The human infertility single-cell testis atlas (HISTA): an interactive molecular scRNA-Seq reference of the human testis.

BACKGROUND: Single-cell RNA-seq (scRNA-Seq) has been widely adopted to study gene expression of the human testis. Several datasets of scRNA-Seq from human testis have been generated from different groups processed with different informatics pipelines. An integrated atlas of scRNA-Seq expression constructed from multiple donors, developmental ages, and fertility states would be widely useful for the testis research community. OBJECTIVE: To describe the generation and use of the human infertility single-cell testis atlas (HISTA), an interactive web tool for understanding human spermatogenesis through scRNA-Seq analysis. METHODS: We obtained scRNA-Seq datasets derived from 12 donors, including healthy adult controls, juveniles, and several infertility cases, and reprocessed these data using methods to remove batch effects. Using Shiny, an open-source environment for data visualization, we created numerous interactive tools for exploring the data, some of which support simple statistical hypothesis testing. We used the resulting HISTA browser and its underlying data to demonstrate HISTA's value for testis researchers. RESULTS: A primary application of HISTA is to search by a single gene or a set of genes; thus, we present various analyses that quantify and visualize gene expression across the testis cells and pathology. HISTA also contains machine-learning-derived gene modules ("components") that capture the entire transcriptional landscape of the testis tissue. We show how the use of these components can simplify the highly complex data in HISTA and assist with the interpretation of genes with unknown functions. Finally, we demonstrate the diverse ways HISTA can be used for new data analysis, including hypothesis testing. DISCUSSION AND CONCLUSIONS: HISTA is a research environment that can help scientists organize and understand the high-dimensional transcriptional landscape of the human testis. HISTA has already contributed to published testis research and can be updated as needed with input from the research community or downloaded and modified for individual needs.

TAD evolutionary and functional characterization reveals diversity in mammalian TAD boundary properties and function.

Topological associating domains (TADs) are self-interacting genomic units crucial for shaping gene regulation patterns. Despite their importance, the extent of their evolutionary conservation and its functional implications remain largely unknown. In this study, we generate Hi-C and ChIP-seq data and compare TAD organization across four primate and four rodent species and characterize the genetic and epigenetic properties of TAD boundaries in correspondence to their evolutionary conservation. We find 14% of all human TAD boundaries to be shared among all eight species (ultraconserved), while 15% are human-specific. Ultraconserved TAD boundaries have stronger insulation strength, CTCF binding, and enrichment of older retrotransposons compared to species-specific boundaries. CRISPR-Cas9 knockouts of an ultraconserved boundary in a mouse model lead to tissue-specific gene expression changes and morphological phenotypes. Deletion of a human-specific boundary near the autism-related AUTS2 gene results in the upregulation of this gene in neurons. Overall, our study provides pertinent TAD boundary evolutionary conservation annotations and showcases the functional importance of TAD evolution.

Expansion of the area of occurrence of the genus Anochetus Mayr 1861 (Hymenoptera: Formicidae).

The study was carried out on an agroecological property located in Cologne São Manoel, 8th district of Pelotas, RS, Brazil. Anochetus neglectus Emery, 1894 was collected in an area of peach orchard that has been under an agroecological system for 18 years. This record expands the area of ​​occurrence and distribution of this rare species in the Neotropical Region.

A naturally occurring variant of MBD4 causes maternal germline hypermutation in primates.

As part of an ongoing genome sequencing project at the Oregon National Primate Research Center, we identified a rhesus macaque with a rare homozygous frameshift mutation in the gene methyl-CpG binding domain 4, DNA glycosylase (MBD4). MBD4 is responsible for the repair of C > T deamination mutations at CpG dinucleotides and has been linked to somatic hypermutation and cancer predisposition in humans. We show here that MBD4-associated hypermutation also affects the germline: The six offspring of the MBD4-null dam have a fourfold to sixfold increase in de novo mutation burden. This excess burden was predominantly C > T mutations at CpG dinucleotides consistent with MBD4 loss of function in the dam. There was also a significant excess of C > T at CpA sites, indicating an important, unappreciated role for MBD4 to repair deamination in CpA contexts. The MBD4-null dam developed sustained eosinophilia later in life, but we saw no other signs of neoplastic processes associated with MBD4 loss of function in humans nor any obvious disease in the hypermutated offspring. This work provides the first evidence for a genetic factor causing hypermutation in the maternal germline of a mammal and adds to the very small list of naturally occurring variants known to modulate germline mutation rates in mammals.

TAD Evolutionary and functional characterization reveals diversity in mammalian TAD boundary properties and function.

Topological associating domains (TADs) are self-interacting genomic units crucial for shaping gene regulation patterns. Despite their importance, the extent of their evolutionary conservation and its functional implications remain largely unknown. In this study, we generate Hi-C and ChIP-seq data and compare TAD organization across four primate and four rodent species, and characterize the genetic and epigenetic properties of TAD boundaries in correspondence to their evolutionary conservation. We find that only 14% of all human TAD boundaries are shared among all eight species (ultraconserved), while 15% are human-specific. Ultraconserved TAD boundaries have stronger insulation strength, CTCF binding, and enrichment of older retrotransposons, compared to species-specific boundaries. CRISPR-Cas9 knockouts of two ultraconserved boundaries in mouse models leads to tissue-specific gene expression changes and morphological phenotypes. Deletion of a human-specific boundary near the autism-related AUTS2 gene results in upregulation of this gene in neurons. Overall, our study provides pertinent TAD boundary evolutionary conservation annotations, and showcase the functional importance of TAD evolution.

SATINN: an automated neural network-based classification of testicular sections allows for high-throughput histopathology of mouse mutants.

MOTIVATION: The mammalian testis is a complex organ with a cellular composition that changes smoothly and cyclically in normal adults. While testis histology is already an invaluable tool for identifying and describing developmental differences in evolution and disease, methods for standardized, digital image analysis of testis are needed to expand the utility of this approach. RESULTS: We developed SATINN (Software for Analysis of Testis Images with Neural Networks), a multi-level framework for automated analysis of multiplexed immunofluorescence images from mouse testis. This approach uses residual learning to train convolutional neural networks (CNNs) to classify nuclei from seminiferous tubules into seven distinct cell types with an accuracy of 81.7%. These cell classifications are then used in a second-level tubule CNN, which places seminiferous tubules into one of 12 distinct tubule stages with 57.3% direct accuracy and 94.9% within ±1 stage. We further describe numerous cell- and tubule-level statistics that can be derived from wild-type testis. Finally, we demonstrate how the classifiers and derived statistics can be used to rapidly and precisely describe pathology by applying our methods to image data from two mutant mouse lines. Our results demonstrate the feasibility and potential of using computer-assisted analysis for testis histology, an area poised to evolve rapidly on the back of emerging, spatially resolved genomic and proteomic technologies. AVAILABILITY AND IMPLEMENTATION: The source code to reproduce the results described here and a SATINN standalone application with graphic-user interface are available from http://github.com/conradlab/SATINN. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Microfluidic-assisted electrospinning, an alternative to coaxial, as a controlled dual drug release system to treat inflammatory arthritic diseases.

Inflammatory arthritic diseases are characterized by a persistent inflammation of the synovial tissues where tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) pro-inflammatory cytokines are over-expressed, leading to progressive musculoskeletal disability. Methotrexate (MTX), a disease-modifying-anti-rheumatic drug (DMARD) commonly applied in their treatment, can be used in combination with biological-DMARDs as anti-TNFα antibody to improve the treatments efficacy. However, their systemic administration comes with severe side-effects and limited therapeutic efficacy due to their off-target distribution and short half-life. To overcome such limitations, encapsulation of clinically relevant concentrations of MTX and anti-TNFα antibody into polycaprolactone (PCL) or poly(vinyl-alcohol) (PVA) microfluidic-assisted or coaxial electrospun fibrous meshes is proposed as local controlled dual drug release systems. Release studies show that microfluidic-assisted electrospinning meshes encapsulating both drugs achieved higher concentrations than coaxials. Biological assays using human articular chondrocytes (hACs) and monocytic cells (THP-1 cell line) demonstrate that fibrous meshes encapsulating the drugs are non-toxic. The systems' efficacy is proved by a significant decrease of TNFα and IL-6 concentrations in conditioned medium of lipopolysaccharide (LPS)-stimulated THP-1 cells, especially in the presence of microfluidic-assisted electrospun meshes, when compared with THP-1 conditioned medium (59.5% and 83.9% less, respectively). Therefore, microfluidic-assisted electrospinning fibrous meshes with encapsulating drugs represent an alternative to coaxial, as a local therapy for inflammatory arthritis diseases.

Leishmania infantum infection rate in dogs housed in open-admission shelters is higher than of domiciled dogs in an endemic area of canine visceral leishmaniasis. Epidemiological implications.

Canine visceral leishmaniasis (CVL) is caused by Leishmania infantum and is endemic in many areas of southeastern Brazil. We have hypothesized that the prevalence of infection by L. infantum in dogs housed in open-admission animal shelters is beyond the range of 3.4 - 9.6% reported among dogs domiciled in similar CVL-endemic areas. Hence, this study aimed to determine the rate of L. infantum infection among dogs maintained in shelters and to investigate the epidemiology of CVL in such environments by analyzing hematological and biochemical parameters. A total of 627 dogs from 17 different shelters across the State of Minas Gerais were screened using the Dual-Path Platform test and enzyme-linked immunosorbent assay and 211 (33.6%) were found to be seropositive in both tests. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed on skin, bone marrow and lymphoid tissues of 118 seropositive dogs with inconclusive CVL clinical diagnosis and, of these, 78 (66.1%) were PCR+ for L. infantum and 7 (5.9%) were PCR+ for L. amazonensis. One dog presented a PCR-RFLP profile that was consistent with co-infection by both parasites. Leishmania amazonensis DNA was detected in skin samples of six single-infected dogs and this constitutes a novel finding. Dogs infected only with L. amazonensis were less debilitated than those infected by L. infantum, which showed typical clinical manifestations of CVL. The co-infected dog showed only mild clinical signs. The results presented herein not only support our original hypothesis but also suggest that dogs are potential reservoirs of L. amazonensis. Public health authorities should acknowledge their responsibility towards animals in collective shelters, recognize that they are potential foci of zoonotic diseases, and establish proper functioning directives to minimize transmission to humans and to other dogs.

Precision biomaterials in cancer theranostics and modelling.

Despite significant achievements in the understanding and treatment of cancer, it remains a major burden. Traditional therapeutic approaches based on the 'one-size-fits-all' paradigm are becoming obsolete, as demonstrated by the increasing number of patients failing to respond to treatments. In contrast, more precise approaches based on individualized genetic profiling of tumors have already demonstrated their potential. However, even more personalized treatments display shortcomings mainly associated with systemic delivery, such as low local drug efficacy or specificity. A large amount of effort is currently being invested in developing precision medicine-based strategies for improving the efficiency of cancer theranostics and modelling, which are envisioned to be more accurate, standardized, localized, and less expensive. To this end, interdisciplinary research fields, such as biomedicine, material sciences, pharmacology, chemistry, tissue engineering, and nanotechnology, must converge for boosting the precision cancer ecosystem. In this regard, precision biomaterials have emerged as a promising strategy to detect, model, and treat cancer more efficiently. These are defined as those biomaterials precisely engineered with specific theranostic functions and bioactive components, with the possibility to be tailored to the cancer patient needs, thus having a vast potential in the increasing demand for more efficient treatments. In this review, we discuss the latest advances in the field of precision biomaterials in cancer research, which are expected to revolutionize disease management, focusing on their uses for cancer modelling, detection, and therapeutic applications. We finally comment on the needed requirements to accelerate their application in the clinic to improve cancer patient prognosis.

What is the influence of agroecological and conventional crops under ant assemblages?

The objective of the study was to compare the richness and diversity of ant assemblages in an agroecological system under peach orchard, conventional system under peach orchard cultivation and native vegetation in rural properties located in a Pampa Biome. The study was conducted in four samplings in 2017: 1st and 09th March (summer); 24th and 31st July (winter); and four samplings in 2018: 23rd and 30th January (summer); 31st July and 07th August (winter). Pitfall traps were used. The assemblages were characterized and compared using richness, number of occurrences of ants, Shannon diversity (H'), equitability, rarefaction analysis and Chao 1. The association of the species with the samples was evaluated by a Principal Component Analysis (PCA). The agroecological system had the highest number of occurrences, while the conventional orchard the lowest number. Richness and abundance were greatest during the summer. The conventional peach orchard obtained the lowest H' for both seasons when compared to the agroecological orchard and native vegetation. The PCA explained 77.40% of the occurrence of ants in the environments and in the seasons. The results found demonstrated that conservationist systems tend to harbor greater wealth and diversity of ant assemblages, as well as occurring in native áreas.

Comparative single-cell analysis of biopsies clarifies pathogenic mechanisms in Klinefelter syndrome.

Klinefelter syndrome (KS), also known as 47, XXY, is characterized by a distinct set of physiological abnormalities, commonly including infertility. The molecular basis for Klinefelter-related infertility is still unclear, largely because of the cellular complexity of the testis and the intricate endocrine and paracrine signaling that regulates spermatogenesis. Here, we demonstrate an analysis framework for dissecting human testis pathology that uses comparative analysis of single-cell RNA-sequencing data from the biopsies of 12 human donors. By comparing donors from a range of ages and forms of infertility, we generate gene expression signatures that characterize normal testicular function and distinguish clinically distinct forms of male infertility. Unexpectedly, we identified a subpopulation of Sertoli cells within multiple individuals with KS that lack transcription from the XIST locus, and the consequence of this is increased X-linked gene expression compared to all other KS cell populations. By systematic assessment of known cell signaling pathways, we identify 72 pathways potentially active in testis, dozens of which appear upregulated in KS. Altogether our data support a model of pathogenic changes in interstitial cells cascading from loss of X inactivation in pubertal Sertoli cells and nominate dosage-sensitive factors secreted by Sertoli cells that may contribute to the process. Our findings demonstrate the value of comparative patient analysis in mapping genetic mechanisms of disease and identify an epigenetic phenomenon in KS Sertoli cells that may prove important for understanding causes of infertility and sex chromosome evolution.

Tumor-Associated Protrusion Fluctuations as a Signature of Cancer Invasiveness.

The generation of invasive fluctuating protrusions is a distinctive feature of tumor dissemination. During the invasion, individual cancer cells modulate the morphodynamics of protrusions to optimize their migration efficiency. However, it remains unclear how protrusion fluctuations govern the invasion of more complex multi-cellular structures, such as tumors, and their correlation with the tumor metastatic potential. Herein, a reductionist approach based on 3D tumor cell micro-spheroids with different invasion capabilities is used as a model to decipher the role of tumor-associated fluctuating protrusions in cancer progression. To quantify fluctuations, a set of key biophysical parameters that precisely correlate with the invasive potential of tumors is defined. It is shown that different pharmacological drugs and cytokines are capable of modulating protrusion activity, significantly altering protrusion fluctuations, and tumor invasiveness. This correlation is used to define a novel quantitative invasion index encoding the key biophysical parameters of fluctuations and the relative levels of cell-cell/matrix interactions, which is capable of assessing the tumor's metastatic capability solely based on its magnitude. Overall, this study provides new insights into how protrusion fluctuations regulate tumor cell invasion, suggesting that they may be employed as a novel early indicator, or biophysical signature, of the metastatic potential of tumors.

Effects of additives in wet brewery residue silage on lamb carcass traits and meat quality.

The objective was to evaluate the use of wet brewery residue (WBR) silage additives on carcass characteristics and sheep meat quality. Thirty-two Santa Inês male sheep uncastrated with initial body weight of 22.61 ± 7.2 kg were allocated to a completely randomized design with four treatments: (1) WBR silage without additive (WBRS), (2) WBR silage with milled corn (WBRS + MC), (3) WBR silage with wheat bran (WBRS + WB), and (4) WBR silage with cassava flour (WBRS + CF) and eight replicates. WBRS + WB resulted in lower cold carcass weight than WBRS + CF; however, this reduction was not sufficient to alter the carcass commercial yield or loin-eye area. The leg cut of animals fed WBRS + WB showed less value than those animals fed with WBRS + CS. The meat lightness of WBRS was higher that of WBRS + MC, WBRS + WB, and WBRS + CF. The cooking loss for WBRS + WB was less than those animals fed with WBRS + CS. However, meat protein, meat cholesterol, and shear force were similar among treatments (17.69%, 42.46 mg/100 g of meat, and 2.48 kgf/cm2, respectively). The use of additives in wet brewery residue silage does not improve carcass characteristics or the quality of sheep meat, and it is therefore recommended to use WBR silage without additives.

Magnetic Nanoparticles for Biomedical Applications: From the Soul of the Earth to the Deep History of Ourselves.

Precisely engineered magnetic nanoparticles (MNPs) have been widely explored for applications including theragnostic platforms, drug delivery systems, biomaterial/device coatings, tissue engineering scaffolds, performance-enhanced therapeutic alternatives, and even in SARS-CoV-2 detection strips. Such popularity is due to their unique, challenging, and tailorable physicochemical/magnetic properties. Given the wide biomedical-related potential applications of MNPs, significant achievements have been reached and published (exponentially) in the last five years, both in synthesis and application tailoring. Within this review, and in addition to essential works in this field, we have focused on the latest representative reports regarding the biomedical use of MNPs including characteristics related to their oriented synthesis, tailored geometry, and designed multibiofunctionality. Further, actual trends, needs, and limitations of magnetic-based nanostructures for biomedical applications will also be discussed.

Canine cutaneous neoplasms in the metropolitan region of Goiânia, Goiás state, Brazil / Neoplasias cutâneas caninas na região metropolitana de Goiânia, Goiás, Brasil

ABSTRACT: The present study aimed to describe the occurrence and epidemiological features of skin neoplasms diagnosed in dogs in the metropolitan region of Goiânia, Goiás state, Brazil. Diagnoses from dog biopsies from 2011 to 2016 provided by a private veterinary pathology laboratory were analyzed. The main diagnoses were mast cell tumor, hemangiosarcoma, squamous cell carcinoma, malignant melanoma, and hemangioma. Highest frequency of neoplasms was found in female dogs, dogs aged > 8 years, and purebred dogs, particularly the American Pit Bull Terriers and the Poodles. Most common sites affected by the neoplasms were the limb and the head. Using multiple correspondence analysis, groups of neoplasms were found to be associated with different epidemiological features and the size of the neoplasms was associated with the biological behavior. The results of this study described predispositions and verified the importance of different types of skin neoplasms in dogs in the region being studied.(AU)

O objetivo deste estudo foi determinar a prevalência e as características epidemiológicas das neoplasias cutâneas em cães na região metropolitana de Goiânia, Goiás. Foram analisados os diagnósticos de um laboratório do setor privado de 2011 a 2016. Mastocitoma, hemangiossarcoma, carcinoma de células escamosas, melanoma maligno e hemangioma representaram os principais diagnósticos. A maioria dos casos ocorreram em cães de raças definidas, fêmeas e com idade >8 anos. American Pit Bull Terrier e Poodle foram as raças mais encontradas. As neoplasias acometeram principalmente regiões de membros e cabeça. Pela análise de correspondência múltipla, associou-se os grupos de neoplasias com diferentes características epidemiológicas e o tamanho da neoplasia com o comportamento biológico. A comparação dos resultados com pesquisas prévias possibilitou confirmar predisposições previamente descritas e verificar a importância dos diferentes tipos de neoplasias cutâneas em cães na região estudada.(AU)

Rare mutations in the complement regulatory gene CSMD1 are associated with male and female infertility.

Infertility in men and women is a complex genetic trait with shared biological bases between the sexes. Here, we perform a series of rare variant analyses across 73,185 women and men to identify genes that contribute to primary gonadal dysfunction. We report CSMD1, a complement regulatory protein on chromosome 8p23, as a strong candidate locus in both sexes. We show that CSMD1 is enriched at the germ-cell/somatic-cell interface in both male and female gonads. Csmd1-knockout males show increased rates of infertility with significantly increased complement C3 protein deposition in the testes, accompanied by severe histological degeneration. Knockout females show significant reduction in ovarian quality and breeding success, as well as mammary branching impairment. Double knockout of Csmd1 and C3 causes non-additive reduction in breeding success, suggesting that CSMD1 and the complement pathway play an important role in the normal postnatal development of the gonads in both sexes.

Commercially Relevant Orthogonal Multi-Component Supramolecular Hydrogels for Programmed Cell Growth.

This study reports the ability of synthetically simple, commercially viable sugar-derived 1,3:2,4-dibenzylidenesorbitol-4',4"-diacylhydrazide (DBS-CONHNH2 ) to support cell growth. Simple mixing and orthogonal self-sorting can formulate heparin, agarose, and heparin-binding micelles into these gels-easily incorporating additional function. Interestingly, the components used in the gel formulation, direct the ability of cells to grow, meaning the chemical programming of these multi-component gels is directly translated to the biological systems in contact with them. This simple approach has potential for future development in regenerative medicine.

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry.

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.