Dermal Fibroblast Cell Line from a Patient with the Huntington's Disease as a Promising Model for Studying Disease Pathogenesis: Production and Characterization.

Huntington's disease (HD) is an incurable hereditary disease caused by expansion of the CAG repeats in the HTT gene encoding the mutant huntingtin protein (mHTT). Despite numerous studies in cellular and animal models, the mechanisms underlying the biological role of mHTT and its toxicity to striatal neurons have not yet been established and no effective therapy for HD patients has been developed so far. We produced and characterized a new line of dermal fibroblasts (HDDF, Huntington's disease dermal fibroblasts) from a patient with a confirmed HD diagnosis. We also studied the growth characteristics of HDDF cells, stained them for canonical markers, karyotyped these cells, and investigated their phenotype. HDDF cells was successfully reprogrammed into induced striatal neurons via transdifferentiation. The new fibroblast line can be used as a cell model to study the biological role of mHTT and manifestations of HD pathogenesis in both fibroblasts and induced neuronal cells obtained from them by reprogramming techniques.

Focal Adhesion Maturation Responsible for Behavioral Changes in Human Corneal Stromal Fibroblasts on Fibrillar Substrates.

The functioning of the human cornea heavily relies on the maintenance of its extracellular matrix (ECM) mechanical properties. Within this context, corneal stromal fibroblasts (CSFs) are essential, as they are responsible for remodeling the corneal ECM. In this study, we used a decellularized human amniotic membrane (dHAM) and a custom fibrillar collagen film (FCF) to explore the effects of fibrillar materials on human CSFs. Our findings indicate that substrates like FCF can enhance the early development of focal adhesions (FAs), leading to the activation and propagation of mechanotransduction signals. This is primarily achieved through FAK autophosphorylation and YAP1 nuclear translocation pathways. Remarkably, inhibiting FAK autophosphorylation negated the observed changes. Proteome analysis further confirmed the central role of FAs in mechanotransduction propagation in CSFs cultured on FCF. This analysis also highlighted complex signaling pathways, including chromatin epigenetic modifications, in response to fibrillar substrates. Overall, our research highlights the potential pathways through which CSFs undergo behavioral changes when exposed to fibrillar substrates, identifying FAs as essential mechanotransducers.

Protocol Optimization for Direct Reprogramming of Primary Human Fibroblast into Induced Striatal Neurons.

The modeling of neuropathology on induced neurons obtained by cell reprogramming technologies can fill a gap between clinical trials and studies on model organisms for the development of treatment strategies for neurodegenerative diseases. Patient-specific models based on patients' cells play an important role in such studies. There are two ways to obtain induced neuronal cells. One is based on induced pluripotent stem cells. The other is based on direct reprogramming, which allows us to obtain mature neuronal cells from adult somatic cells, such as dermal fibroblasts. Moreover, the latter method makes it possible to better preserve the age-related aspects of neuropathology, which is valuable for diseases that occur with age. However, direct methods of reprogramming have a significant drawback associated with low cell viability during procedures. Furthermore, the number of reprogrammable neurons available for morphological and functional studies is limited by the initial number of somatic cells. In this article, we propose modifications of a previously developed direct reprogramming method, based on the combination of microRNA and transcription factors, which allowed us to obtain a population of functionally active induced striatal neurons (iSNs) with a high efficiency. We also overcame the problem of the presence of multinucleated neurons associated with the cellular division of starting fibroblasts. Synchronization cells in the G1 phase increased the homogeneity of the fibroblast population, increased the survival rate of induced neurons, and eliminated the presence of multinucleated cells at the end of the reprogramming procedure. We have demonstrated that iSNs are functionally active and able to form synaptic connections in co-cultures with mouse cortical neurons. The proposed modifications can also be used to obtain a population of other induced neuronal types, such as motor and dopaminergic ones, by selecting transcription factors that determine differentiation into a region-specific neuron.

Corneal Reconstruction with EGFP-Labelled Limbal Mesenchymal Stem Cells in a Rabbit Model of Limbal Stem Cell Deficiency.

Ocular surface reconstruction is essential for treating corneal epithelial defects and vision recovery. Stem cell-based therapy demonstrates promising results but requires further research to elucidate stem cell survival, growth, and differentiation after transplantation in vivo. This study examined the corneal reconstruction promoted by EGFP-labeled limbal mesenchymal stem cells (L-MSCs-EGFP) and their fate after transplantation. EGFP labeling allowed us to evaluate the migration and survival rates of the transferred cells. L-MSCs-EGFP seeded onto decellularized human amniotic membrane (dHAM) were transplanted into rabbits with a modeled limbal stem cell deficiency. The localization and viability of the transplanted cells in animal tissue were analyzed using histology, immunohistochemistry, and confocal microscopy up to 3 months after transplantation. EGFP-labeled cells remained viable for the first 14 days after transplantation. By the 90th day, epithelialization of the rabbit corneas reached 90%, but the presence of viable labeled cells was not observed within the newly formed epithelium. Although labeled cells demonstrated low survivability in host tissue, the squamous corneal-like epithelium was partially restored by the 30th day after transplantation of the tissue-engineered graft. Overall, this study paves the way for further optimization of transplantation conditions and studying the mechanisms of corneal tissue restoration.

Uncovering Earth's virome.

Viruses are the most abundant biological entities on Earth, but challenges in detecting, isolating, and classifying unknown viruses have prevented exhaustive surveys of the global virome. Here we analysed over 5 Tb of metagenomic sequence data from 3,042 geographically diverse samples to assess the global distribution, phylogenetic diversity, and host specificity of viruses. We discovered over 125,000 partial DNA viral genomes, including the largest phage yet identified, and increased the number of known viral genes by 16-fold. Half of the predicted partial viral genomes were clustered into genetically distinct groups, most of which included genes unrelated to those in known viruses. Using CRISPR spacers and transfer RNA matches to link viral groups to microbial host(s), we doubled the number of microbial phyla known to be infected by viruses, and identified viruses that can infect organisms from different phyla. Analysis of viral distribution across diverse ecosystems revealed strong habitat-type specificity for the vast majority of viruses, but also identified some cosmopolitan groups. Our results highlight an extensive global viral diversity and provide detailed insight into viral habitat distribution and host­virus interactions.

Efficacy and safety of nivolumab combined with brentuximab vedotin after nivolumab monotherapy failure in patients with relapsed and refractory classic Hodgkin lymphoma.

OBJECTIVES: Therapy of patients with relapsed and refractory classic Hodgkin lymphoma (r/r cHL) after PD-1 inhibitors failure remains an unresolved issue. The aim of this study was to evaluate the efficacy and safety of the combination of nivolumab with brentuximab vedotin (Nivo + BV) after nivolumab monotherapy failure. METHODS: This study retrospectively analyzed 21 patients with r/r cHL who were treated with the combination of Nivo + BV after Nivo failure. The response was evaluated by PET-CT scan according to the LYRIC criteria. Adverse events (AEs) were assessed according to NCI CTCAE v.4.03. RESULTS: Median follow-up was 19 (9-47) months. The ORR was 57%. The median OS was not reached, 24 month OS was 80% (95% CI 50-93%). Median PFS was 12 months with 24 month PFS of 31% (95% CI 12-53%). Any grade AEs were observed in 12 patients (63%), 3-4 grade AEs in 2 patients (10%). Allogeneic hematopoietic stem cell transplantation (allo-HSCT) after Nivo + BV was performed in 8 (38%) patients. The median time between Nivo + BV and allo-HSCT was 8 (5-21) months. CONCLUSIONS: Combination of Nivo + BV in r/r cHL after nivolumab monotherapy failure is potentially an effective and safe approach.

Nivolumab discontinuation and retreatment in patients with relapsed or refractory Hodgkin lymphoma.

Immune checkpoint inhibitors (ICI) have demonstrated high therapeutic efficacy in relapsed or refractory classical Hodgkin lymphoma (r/r cHL). Nevertheless, despite the accumulated data, the question of the ICI therapy duration and efficacy of nivolumab retreatment remains unresolved. In this retrospective study, in a cohort of 23 adult patients with r/r cHL who discontinued nivolumab in complete response (CR), the possibility of durable remission achievement (2-year PFS was 55.1%) was demonstrated. Retreatment with nivolumab has demonstrated efficacy with high overall response rate (ORR) and CR (67% and 33.3% respectively). At the final analysis, all patients were alive with median PFS of 16.5 months. Grade 3-4 adverse events (AEs) were reported in 36% of patients, and there was no deterioration in terms of nivolumab retreatment-associated complications.

Different Conditions for the Modification of Polycaprolactone Films with L-Arginine.

Poly-ε-caprolactone (PCL) is a biodegradable polymer used in regenerative medicine. Mesenchymal stem cells (MSCs) play an important role in the regeneration of different tissues. The hydrophobicity and neutrality of a PCL surface reduce MSCs' adhesion and proliferation. In this study, PCL films were treated with arginine to improve surface hydrophilicity. The influences of arginine concentration, temperature, and solvent on PCL surface properties were investigated. PCL films treated with a solution of arginine in isopropyl alcohol were found to have the maximum number of amino groups. The greatest number of cells, 2 h after seeding, adhered to such films. It was shown that amino groups affect the interaction of cells with a modified surface and the hydrolysis reaction after treatment with isopropyl alcohol promotes the formation of adhesive focal contacts. Hence, our results illustrate that functional groups on the PCL surface after arginine solution treatment regulate MSC adhesion and focal contact formation.

New data on spermatogenic cyst formation and cellular composition of the testis in a marine gastropod, Littorina saxatilis.

Knowledge of the testis structure is important for gastropod taxonomy and phylogeny, particularly for the comparative analysis of sympatric Littorina species. Observing fresh tissue and squashing fixed tissue with gradually increasing pressure, we have recently described a peculiar type of cystic spermatogenesis, rare in mollusks. It has not been documented in most mollusks until now. The testis of adult males consists of numerous lobules filled with multicellular cysts containing germline cells at different stages of differentiation. Each cyst is formed by one cyst cell of somatic origin. Here, we provide evidence for the existence of two ways of cyst formation in Littorina saxatilis. One of them begins with a goniablast cyst formation; it somewhat resembles cyst formation in Drosophila testes. The second way begins with capture of a free spermatogonium by the polyploid cyst cell which is capable to move along the gonad tissues. This way of cyst formation has not been described previously. Our data expand the understanding of the diversity of spermatogenesis types in invertebrates.

Proteomic Profiling of the Human Fetal Multipotent Mesenchymal Stromal Cells Secretome.

Secretome of multipotent mesenchymal stromal cells (MSCs) is actively used in biomedical applications such as alveolar bone regeneration, treatment of cardiovascular disease, and neurodegenerative disorders. Nevertheless, hMSCs have low proliferative potential and production of the industrial quantity of their secretome might be challenging. Human fetal multipotent mesenchymal stromal cells (FetMSCs) isolated from early human embryo bone marrow are easy to expand and might be a potential source for pharmaceutical substances production based on their secretome. However, the secretome of FetMSCs was not previously analyzed. Here, we describe the secretome of FetMSCs using LC-MALDI shotgun proteomics. We identified 236 proteins. Functional annotation of the identified proteins revealed their involvement in angiogenesis, ossification, regulation of apoptosis, and immune response processes, which made it promising for biomedical applications. The proteins identified in the FetMSCs secretome are involved in the same biological processes as proteins from previously described adult hMSCs secretomes. Nevertheless, many of the common hMSCs secretome components (such as VEGF, FGF, Wnt and TGF-ß) have not been identified in the FetMSCs secretome.

Spermatogenesis and lobular cyst type of testes organization in marine gastropod Littorina saxatilis (Olivi 1792).

Although individual stages of spermatogenesis in Littorina saxatilis are well studied at the electron microscopic level, the gonad structure and the spatial localization of gametes at different stages of maturation remain unclear. Using differential-interference contrast (DIC) for observations of fresh tissue we show that the mature testis consists of numerous ovoid lobules forming larger lobes. The lobules of intact mature testes of L. saxatilis are filled with randomly arranged multicellular cysts containing gametes at different stages of maturation. Gametes within a cyst are highly synchronized in respect of the differentiation degree. At the same time, no spatial gradient in the arrangement of cysts according to the maturation degree of gametes in them was observed in any of the studied lobules. The male gonads contain cysts with early spermatids, mid, late spermatids, and spermatozoa. Using silver-staining, DAPI, and chromomycin A3 (CMA3) staining, we identify 20 main types of nucleus organization in differentiating sperm. Premature and mature male gonads contain cysts with a mosaic arrangement as well as rare solitary cyst cells, goniablast cysts, or separate spermatogonia in between them. Our data indicate that the testis structure in L. saxatilis cannot be attributed to the tubular type, as previously thought. It corresponds to the lobular cyst type but individual lobules contain cysts with gametes at the same stage of development. It is similar to the testis structure of several fishes, amphibians, and Drosophila melanogaster. This type of the gonad organization has never been described in gastropods before.

Draft Genome of Burkholderia cenocepacia TAtl-371, a Strain from the Burkholderia cepacia Complex Retains Antagonism in Different Carbon and Nitrogen Sources.

Burkholderia cenocepacia TAtl-371 was isolated from the rhizosphere of a tomato plant growing in Atlatlahucan, Morelos, Mexico. This strain exhibited a broad antimicrobial spectrum against bacteria, yeast, and fungi. Here, we report and describe the improved, high-quality permanent draft genome of B. cenocepacia TAtl-371, which was sequenced using a combination of PacBio RS and PacBio RS II sequencing methods. The 7,496,106 bp genome of the TAtl-371 strain is arranged in three scaffolds, contains 6722 protein-coding genes, and 99 RNA only-encoding genes. Genome analysis revealed genes related to biosynthesis of antimicrobials such as non-ribosomal peptides, siderophores, chitinases, and bacteriocins. Moreover, analysis of bacterial growth on different carbon and nitrogen sources shows that the strain retains its antimicrobial ability.

LOSP: A putative marker of parasperm lineage in male reproductive system of the prosobranch mollusk Littorina obtusata.

Reproductive isolation is the key attribute of biological species and establishment of the reproductive barriers is an essential event for speciation. Among the mechanisms of reproductive isolation, gamete incompatibility due to the variability of gamete interaction proteins may drive fast divergence even in sympatry. However, the number of available models to study this phenomenon is limited. In case of internally fertilized invertebrates, models to study gamete incompatibility and sperm competition mechanisms are restricted to a single taxon: insects. Here, we propose a group of closely related Littorina species as a new model for such studies. Particularly since periwinkles are already thoroughly studied in terms of morphology, physiology, ecology, phylogeny, and ecological speciation. Earlier, we have identified the first species-specific Littorina sperm protein (LOSP) with no known conservative domains or homologies. LOSP is relatively abundant component of sperm extracts and might be involved in gamete incompatibility. Here, we characterize its definitive localization and mRNA expression pattern in the male reproductive system by immunocytochemistry and RNA in situ hybridization. We demonstrate that LOSP distribution is limited to the parasperm cells. Losp gene expression occurs only at the early stages of parasperm development. The protein is stored within granules of mature parasperm and, most likely, is released after ejaculation inside female reproductive system. Thus, LOSP is the only described molluscan paraspermal protein to date, and there is a possibility for LOSP to be involved in gamete incompatibility since heterospermy is a common phenomenon among Littorina.

Supporting community annotation and user collaboration in the integrated microbial genomes (IMG) system.

BACKGROUND: The exponential growth of genomic data from next generation technologies renders traditional manual expert curation effort unsustainable. Many genomic systems have included community annotation tools to address the problem. Most of these systems adopted a "Wiki-based" approach to take advantage of existing wiki technologies, but encountered obstacles in issues such as usability, authorship recognition, information reliability and incentive for community participation. RESULTS: Here, we present a different approach, relying on tightly integrated method rather than "Wiki-based" method, to support community annotation and user collaboration in the Integrated Microbial Genomes (IMG) system. The IMG approach allows users to use existing IMG data warehouse and analysis tools to add gene, pathway and biosynthetic cluster annotations, to analyze/reorganize contigs, genes and functions using workspace datasets, and to share private user annotations and workspace datasets with collaborators. We show that the annotation effort using IMG can be part of the research process to overcome the user incentive and authorship recognition problems thus fostering collaboration among domain experts. The usability and reliability issues are addressed by the integration of curated information and analysis tools in IMG, together with DOE Joint Genome Institute (JGI) expert review. CONCLUSION: By incorporating annotation operations into IMG, we provide an integrated environment for users to perform deeper and extended data analysis and annotation in a single system that can lead to publications and community knowledge sharing as shown in the case studies.

Characterization and Physiological Differences of Two Primary Cultures of Human Normal and Hypertrophic Scar Dermal Fibroblasts: A Pilot Study.

Background/Objectives: Dermal fibroblasts (DFs) are key participants in skin hypertrophic scarring, and their properties are being studied to identify the molecular and cellular mechanisms underlying the pathogenesis of skin scarring. Methods: In the present work, we performed a comparative analysis of DFs isolated from normal skin (normal dermal fibroblasts, NDFs), and hypertrophic scar skin (hypertrophic scar fibroblasts, HTSFs). The fibroblasts were karyotyped and phenotyped, and experiments on growth rate, wound healing, and single-cell motility were conducted. Results: Comparative analysis revealed a minor karyotype difference between cells. However, HTSFs are characterized by higher proliferation level and motility compared to NDFs. These significant differences may be associated with quantitative and qualitative differences in the cell secretome. A proteomic comparison of NDF and HTSF found that differences were associated with metabolic proteins reflecting physiological differences between the two cells lines. Numerous unique proteins were found only in the vesicular phase of vHTSFs. Some proteins involved in cell proliferation (protein-glutamine gamma-glutamyltransferase K) and cell motility (catenin delta-1), which regulate gene transcription and the activity of Rho family GTPases and downstream cytoskeletal dynamics, were identified. A number of proteins which potentially play a role in fibrosis and inflammation (mucin-5B, CD97, adhesion G protein-coupled receptor E2, antileukoproteinase, protein S100-A8 and S100-A9, protein caspase recruitment domain-containing protein 14) were detected in vHTSFs. Conclusions: A comparative analysis of primary cell cultures revealed their various properties, especially in the cell secretome. These proteins may be considered promising target molecules for developing treatment or prevention strategies for pathological skin scarring.

Membrane-bound Heat Shock Protein mHsp70 Is Required for Migration and Invasion of Brain Tumors.

Molecular chaperones, especially 70 kDa heat shock protein, in addition to their intracellular localization in cancer cells, can be exposed on the surface of the plasma membrane. We report that the membrane-associated chaperone mHsp70 of malignant brain tumors is required for high migratory and invasive activity of cancer cells. Live-cell inverted confocal microscopy of tumor samples from adult (n = 23) and pediatric (n = 9) neurooncologic patients showed pronounced protein expression on the membrane, especially in the perifocal zone. Mass spectrometry analysis of lipid rafts isolated from tumor cells confirmed the presence of the protein in the chaperone cluster (including representatives of other families, such as Hsp70, Hsc70, Hsp105, and Hsp90), which in turn, during interactome analysis, was associated with proteins involved in cell migration (e.g., Rac1, RhoC, and myosin-9). The use of small-molecule inhibitors of HSP70 (PES and JG98) led to a substantial decrease in the invasive potential of cells isolated from a tumor sample of patients, which indicates the role of the chaperone in invasion. Moreover, the use of HSP70 inhibitors in animal models of orthotopic brain tumors significantly delayed tumor progression, which was accompanied by an increase in overall survival. Data demonstrate that chaperone inhibitors, particularly JG98, disrupt the function of mHsp70, thereby providing an opportunity to better understand the diverse functions of this protein and offer aid in the development of novel cancer therapies. SIGNIFICANCE: Membrane-bound mHsp70 is required for brain tumor cell migration and invasion and therefore could be employed as a target for anticancer therapies.

Mesenchymal stem cells-derived extracellular vesicles for therapeutics of renal tuberculosis.

Extrapulmonary tuberculosis with a renal involvement can be a manifestation of a disseminated infection that requires therapeutic intervention, particularly with a decrease in efficacy of conventional regimens. In the present study, we investigated the therapeutic potency of mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) in the complex anti-tuberculosis treatment (ATT). A rabbit model of renal tuberculosis (rTB) was constructed by injecting of the standard strain Mycobacterium tuberculosis H37Rv into the cortical layer of the kidney parenchyma. Isolated rabbit MSC-EVs were intravenously administered once as an addition to standard ATT (isoniazid, pyrazinamide, and ethambutol). The therapeutic efficacy was assessed by analyzing changes of blood biochemical biomarkers and levels of anti- and pro-inflammatory cytokines as well as by renal computed tomography with subsequent histological and morphometric examination. The therapeutic effect of therapy with MSC-EVs was shown by ELISA method that confirmed a statistically significant increase of the anti-inflammatory and decrease of pro-inflammatory cytokines as compared to conventional treatment. In addition, there is a positive trend in increase of ALP level, animal weigh, and normalization of ADA activity that can indicate an improvement of kidney state. A significant reduction of the area of specific and interstitial inflammation indicated positive affect of MSC-EVs that suggests a shorter duration of ATT. The number of MSC-EVs proteins (as identified by mass-spectometry analysis) with anti-microbial, anti-inflammatory and immunoregulatory functions reduced the level of the inflammatory response and the severity of kidney damage (further proved by morphometric analysis). In conclusion, MSC-EVs can be a promising tool for the complex treatment of various infectious diseases, in particularly rTB.

GenePRIMP: a gene prediction improvement pipeline for prokaryotic genomes.

We present 'gene prediction improvement pipeline' (GenePRIMP; http://geneprimp.jgi-psf.org/), a computational process that performs evidence-based evaluation of gene models in prokaryotic genomes and reports anomalies including inconsistent start sites, missed genes and split genes. We found that manual curation of gene models using the anomaly reports generated by GenePRIMP improved their quality, and demonstrate the applicability of GenePRIMP in improving finishing quality and comparing different genome-sequencing and annotation technologies.

Micro-spatial distribution of two sibling periwinkle species across the intertidal indicates hybrdization.

Populations of periwinkles Littorina saxatilis (Olivi 1792) and L. arcana Hannaford Ellis, 1978 are well suited for microevolutionary studies, being at the same time closely related and intraspecifically diverse. The divergence between these two sibling species, sympatric over large parts of their distribution areas, is small, the only morphological difference being the pallial gland complex structure in females. Molecular identification is possible with the use of a RAPD nuclear marker (cloned A2.8 DNA fragment) typical for L. arcana. However, in some individuals from sympatric populations molecular and morphological criteria suggest conflicting species affiliation, which may be explained either by hybridization or by shared ancestral polymorphism. We tested the hybridization hypotheses examining the micro-spatial distribution of these two species across the intertidal zone in two distant sites at the Barents Sea. We found that (a) the frequency of putative hybrids in sympatric populations was proportional to the frequency of L. arcana; (b) L. saxatilis bearing A2.8 DNA fragment were almost absent in the lower part of the intertidal zone, where L. arcana was absent too; (c) there was a close positive correlation between the distribution of potential parent molluscs and putative hybrids. Moreover, logistic regression models showed a good agreement between the distribution of putative hybrid frequencies and that of parental species frequencies. All our observations taken together support the hypothesis of hybridization between L. saxatilis and L. arcana. Elucidating the mechanisms that support the species status of these sympatric populations is necessary.