Progressive ataxia of Charolais cattle highlights a role of KIF1C in sustainable myelination.

Hereditary spastic paraplegias (HSPs) are clinically and genetically heterogeneous human neurodegenerative diseases. Amongst the identified genetic causes, mutations in genes encoding motor proteins such as kinesins have been involved in various HSP clinical isoforms. Mutations in KIF1C are responsible for autosomal recessive spastic paraplegia type 58 (SPG58) and spastic ataxia 2 (SPAX2). Bovines also develop neurodegenerative diseases, some of them having a genetic aetiology. Bovine progressive ataxia was first described in the Charolais breed in the early 1970s in England and further cases in this breed were subsequently reported worldwide. We can now report that progressive ataxia of Charolais cattle results from a homozygous single nucleotide polymorphism in the coding region of the KIF1C gene. In this study, we show that the mutation at the heterozygous state is associated with a better score for muscular development, explaining its balancing selection for several decades, and the resulting high frequency (13%) of the allele in the French Charolais breed. We demonstrate that the KIF1C bovine mutation leads to a functional knock-out, therefore mimicking mutations in humans affected by SPG58/SPAX2. The functional consequences of KIF1C loss of function in cattle were also histologically reevaluated. We showed by an immunochemistry approach that demyelinating plaques were due to altered oligodendrocyte membrane protrusion, and we highlight an abnormal accumulation of actin in the core of demyelinating plaques, which is normally concentrated at the leading edge of oligodendrocytes during axon wrapping. We also observed that the lesions were associated with abnormal extension of paranodal sections. Moreover, this model highlights the role of KIF1C protein in preserving the structural integrity and function of myelin, since the clinical signs and lesions arise in young-adult Charolais cattle. Finally, this model provides useful information for SPG58/SPAX2 disease and other demyelinating lesions.

Attenuated sensing of SHH by Ptch1 underlies evolution of bovine limbs.

The large spectrum of limb morphologies reflects the wide evolutionary diversification of the basic pentadactyl pattern in tetrapods. In even-toed ungulates (artiodactyls, including cattle), limbs are adapted for running as a consequence of progressive reduction of their distal skeleton to symmetrical and elongated middle digits with hoofed phalanges. Here we analyse bovine embryos to establish that polarized gene expression is progressively lost during limb development in comparison to the mouse. Notably, the transcriptional upregulation of the Ptch1 gene, which encodes a Sonic hedgehog (SHH) receptor, is disrupted specifically in the bovine limb bud mesenchyme. This is due to evolutionary alteration of a Ptch1 cis-regulatory module, which no longer responds to graded SHH signalling during bovine handplate development. Our study provides a molecular explanation for the loss of digit asymmetry in bovine limb buds and suggests that modifications affecting the Ptch1 cis-regulatory landscape have contributed to evolutionary diversification of artiodactyl limbs.

Bovine and murine models highlight novel roles for SLC25A46 in mitochondrial dynamics and metabolism, with implications for human and animal health.

Neuropathies are neurodegenerative diseases affecting humans and other mammals. Many genetic causes have been identified so far, including mutations of genes encoding proteins involved in mitochondrial dynamics. Recently, the "Turning calves syndrome", a novel sensorimotor polyneuropathy was described in the French Rouge-des-Prés cattle breed. In the present study, we determined that this hereditary disease resulted from a single nucleotide substitution in SLC25A46, a gene encoding a protein of the mitochondrial carrier family. This mutation caused an apparent damaging amino-acid substitution. To better understand the function of this protein, we knocked out the Slc25a46 gene in a mouse model. This alteration affected not only the nervous system but also altered general metabolism, resulting in premature mortality. Based on optic microscopy examination, electron microscopy and on biochemical, metabolic and proteomic analyses, we showed that the Slc25a46 disruption caused a fusion/fission imbalance and an abnormal mitochondrial architecture that disturbed mitochondrial metabolism. These data extended the range of phenotypes associated with Slc25a46 dysfunction. Moreover, this Slc25a46 knock-out mouse model should be useful to further elucidate the role of SLC25A46 in mitochondrial dynamics.

Massive depletion of bovine leukemia virus proviral clones located in genomic transcriptionally active sites during primary infection.

Deltaretroviruses such as human T-lymphotropic virus type 1 (HTLV-1) and bovine leukemia virus (BLV) induce a persistent infection that remains generally asymptomatic but can also lead to leukemia or lymphoma. These viruses replicate by infecting new lymphocytes (i.e. the infectious cycle) or via clonal expansion of the infected cells (mitotic cycle). The relative importance of these two cycles in viral replication varies during infection. The majority of infected clones are created early before the onset of an efficient immune response. Later on, the main replication route is mitotic expansion of pre-existing infected clones. Due to the paucity of available samples and for ethical reasons, only scarce data is available on early infection by HTLV-1. Therefore, we addressed this question in a comparative BLV model. We used high-throughput sequencing to map and quantify the insertion sites of the provirus in order to monitor the clonality of the BLV-infected cells population (i.e. the number of distinct clones and abundance of each clone). We found that BLV propagation shifts from cell neoinfection to clonal proliferation in about 2 months from inoculation. Initially, BLV proviral integration significantly favors transcribed regions of the genome. Negative selection then eliminates 97% of the clones detected at seroconversion and disfavors BLV-infected cells carrying a provirus located close to a promoter or a gene. Nevertheless, among the surviving proviruses, clone abundance positively correlates with proximity of the provirus to a transcribed region. Two opposite forces thus operate during primary infection and dictate the fate of long term clonal composition: (1) initial integration inside genes or promoters and (2) host negative selection disfavoring proviruses located next to transcribed regions. The result of this initial response will contribute to the proviral load set point value as clonal abundance will benefit from carrying a provirus in transcribed regions.

Concordance analysis for QTL detection in dairy cattle: a case study of leg morphology.

BACKGROUND: The present availability of sequence data gives new opportunities to narrow down from QTL (quantitative trait locus) regions to causative mutations. Our objective was to decrease the number of candidate causative mutations in a QTL region. For this, a concordance analysis was applied for a leg conformation trait in dairy cattle. Several QTL were detected for which the QTL status (homozygous or heterozygous for the QTL) was inferred for each individual. Subsequently, the inferred QTL status was used in a concordance analysis to reduce the number of candidate mutations. METHODS: Twenty QTL for rear leg set side view were mapped using Bayes C. Marker effects estimated during QTL mapping were used to infer the QTL status for each individual. Subsequently, polymorphisms present in the QTL regions were extracted from the whole-genome sequences of 71 Holstein bulls. Only polymorphisms for which the status was concordant with the QTL status were kept as candidate causative mutations. RESULTS: QTL status could be inferred for 15 of the 20 QTL. The number of concordant polymorphisms differed between QTL and depended on the number of QTL statuses that could be inferred and the linkage disequilibrium in the QTL region. For some QTL, the concordance analysis was efficient and narrowed down to a limited number of candidate mutations located in one or two genes, while for other QTL a large number of genes contained concordant polymorphisms. CONCLUSIONS: For regions for which the concordance analysis could be performed, we were able to reduce the number of candidate mutations. For part of the QTL, the concordant analyses narrowed QTL regions down to a limited number of genes, of which some are known for their role in limb or skeletal development in humans and mice. Mutations in these genes are good candidates for QTN (quantitative trait nucleotides) influencing rear leg set side view.

Sample Size Considerations for Fine-Tuning Large Language Models for Named Entity Recognition Tasks: Methodological Study.

BACKGROUND: Large language models (LLMs) have the potential to support promising new applications in health informatics. However, practical data on sample size considerations for fine-tuning LLMs to perform specific tasks in biomedical and health policy contexts are lacking. OBJECTIVE: This study aims to evaluate sample size and sample selection techniques for fine-tuning LLMs to support improved named entity recognition (NER) for a custom data set of conflicts of interest disclosure statements. METHODS: A random sample of 200 disclosure statements was prepared for annotation. All "PERSON" and "ORG" entities were identified by each of the 2 raters, and once appropriate agreement was established, the annotators independently annotated an additional 290 disclosure statements. From the 490 annotated documents, 2500 stratified random samples in different size ranges were drawn. The 2500 training set subsamples were used to fine-tune a selection of language models across 2 model architectures (Bidirectional Encoder Representations from Transformers [BERT] and Generative Pre-trained Transformer [GPT]) for improved NER, and multiple regression was used to assess the relationship between sample size (sentences), entity density (entities per sentence [EPS]), and trained model performance (F1-score). Additionally, single-predictor threshold regression models were used to evaluate the possibility of diminishing marginal returns from increased sample size or entity density. RESULTS: Fine-tuned models ranged in topline NER performance from F1-score=0.79 to F1-score=0.96 across architectures. Two-predictor multiple linear regression models were statistically significant with multiple R2 ranging from 0.6057 to 0.7896 (all P<.001). EPS and the number of sentences were significant predictors of F1-scores in all cases ( P<.001), except for the GPT-2_large model, where EPS was not a significant predictor (P=.184). Model thresholds indicate points of diminishing marginal return from increased training data set sample size measured by the number of sentences, with point estimates ranging from 439 sentences for RoBERTa_large to 527 sentences for GPT-2_large. Likewise, the threshold regression models indicate a diminishing marginal return for EPS with point estimates between 1.36 and 1.38. CONCLUSIONS: Relatively modest sample sizes can be used to fine-tune LLMs for NER tasks applied to biomedical text, and training data entity density should representatively approximate entity density in production data. Training data quality and a model architecture's intended use (text generation vs text processing or classification) may be as, or more, important as training data volume and model parameter size.

Safety of long-term treatment of HAM/TSP patients with valproic acid.

HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurodegenerative disease of the central nervous system induced by human T-lymphotropic virus type 1. As a potential therapeutic approach, we previously suggested reducing the proviral load by modulating lysine deacetylase activity using valproic acid (VPA) and exposing virus-positive cells to the host immune response. We conducted a single-center, 2-year, open-label trial, with 19 HAM/TSP volunteers treated with oral VPA. Proviral load, CD38/HLA-DR expression, and CD8(+) lysis efficiency were not significantly affected by VPA. Mean scores of HAM/TSP disability did not differ between baseline and final visit. Walking Time Test increased significantly (> 20%) in 3 patients and was in keeping with minor VPA side effects (drowsiness and tremor). Walking Time Test improved rapidly after VPA discontinuation. We conclude that long-term treatment with VPA is safe in HAM/TSP.

Modelling metastasis in zebrafish unveils regulatory interactions of cancer-associated fibroblasts with circulating tumour cells.

The dynamic intercommunication between tumour cells and cells from the microenvironment, such as cancer-associated fibroblast (CAFs), is a key factor driving breast cancer (BC) metastasis. Clusters of circulating tumour cells (CTCs), known to bare a higher efficiency at establishing metastases, are found in the blood of BC patients, often accompanied by CAFs in heterotypic CTC-clusters. Previously we have shown the utility of CTC-clusters models and the zebrafish embryo as a model of metastasis to understand the biology of breast cancer CTC-clusters. In this work, we use the zebrafish embryo to study the interactions between CTCs in homotypic clusters and CTC-CAFs in heterotypic CTC-clusters to identify potential pro-metastatic traits derived from CTC-CAF communication. We found that upon dissemination CAFs seem to exert a pro-survival and pro-proliferative effect on the CTCs, but only when CTCs and CAFs remain joined as cell clusters. Our data indicate that the clustering of CTC and CAF allows the establishment of physical interactions that when maintained over time favour the selection of CTCs with a higher capacity to survive and proliferate upon dissemination. Importantly, this effect seems to be dependent on the survival of disseminated CAFs and was not observed in the presence of normal fibroblasts. Moreover, we show that CAFs can exert regulatory effects on the CTCs without being involved in promoting tumour cell invasion. Lastly, we show that the physical communication between BC cells and CAFs leads to the production of soluble factors involved in BC cell survival and proliferation. These findings suggest the existence of a CAF-regulatory effect on CTC survival and proliferation sustained by cell-to-cell contacts and highlight the need to understand the molecular mechanisms that mediate the interaction between the CTCs and CAFs in clusters enhancing the metastatic capacity of CTCs.

African 2, a clonal complex of Mycobacterium bovis epidemiologically important in East Africa.

We have identified a clonal complex of Mycobacterium bovis isolated at high frequency from cattle in Uganda, Burundi, Tanzania, and Ethiopia. We have named this related group of M. bovis strains the African 2 (Af2) clonal complex of M. bovis. Af2 strains are defined by a specific chromosomal deletion (RDAf2) and can be identified by the absence of spacers 3 to 7 in their spoligotype patterns. Deletion analysis of M. bovis isolates from Algeria, Mali, Chad, Nigeria, Cameroon, South Africa, and Mozambique did not identify any strains of the Af2 clonal complex, suggesting that this clonal complex of M. bovis is localized in East Africa. The specific spoligotype pattern of the Af2 clonal complex was rarely identified among isolates from outside Africa, and the few isolates that were found and tested were intact at the RDAf2 locus. We conclude that the Af2 clonal complex is localized to cattle in East Africa. We found that strains of the Af2 clonal complex of M. bovis have, in general, four or more copies of the insertion sequence IS6110, in contrast to the majority of M. bovis strains isolated from cattle, which are thought to carry only one or a few copies.

Mycobacterium caprae infection in livestock and wildlife, Spain.

Mycobacterium caprae is a pathogen that can infect animals and humans. To better understand the epidemiology of M. caprae, we spoligotyped 791 animal isolates. Results suggest infection is widespread in Spain, affecting 6 domestic and wild animal species. The epidemiology is driven by infections in caprids, although the organism has emerged in cattle.

MINTIA: a metagenomic INserT integrated assembly and annotation tool.

The earth harbors trillions of bacterial species adapted to very diverse ecosystems thanks to specific metabolic function acquisition. Most of the genes responsible for these functions belong to uncultured bacteria and are still to be discovered. Functional metagenomics based on activity screening is a classical way to retrieve these genes from microbiomes. This approach is based on the insertion of large metagenomic DNA fragments into a vector and transformation of a host to express heterologous genes. Metagenomic libraries are then screened for activities of interest, and the metagenomic DNA inserts of active clones are extracted to be sequenced and analysed to identify genes that are responsible for the detected activity. Hundreds of metagenomics sequences found using this strategy have already been published in public databases. Here we present the MINTIA software package enabling biologists to easily generate and analyze large metagenomic sequence sets, retrieved after activity-based screening. It filters reads, performs assembly, removes cloning vector, annotates open reading frames and generates user friendly reports as well as files ready for submission to international sequence repositories. The software package can be downloaded from https://github.com/Bios4Biol/MINTIA.

Geographical distribution and ecological niche modeling of the etiological agents of human sporotrichosis in Venezuela.

The geographical distribution and ecological niche of the two circulating species of the Sporothrix genus in Venezuela was established. For this, 68 isolates of Sporothrix spp. from patients of different regions of the country were analyzed. A molecular taxonomy analysis was conducted using a fragment of the calmodulin gene (CAL), and ITS regions, confirming the presence of S. schenckii (62%) and S. globosa (38%). Computational models of ecological niche for each species were obtained by the maximum entropy method using the MaxEnt software, which predicted the best environmental conditions for the presence of the two species. These models predict that the main variables influencing the presence of S. schenckii were altitude and annual mean temperature, while for S. globosa, the more influent variable was the land use, with 82% of S. globosa located at urban areas vs 56% for S. schenckii. The results here presented could contribute to understand the specific environmental factors that might modulate the occurrence of Sporothrix spp. as well as its transmission. To our knowledge, our analyses show for the first time Sporothrix spp.-specific ecological niche data, a valuable tool to promote evidence-based public health policymaking within endemic areas of sporotrichosis.

Small genes under sporulation control in the Bacillus subtilis genome.

Using an oligonucleotide microarray, we searched for previously unrecognized transcription units in intergenic regions in the genome of Bacillus subtilis, with an emphasis on identifying small genes activated during spore formation. Nineteen transcription units were identified, 11 of which were shown to depend on one or more sporulation-regulatory proteins for their expression. A high proportion of the transcription units contained small, functional open reading frames (ORFs). One such newly identified ORF is a member of a family of six structurally similar genes that are transcribed under the control of sporulation transcription factor σ(E) or σ(K). A multiple mutant lacking all six genes was found to sporulate with slightly higher efficiency than the wild type, suggesting that under standard laboratory conditions the expression of these genes imposes a small cost on the production of heat-resistant spores. Finally, three of the transcription units specified small, noncoding RNAs; one of these was under the control of the sporulation transcription factor σ(E), and another was under the control of the motility sigma factor σ(D).

Bovine leukemia virus can be classified into seven genotypes: evidence for the existence of two novel clades.

Previous studies have classified the env sequences of bovine leukemia virus (BLV) provirus from different locations worldwide into between two and four genetic groupings. These different studies gave unique names to the identified groups and no study has yet integrated all the available sequences. Thus, we hypothesized that many of the different groups previously identified actually correspond to a limited group of genotypes that are unevenly distributed worldwide. To examine this hypothesis, we sequenced the env gene from 28 BLV field strains and compared these sequences to 46 env sequences that represent all the genetic groupings already identified. By using phylogenetic analyses, we recovered six clades, or genotypes, that we have called genotypes 1, 2, 3, 4, 5 and 6. Genotypes 1-5 have counterparts among the sequence groupings identified previously. One env sequence did not cluster with any of the others and was highly divergent when compared with the six genotypes identified here. Thus, an extra genotype, which we named 7, may exist. Similarity comparisons were highly congruent with phylogenetic analyses. Furthermore, our analyses confirmed the existence of geographical clusters.

Single nucleotide polymorphisms in the IS900 sequence of Mycobacterium avium subsp. paratuberculosis are strain type specific.

Insertion sequence IS900 is used as a target for the identification of Mycobacterium avium subsp. paratuberculosis. Previous reports have revealed single nucleotide polymorphisms within IS900. This study, which analyzed the IS900 sequences of a panel of isolates representing M. avium subsp. paratuberculosis strain types I, II, and III, revealed conserved type-specific polymorphisms that could be utilized as a tool for diagnostic and epidemiological purposes.

Effective lead selection for improved protein production in Aspergillus niger based on integrated genomics.

The filamentous fungus Aspergillus niger is widely exploited for industrial production of enzymes and organic acids. An integrated genomics approach was developed to determine cellular responses of A. niger to protein production in well-controlled fermentations. Different protein extraction methods in combination with automated sample processing and protein identification allowed quantitative analysis of 898 proteins. Three different enzyme overproducing strains were compared to their isogenic fungal host strains. Clear differences in response to the amount and nature of the overproduced enzymes were observed. The corresponding genes of the differentially expressed proteins were studied using transcriptomics. Genes that were up-regulated both at the proteome and transcriptome level were selected as leads for generic strain improvement. Up-regulated proteins included proteins involved in carbon and nitrogen metabolism as well as (oxidative) stress response, and proteins involved in protein folding and endoplasmic reticulum-associated degradation (ERAD). Reduction of protein degradation through the removal of the ERAD factor doaA combined with overexpression of the oligosaccharyl transferase sttC in A. niger overproducing beta-glucuronidase (GUS) strains indeed resulted in a small increase in GUS expression.

A Francisella novicida Mutant, Lacking the Soluble Lytic Transglycosylase Slt, Exhibits Defects in Both Growth and Virulence.

Francisella tularensis is the causative agent of tularemia and has gained recent interest as it poses a significant biothreat risk. F. novicida is commonly used as a laboratory surrogate for tularemia research due to genetic similarity and susceptibility of mice to infection. Currently, there is no FDA-approved tularemia vaccine, and identifying therapeutic targets remains a critical gap in strategies for combating this pathogen. Here, we investigate the soluble lytic transglycosylase or Slt in F. novicida, which belongs to a class of peptidoglycan-modifying enzymes known to be involved in cell division. We assess the role of Slt in biology and virulence of the organism as well as the vaccine potential of the slt mutant. We show that the F. novicida slt mutant has a significant growth defect in acidic pH conditions. Further microscopic analysis revealed significantly altered cell morphology compared to wild-type, including larger cell size, extensive membrane protrusions, and cell clumping and fusion, which was partially restored by growth in neutral pH or genetic complementation. Viability of the mutant was also significantly decreased during growth in acidic medium, but not at neutral pH. Furthermore, the slt mutant exhibited significant attenuation in a murine model of intranasal infection and virulence could be restored by genetic complementation. Moreover, we could protect mice using the slt mutant as a live vaccine strain against challenge with the parent strain; however, we were not able to protect against challenge with the fully virulent F. tularensis Schu S4 strain. These studies demonstrate a critical role for the Slt enzyme in maintaining proper cell division and morphology in acidic conditions, as well as replication and virulence in vivo. Our results suggest that although the current vaccination strategy with F. novicida slt mutant would not protect against Schu S4 challenges, the Slt enzyme could be an ideal target for future therapeutic development.

The origins of 168, W23, and other Bacillus subtilis legacy strains.

Bacillus subtilis is both a model organism for basic research and an industrial workhorse, yet there are major gaps in our understanding of the genomic heritage and provenance of many widely used strains. We analyzed 17 legacy strains dating to the early years of B. subtilis genetics. For three--NCIB 3610T, PY79, and SMY--we performed comparative genome sequencing. For the remainder, we used conventional sequencing to sample genomic regions expected to show sequence heterogeneity. Sequence comparisons showed that 168, its siblings (122, 160, and 166), and the type strains NCIB 3610 and ATCC 6051 are highly similar and are likely descendants of the original Marburg strain, although the 168 lineage shows genetic evidence of early domestication. Strains 23, W23, and W23SR are identical in sequence to each other but only 94.6% identical to the Marburg group in the sequenced regions. Strain 23, the probable W23 parent, likely arose from a contaminant in the mutagenesis experiments that produced 168. The remaining strains are all genomic hybrids, showing one or more "W23 islands" in a 168 genomic backbone. Each traces its origin to transformations of 168 derivatives with DNA from 23 or W23. The common prototrophic lab strain PY79 possesses substantial W23 islands at its trp and sac loci, along with large deletions that have reduced its genome 4.3%. SMY, reputed to be the parent of 168, is actually a 168-W23 hybrid that likely shares a recent ancestor with PY79. These data provide greater insight into the genomic history of these B. subtilis legacy strains.

Parental awareness of schoolbag carriage: A comparative study of Irish and United States parents.

BACKGROUND: Given the global nature of schoolbag carriage, there has been extensive research on schoolbag weight and use with resultant guidance on many aspects of carrying a schoolbag. However, there is limited evidence of knowledge translation or parents' awareness of schoolbag carriage. OBJECTIVE: This study investigated parental awareness of factors related to schoolbag carriage. METHOD: A cross-sectional survey using an anonymous 30-item questionnaire and purposive sampling was used. Questionnaires were distributed to parents of primary school children through the schools. Descriptive statistics of frequencies and percentages were used and associations were tested using Chi-square analysis in SPSS v23. RESULTS: A total of 700 parents in Ireland (Ire) and the United States (US) participated in the study (n = 444 [Ire] and n = 256 [US]). Generally, parents had satisfactory awareness of appropriate schoolbag type and carriage. The majority of children owned a backpack (89.9% [Ire] vs. 93.7% [US]), although fewer parents considered this to be the most suitable bag for their child (69.6% [Ire] vs. 88.2% [US]). More Irish parents (29.2%) favoured a wheeled schoolbag compared to US parents (6.2%) (p < 0.001). The majority (70.8% [Ire] vs. 55.7% [US]) wanted more information. The preferred platforms for receiving information were a handout (78.1% [Ire] vs. 71.6% [US]) and on-line (44.6% [Ire] vs. 53.9% [US]). CONCLUSIONS: Despite gaps identified, parents had good awareness of factors relating to schoolbag carriage, but this study shows that they would like more information. The preferred platform for knowledge translation was a handout. Parents are the best advocates for safety promotion and represent the group most likely to improve schoolbag carriage among children.