces, and from the INERA Yangambi Coffee Collection. We assessed genetic clustering patterns, genetic diversity, and genetic differentiation between populations.

Key resultsGenetic differentiation was relatively strong between wild and cultivated C. canephora shrubs, and both gene pools harbored multiple unique alleles. Strong genetic differentiation was also observed between wild populations. The level of genetic diversity in wild populations was similar to that of the INERA Yangambi Coffee Collection, but local wild genotypes were mostly missing from that collection. Shrubs grown in the backyards were genetically similar to the breeding material from INERA Yangambi.

ConclusionsMost C. canephora that is grown in local backyards originated from INERA breeding programs, while a few shrubs were obtained directly from surrounding forests. The INERA Yangambi Coffee Collection could benefit from an enrichment with local wild genotypes, to increase the genetic resources available for breeding purposes, as well as to support ex situ conservation.


title: OPAM, an Open source, 3D printed Low-cost Micro-Manipulator for Single Cell Manipulation

abstract: We introduce OPAM, an Open source, low-cost (under $150), 3D-Printed, stepper motor driven, Arduino based, single cell Micromanipulator (OPAM). Modification of a commercial stepper motor led to dramatically increased stability and maneuverability of the motor, based on which the micromanipulator was designed. All components of this micromanipulator can be 3D printed using an entry-level 3D printer and assembled with ease. With this single cell manipulator, successful targeted single cell capture and transfer was confirmed under the microscope, which showed great promise for single cell related experiments.

category: bioengineering