ALBERT

Description: Skeletal muscle is an adaptive tissue with the ability to regenerate in response to exercise training. Cross-sectional area (CSA) quantification, as a main parameter to assess muscle regeneration capability, is highly tedious and time-consuming, necessitating an accurate and automated approach to analysis. Although several excellent programs are available to automate analysis of muscle histology, they fail to efficiently and accurately measure CSA in regenerating myofibers in response to exercise training. Here, we have developed a novel fully-automated image segmentation method based on neutrosophic set algorithms to analyse whole skeletal muscle cross sections in exercise-induced regenerating myofibers, referred as MyoView, designed to obtain accurate fiber size and distribution measurements. MyoView provides relatively efficient, accurate, and reliable measurements for CSA quantification and detecting different myofibers, myonuclei and satellite cells in response to the post-exercise regenerating process. We showed that MyoView is comparable with manual quantification. We also showed that MyoView is more accurate and efficient to measure CSA in post-exercise regenerating myofibers as compared with Open-CSAM, MuscleJ, SMASH and MyoVision. Furthermore, we demonstrated that to obtain an accurate CSA quantification of exercise-induced regenerating myofibers, whole muscle cross-section analysis is an essential part, especially for the measurement of different fiber-types. We present MyoView as a new tool to quantify CSA, myonuclei and satellite cells in skeletal muscle from any experimental condition including exercise-induced regenerating myofibers.

Description: Rotating forms of suspension culture allow cells to aggregate into spheroids, prevent the de-differentiating influence of 2D culture, and, perhaps most importantly of all, provide physiologically relevant, in vivo levels of shear stress. Rotating suspension culture technology has not been widely implemented, in large part because the vessels are prohibitively expensive, labor-intensive to use, and are difficult to scale for industrial applications. Our solution addresses each of these challenges in a new vessel called a cell spinpod. These small 3.5 mL capacity vessels are constructed from injection-molded thermoplastic polymer components. They contain self-sealing axial silicone rubber ports, and fluoropolymer, breathable membranes. Here we report the two-fluid modeling of the flow and stresses in cell spinpods. Cell spinpods were used to demonstrate the effect of fluid shear stress on renal cell gene expression and cellular functions, particularly membrane and xenobiotic transporters, mitochondrial function, and myeloma light chain, cisplatin and doxorubicin, toxicity. During exposure to myeloma immunoglobulin light chains, rotation increased release of clinically validated nephrotoxicity cytokine markers in a toxin-specific pattern. Addition of cisplatin or doxorubicin nephrotoxins reversed the enhanced glucose and albumin uptake induced by fluid shear stress in rotating cell spinpod cultures. Cell spinpods are a simple, inexpensive, easily automated culture device that enhances cellular functions for in vitro studies of nephrotoxicity.

Description: The COVID-19 outbreak has caused over three million deaths worldwide. Understanding the pathology of the disease and the factors that drive severe and fatal clinical outcomes is of special relevance. Studying the role of the respiratory microbiota in COVID-19 is especially important as the respiratory microbiota is known to interact with the host immune system, contributing to clinical outcomes in chronic and acute respiratory diseases. Here, we characterized the microbiota in the respiratory tract of patients with mild, severe, or fatal COVID-19, and compared it to healthy controls and patients with non-COVID-19-pneumonia. We comparatively studied the microbial composition, diversity, and microbiota structure between the study groups and correlated the results with clinical data. We found differences in the microbial composition for COVID-19 patients, healthy controls, and non-COVID-19 pneumonia controls. In particular, we detected a high number of potentially opportunistic pathogens associated with severe and fatal levels of the disease. Also, we found higher levels of dysbiosis in the respiratory microbiota of patients with COVID-19 compared to the healthy controls. In addition, we detected differences in diversity structure between the microbiota of patients with mild, severe, and fatal COVID-19, as well as the presence of specific bacteria that correlated with clinical variables associated with increased risk of mortality. In summary, our results demonstrate that increased dysbiosis of the respiratory tract microbiota in patients with COVID-19 along with a continuous loss of microbial complexity structure found in mild to fatal COVID-19 cases may potentially alter clinical outcomes in patients. Taken together, our findings identify the respiratory microbiota as a factor potentially associated with the severity of COVID-19.

Description: RUNX2 encodes the master bone transcription factor driving skeletal development in vertebrates, and playing a specific role in craniofacial and skull morphogenesis. The anatomically modern human (AMH) features sequence changes in the RUNX2 locus compared with archaic hominins’ species. We aimed to understand how these changes may have contributed to human skull globularization occurred in recent evolution. We compared in silico AMH and archaic hominins’ genomes, and used mesenchymal stromal cells isolated from skull sutures of craniosynostosis patients for in vitro functional assays. We detected 459 and 470 nucleotide changes in noncoding regions of the AMH RUNX2 locus, compared with the Neandertal and Denisovan genomes, respectively. Three nucleotide changes in the proximal promoter were predicted to alter the binding of the zinc finger protein Znf263 and long-distance interactions with other cis-regulatory regions. By surface plasmon resonance, we selected nucleotide substitutions in the 3’UTRs able to affect miRNA binding affinity. Specifically, miR-3150a-3p and miR-6785-5p expression inversely correlated with RUNX2 expression during in vitro osteogenic differentiation. The expression of two long non-coding RNAs, AL096865.1 and RUNX2-AS1, within the same locus, was modulated during in vitro osteogenic differentiation and correlated with the expression of specific RUNX2 isoforms. Our data suggest that RUNX2 may have undergone adaptive phenotypic evolution caused by epigenetic and post-transcriptional regulatory mechanisms, which may explain the delayed suture fusion leading to the present-day globular skull shape.

Description: The release of Hsp70 chaperone from tumor cells is found to trigger the full-scale anti-cancer immune response. Such release and the proper immune reaction can be induced by the delivery of recombinant Hsp70 to a tumor and we sought to explore how the endogenous Hsp70 can be transported to extracellular space leading to the burst of anti-cancer activity. Hsp70 transport mechanisms were studied by analyzing its intracellular tracks with Rab proteins as well as by using specific inhibitors of membrane domains. To study Hsp70 forms released from cells we employed the assay consisting of two affinity chromatography methods. Hsp70 content in culture medium and extracellular vesicles (EVs) was measured with the aid of ELISA. The properties and composition of EVs were assessed using nanoparticle tracking analysis and immunoblotting. The activity of immune cells was studied using an assay of cytotoxic lymphocytes, and for in vivo studies we employed methods of affinity separation of lymphocyte fractions. Analyzing B16 melanoma cells treated with recombinant Hsp70 we found that the chaperone triggered extracellular transport of its endogenous analog in soluble and enclosed in EVs forms; both species efficiently penetrated adjacent cells and this secondary transport was corroborated with the strong increase of Natural Killer (NK) cell toxicity towards melanoma. When B16 and CT-26 colon cancer cells before their injection in animals were treated with Hsp70-enriched EVs, a powerful anti-cancer effect was observed as shown by a two-fold reduction in tumor growth rate and elevation of life span. We found that the immunomodulatory effect was due to the enhancement of the CD8-positive response and anti-tumor cytokine accumulation; supporting this there was no delay in CT-26 tumor growth when Hsp70-enriched EVs were grafted in nude mice. Importantly, pre-treatment of B16 cells with Hsp70-bearing EVs resulted in a decline of arginase-1-positive macrophages, showing no generation of tumor-associated macrophages. In conclusion, Hsp70-containing EVs generated by specifically treated cancer cells give a full-scale and effective pattern of anti-tumor immune responses.

Description: Lignocellulosic biomass is an important source of renewable energy and a potential replacement for fossil fuels. In this work, the X-ray fluorescence (XRF) method was used to analyze the elemental composition of raw and pretreated lignocellulosic biomass of cassava peels, corn cobs, rice husks, sugarcane bagasse, yam peels, and mixtures of cassava peels and yam peels, corn cobs and rice husks and all five biomass samples combined. The influence of particle size on elemental properties was investigated by screening the selected biomass into two size fractions, of an average of 300 and 435 µm, respectively. The total concentration of Mg, Al, Si, P, S, Cl, Ca, Ti, Cr, Mn, Fe, Co, Cu, Zn, Sn, Ni, Br, Mo, Ba, Hg, and Pb were determined for each of the biomass samples before and after the different pretreatments adopted in this study. From the results of the analysis, there was a significant reduction in the concentration of calcium in all the analyzed biomass after the alkaline pretreatment with rice husks biomass having the lowest concentration of 66 ppm after the alkaline pretreatment. The sulfur content of the acid pretreated biomass increased considerably which is likely due to the sulfuric acid used for the acid pretreatment. The fact that a mixture of biomass feedstock affects the properties of the biomass after pretreatment was validated in the mixed biomass of cassava peels and yam peels biomass as an example. The concentration of Mg in the mixed biomass was 1441 ppm but was 200 ppm and 353 ppm in individual cassava peels and yam peels respectively. The results of this study demonstrated that pretreated mixtures of biomass have varied elemental compositions, which could be an important factor affecting downstream processes, especially if a hybrid feedstock is used in a large-scale application.

Description: AbstractOver the past decade, the problem of finding an efficient gene-targeting marker set or signature for plant trait characterization has remained challenging. Many databases focusing on pathway mining have been released with one major deficiency, as they lack to develop marker sets that target only genes controlling a specific pathway or certain biological process. Herein, we present the PlantPathMarks database (PPMdb) as a comprehensive, web-based, user-friendly, and interactive hub for pathway-based markers in plant genomes. Based on our newly developed pathway gene set mining approach, two novel pathway-based marker systems called pathway gene-targeted markers (PGTMs) and pathway microsatellite-targeted markers (PMTMs) were developed as a novel class of annotation-based markers. In the PPMdb database, 2,690,742 pathway-based markers reflecting 9,894 marker panels were developed across 82 plant genomes. The markers include 691,555 PGTMs and 1,999,187 PMTMs. Across these genomes, 165,378 enzyme-coding genes were mapped against 126 KEGG reference pathway maps. PPMdb is furnished with three interactive visualization tools (Map Browse, JBrowse and Species Comparison) to visualize, map, and compare the developed markers over their KEGG reference pathway maps. All the stored marker panels can be freely downloaded. PPMdb promises to create a radical shift in the paradigm of the area of molecular marker research. The use of PPMdb as a mega-tool represents an impediment for non-bioinformatician plant scientists and breeders. PPMdb is freely available at http://ppmdb.easyomics.org.

Description: In this article developed mathematical model that includes friction occurrence on spline connections is presented. The work also contains results of experimental research on torque capacity of multidisc wet clutch. These results are expressed as a function of contact pressure for different number of friction surfaces. Due to increased interest in research concerning multidisc wet clutches it is essential to determine impact of friction on fit connections on transmitted torque. Analytical calculations that include both known loss coefficient and assumed lack of friction on fit connections are compared to results of experiments. The paper contains detailed description of test stand and methodology of experiment. As a result of conducted tests it was found that correction coefficients known from literature are highly inaccurate. Measured values of torque indicate that transmitted torque reach significantly higher values. It was also revealed that after slippage appeared, the pressure plate usually moved in the direction of exerted clamping force, but movement in reversed direction also took place for some experiments. While movement corresponding to clamping force reached ca. 0.08 mm, in opposite direction amounted to 0.02 mm. Furthermore, studies presented that lapping of adjacent friction surfaces greatly affects differences between respective results obtained for a specific experiment.

Description: Wildfires considerably affect forest ecosystems. However, there is a lack of data on the post-fire status of insect communities in these ecosystems. This paper presents results of a study conducted in 2019 which considered the post-fire status of the insect fauna in a Protected Area, Mordovia State Nature Reserve (Republic of Mordovia, centre of European Russia), considered as regional hotspot of insect diversity in Mordovia. We sampled insects on intact (unburned, control) and fire-damaged (burnt in 2010) sites and compared the alpha-diversity between sites. In total, we sampled and analysed 16,861 specimens belonging to 11 insect orders, 51 families and 190 species. The largest orders represented in the samples were Coleoptera (95 species), Diptera (54 species), Hymenoptera (21 species), and Neuroptera (11 species). Other insect orders were represented by between one and four species. The largest four orders (Coleoptera, Lepidoptera, Diptera and Hymenoptera) represented 96.7% of all studied specimens. We found that in the ninth year after low intensity surface fire damage, the insect diversity had returned to a similar level to that of the control (unburned) sites. Sites damaged by crown wildfire differed considerably from other sites in terms of a negative impact on both species diversity and the number of specimens. This indicates the serious effect of the crown fires on the biodiversity and consequent long-term recovery of the damaged ecosystem.