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Cancer cells are softer than the normal cells, and metastatic cells are even softer. These changes in biomechanical properties contribute to cancer progression by facilitating cell movement through physically constraining environments. To identify properties that enabled passage through physical constraints, cells that were more efficient at moving through narrow membrane micropores were selected from established cell lines. By examining micropore-selected human MDA MB 231 breast cancer and MDA MB 435 melanoma cancer cells, membrane fluidity and nuclear elasticity were excluded as primary contributors. Instead, reduced actin cytoskeleton anisotropy, focal adhesion density and cell stiffness were characteristics associated with efficient passage through constraints. By comparing transcriptomic profiles between the parental and selected populations, increased Ras/MAPK signalling was linked with cytoskeleton rearrangements and cell softening. MEK inhibitor treatment reversed the transcriptional, cytoskeleton, focal adhesion and elasticity changes. Conversely, expression of oncogenic KRas in parental MDA MB 231 cells, or oncogenic BRaf in parental MDA MB 435 cells, significantly reduced cell stiffness. These results reveal that MAPK signalling, in addition to tumour cell proliferation, has a significant role in regulating cell biomechanics. This article has an associated First Person interview with the first author of the paper., Highlighted Article: Selection for tumour cells that efficiently pass through narrow diameter microporous membranes reveals a prominent role for MAPK signalling in regulating cell elasticity.

Fucoidans, a diverse group of sulphated polysaccharides from brown algae, are biopolymers with many documented activities in vitro. While several factors are known to influence these activities, the degree of sulphation of the polymer is often regarded as a key parameter. The degree of sulphation on a biopolymer may be seasonally dependent, which, in turn, may affect the bioactivities of fucoidans. In this study, the effect of season and geographical location on the fucoidan sulphur content (as a proxy for degree of sulphation), was investigated. Crude fucoidan extracts were obtained using microwave-assisted extraction on eight different brown algae species, which were then analysed using elemental analysis. A significant seasonal dependence on the sulphur content in fucoidan extracts from Fucus serratus was observed. The difference in sulphur content between the investigated Fucus species is likely due to their different reproduction strategies. Of the eight species used in this study, Ascophyllum nodosum and Fucus evanescens contained the most sulphated fucoidans, while the species Dictyosiphon foeniculaceus contained lightly sulphated fucoidans. This suggests that species which thrive in saline waters may contain more sulphated fucoidans as a means of mitigating osmotic stress. No significant effects on the sulphur content in fucoidans from Fucus vesiculosus was observed between the three investigated countries. From these findings, it is apparent that seasonality is an important parameter, which must be considered for large scale production of highly sulphated fucoidans.

This paper aims to review the recent progress in the research carried out by scientists worldwide regarding American Footballers' head injuries and head protective equipment, focusing on the role of computation methods, mainly finite element method application to American Football helmet design and testing as well as head injury biomechanics. The helmet technology has been constantly improved, and it is driven by market competition, medical records, coaches and athletes' self-awareness. With finite element analysis and computational resources development, it is possible to develop more accurate brain models to recreate American Footballers' head impacts. This method seems to be an excellent simulation tool to verify the helmet's ability to absorb energy and enable the researchers to have an insight into head kinematics and tissue-level injuries. The work is focused on head injuries in American Football as the sport becomes more popular across the globe. Additionally, a reference to the development and newest technology is presented. The review's proposed approach gathers studies presented within the last decade regarding the coupling of finite element brain models with helmets in standardised or on-field conditions. The synthesis of the existing state of the art may enhance the researchers to continue investigating the athlete's trauma and improve the protective gear technology to minimise head injuries. The authors presented numerous studies regarding concussions and the newest findings from the last decade, including Finite Element Head models (FEHm) with American Football helmet simulations. All the studies were searched through Google Scholar, Scopus and ResearchGate databases.

BACKGROUND Transplant glomerulopathy (TG) is a pathological feature of chronic active antibody-mediated rejection (cAMR) and is associated with renal allograft failure. The specific role of B cells in the pathogenesis of TG is unclear. METHODS We used a minor mismatched rat kidney transplant model with B cell-deficient recipients, generated by clustered regularly interspaced short palindromic repeats/Cas9 technology, to investigate the impact of B-cell depletion on the pathogenesis of TG. We hypothesized that B-cell deficiency would prevent TG in the rat kidney transplant model of cAMR. Treatment groups included syngeneic, allogeneic, sensitized allogeneic, and B cell-deficient allogeneic transplant recipients. RESULTS B cell-deficient recipients demonstrated reduced TG lesions, decreased microvascular inflammation, reduced allograft infiltrating macrophages, and reduced interferon gamma transcripts within the allograft. Allograft transcript levels of interferon gamma, monocyte chemoattractant protein-1, and interleukin-1β correlated with numbers of intragraft macrophages. B cell-deficient recipients lacked circulating donor-specific antibodies and had an increased splenic regulatory T-cell population. CONCLUSIONS In this model of cAMR, B-cell depletion attenuated the development of TG with effects on T cell and innate immunity.

Identification and quantification of low-frequency mutations remain challenging despite improvements in the baseline error rate of next-generation sequencing technologies. Here, we describe a method, termed SaferSeqS, that addresses these challenges by (1) efficiently introducing identical molecular barcodes in the Watson and Crick strands of template molecules and (2) enriching target sequences with strand-specific PCR. The method achieves high sensitivity and specificity and detects variants at frequencies below 1 in 100,000 DNA template molecules with a background mutation rate of 100-fold.

Approximately 257 million people chronically infected with hepatitis B virus (HBV) worldwide are at risk of developing hepatocellular carcinoma (HCC). However, despite the availability of potent nucleoside/tide inhibitors, currently there are no curative therapies for chronic HBV infections. To identify potential new antiviral molecules, a select group of compounds previously evaluated in clinical studies were tested against 12 different viruses. Amongst the compounds tested, SRI-32007 (CYT997) demonstrated antiviral activity against HBV (genotype D) in HepG2.2.2.15 cell-based virus yield assay with 50% effective concentration (EC50) and selectivity index (SI) of 60.1 nM and 7.2, respectively. Anti-HBV activity of SRI-32007 was further confirmed against HBV genotype B in huh7 cells with secreted HBe antigen endpoint (EC50 40 nM and SI 250). To determine the stage of HBV life cycle inhibited by SRI-32007, time of addition experiment was conducted in HepG2-NTCP cell-based HBV infectious assay. Results indicated that SRI-32007 retained anti-HBV activity even when added 72 hours postinfection (72 h). Additional mechanism of action studies demonstrated potent inhibition of HBV core promoter activity by SRI-32007 with an EC50 of 40 nM and SI of >250. This study demonstrates anti-HBV activity of a repurposed compound SRI-32007 through inhibition of HBV core promoter activity. Further evaluation of SRI-32007 in HBV animal models is needed to confirm its activity in vivo. Our experiments illustrate the utility of repurposing strategy to identify novel antiviral chemical leads. HBV core promoter inhibitors such as SRI-32007 might enable the development of novel therapeutic strategies to combat HBV infections.

The objective of this study was to determine the relationship between milk β-hydroxybutyrate (BHB), acetone (ACE) as well as parity and lactation stage and milk freezing point (MFP) in Polish Holstein-Friesian cows in early lactation. Additionally, we studied the relationship between milk ketone bodies and daily milk yield (DMY), fat (MF) and protein (MP) content in milk. The data obtained from the Polish Federation of Cattle Breeders and Dairy Farmers, comprised 749,894 test day milk samples, collected between 6 and 60 days in milk (DIM) from 521,049 lactations of 514,066 cows. Milk BHB and ACE were determined using the Fourier transform infrared (FTIR) technology. Four classes of parities were created: first, second, third, and fourth to seventh and two classes of lactation stage: 5–21 and 22–60 DIM. BHB was grouped into five classes: ≤0.05, 0.06–0.10, 0.11–0.20, 0.21–0.50 and >0.50 mmol/L, and ACE was also classified into five classes: ≤0.05, 0.06–0.10, 0.11–0.15, 0.16–0.30 and >0.30 mmol/L. Data on MFP, DMY, and MF and MP content were analyzed using the MIXED procedure of SAS and a linear model in which effects of parity, lactation stage, BHB and ACE classes were included, together with interactions between lactation stage and BHB classes, parity and BHB classes, lactation stage and ACE classes, and parity and ACE classes. The differences among parity, lactation stages, BHB and ACE classes in MFP, DMY, MF and MP were highly significant. There was a clear tendency for decreasing of MFP with increasing of BHB. Such a trend did not occur in case of ACE. DMY and MP decreased and MF increased with increasing BHB or ACE. In conclusion, since MFP can be measured relatively easily and is well related to milk BHB content, it may be used in the prediction of diagnostic models of ketosis based on milk composition.

In eukaryotes, chromatin binding to the inner nuclear membrane (INM) and nuclear pore complexes (NPCs) contributes to spatial organization of the genome and epigenetic programs important for gene expression. In mitosis, chromatin–nuclear envelope (NE) interactions are lost and then formed again as sister chromosomes segregate to postmitotic nuclei. Investigating these processes in S. cerevisiae, we identified temporally and spatially controlled phosphorylation-dependent SUMOylation events that positively regulate postmetaphase chromatin association with the NE. Our work establishes a phosphorylation-mediated targeting mechanism of the SUMO ligase Siz2 to the INM during mitosis, where Siz2 binds to and SUMOylates the VAP protein Scs2. The recruitment of Siz2 through Scs2 is further responsible for a wave of SUMOylation along the INM that supports the assembly and anchorage of subtelomeric chromatin at the INM and localization of an active gene (INO1) to NPCs during the later stages of mitosis and into G1-phase.

Introduction/Background* Adult granulosa cell tumors (AGCT) accounting about 95% GCT (granulosa cell tumors) is characterized by the occurrence of an aggressive form and frequent appearance of recurrence and metastasis (1). Furthermore, reports indicated that the level of visfatin, which is one of adipokine, is elevated in ascites fluid of ovarian cancer patients with diagnosed metastases (2). Moreover, the role of visfatin in cancer metabolic reprogramming was demonstrated (3). Therefore, the aim of this research is to determine whether visfatin by reprogramming metabolism may be involved in invasion of AGCT Methodology Human AGCT-derived cell line KGN (Riken Cell Bank) was cultured in three-dimensional culture. Spheroids were exposed to visfatin (100 ng/ml) for 24 h and invasion was measured using Culturex Spheroid Invasion Matrix (#3500-096-03; Trevigen). Glucose uptake was tested using a Glucose Uptake-Glo Assay (Promega) after treatment with visfatin (100 ng/ml) for 24h. Silencing of NAMPT (visfatin gene) was carried out using siRNA (SMART pool siRNA NAMPT; L-004581-00-0005) and basal glycolysis was measured using Seahorse Glycolytic Rate Assay (Agilent). Result(s)* Firstly, we showed that visfatin increases KGN cells spheroids invasion. Furthermore, we observed stimulating glucose uptake in KGN cells after treatment with visfatin. Whereas the use of STF-31 (a blocker for the GLUT1 and an inhibitor of visfatin) abolished the stimulating effect of visfatin on the invasion, which indicates the important role of glucose metabolism in this process. Moreover, silencing of NAMPT decreased basal glycolysis in KGN cells spheroids. Conclusion* These results stated that visfatin by reprogramming glucose metabolism plays an important role in increasing the ability of AGCT to invasion and because of that it can be a factor to progression of this cancer. This study was funded by the National Science Centre (NCN) Poland (grant number 2019/33/N/NZ5/00471) References Sonoyama A, Kanda M, Ojima Y, et al. J Med Sci 2015;61:E109-14. Li Y, Li X, Liu KR, et al. Eur J Cancer Prev 2015;24;231–9. Audrito V, Manago A, Gaudino F, et al. Semin Cell Dev Biol 2020;98:192–201.

Longevity is one of the functional traits that considerably affect dairy herd profitability. A Weibull proportional hazards model was used to evaluate the impact of difficult calvings and calf stillbirths on cow functional longevity, defined as length of productive life corrected for milk production. The data for analysis comprised calving ease and calf mortality scores of 2,163,426 calvings, 34.4% of which came from primiparous cows. The percentage of male calves was 53.4%. Calving ease was scored as “without assistance” (34.44%), “with assistance” (62.03%), “difficult—hard pull” (3.39%), and “very difficult, including caesarean section” (0.14%). Calf mortality scores were “live born” (94.21%) and “stillborn or died within 24 h” (5.79%). The Weibull proportional hazards model included classes of calving ease or calf mortality scores × parity (1, ≥2) × sex of calf as time-dependent fixed effect. The model also included time-dependent fixed effects of year × season, parity × stage of lactation, annual change in herd size, fat yield and protein yield, time-independent fixed effect of age at first calving, and time dependent random herd × year × season. In first-parity cows, very difficult birth of a bull or heifer increased the relative risk of culling, respectively, 2.18 or 1.26 times as compared with calving without assistance. In later parities, the relative risk of culling related to very difficult calving was 2.0 times (for male calves) and 1.33 times (for female calves) higher than the relative risk of culling associated with calving without assistance. Calf mortality showed a negative impact on longevity in both heifers and cows. First-parity stillbirth increased the relative risk of culling depending on sex of calf by 18% in females and by 15% in males, in later parities the increase of the relative risk of culling was lower (by 7% for females, 9% for males). Difficult calvings and their consequences, especially in primiparous cows, may negatively influence dairy herd profitability by reducing the length of cows’ productive life.

The syndecans represent an ongoing research field focused on their regulatory roles in normal and pathological conditions. Syndecan's role in cancer progression becomes well-documented, implicating their importance in diagnosis and even proposing various cancer potential treatments. Thus, the characterization of the unbinding properties at the single molecules level will appeal to their use as targets for therapeutics. In our study, syndecan-1 and syndecan-4 were measured during the interaction with the vitronectin HEP II binding site. Our findings show that syndecans are calcium ion-dependent molecules that reveal distinct, unbinding properties indicating the alterations in heparin sulfate chain structure, possibly in the chain sequence or sulfation pattern. In that way, we suppose that HS chain affinity to ECM proteins may govern cancer invasion by altering syndecan ability to interact with cancer-related receptors present in the tumor microenvironment, thereby promoting the activation of various signaling cascades regulating tumor cell behavior., Comment: 9 pages, 6 figures

SummaryWe describe the creation and characterization of an isogenic cell line panel representing common cancer pathways, with multiple features optimized for high-throughput screening. More than 1,800 cell lines from three normal human cells were generated using CRISPR-technologies. Surprisingly, we discovered most of these lines did not result in complete gene inactivation, despite integration of sgRNA at the desired genomic site. However, a subset of the lines harbored true, biallelic disruptions of the targeted tumor suppressor gene, yielding a final panel of 100 well-characterize lines covering 19 pathways frequently subject to loss of function in cancers. This panel included genetic markers optimized for sequence-based ratiometric assays for drug-based screening assays. To illustrate the potential utility of this panel, we developed a multiplexed high-throughput screen that identified Wee1 inhibitor MK-1775 as a selective growth inhibitor of cells with inactivation ofTP53. These cell lines and screening approach should prove useful for researchers studying a variety of cellular and biochemical phenomena.

HIV-1 Vpu targets the host cell proteins CD4 and BST-2/Tetherin for degradation, ultimately resulting in enhanced virus spread and host immune evasion. The discovery and characterization of small molecules that antagonize Vpu would further elucidate the contribution of Vpu to pathogenesis and lay the foundation for the study of a new class of novel HIV-1 therapeutics. To identify novel compounds that block Vpu activity, we developed a cell-based ‘gain of function’ assay that produces a positive signal in response to Vpu inhibition. To develop this assay, we took advantage of the viral glycoprotein, GaLV Env. In the presence of Vpu, GaLV Env is not incorporated into viral particles, resulting in non-infectious virions. Vpu inhibition restores infectious particle production. Using this assay, a high throughput screen of >650,000 compounds was performed to identify inhibitors that block the biological activity of Vpu. From this screen, we identified several positive hits but focused on two compounds from one structural family, SRI-41897 and SRI-42371. It was conceivable that the compounds inhibited the formation of infectious virions by targeting host cell proteins instead of Vpu directly, so we developed independent counter-screens for off target interactions of the compounds and found no off target interactions. Additionally, these compounds block Vpu-mediated modulation of CD4, BST-2/Tetherin and antibody dependent cell-mediated toxicity (ADCC). Unfortunately, both SRI-41897 and SRI-42371 were shown to be specific to the N-terminal region of NL4-3 Vpu and did not function against other, more clinically relevant, strains of Vpu.

Otoliths are one of the biominerals whose formation is highly controlled by proteins. The first protein discovered to be involved in otolith biomineralization in zebrafish was starmaker (Stm). Previously, Stm was shown to be responsible for the preferential formation of aragonite, a polymorph of calcium carbonate, in otoliths. In this work, proteomic analysis of adult zebrafish otoliths was performed. Stm is the only highly phosphorylated protein found in our studies. Besides previously studied otolith proteins, we discovered several dozens of unknown proteins that reveal the likely mechanism of biomineralization. A comparison of aragonite and vaterite otoliths showed similarities in protein composition. We observed the presence of Stm in both types of otoliths. In vitro studies of 2 characteristic Stm fragments indicated that the DS-rich region has a special biomineralization activity, especially after phosphorylation.-Kalka, M., Markiewicz, N., Ptak, M., Sone, E. D., Ozyhar, A., Dobryszycki, P., Wojtas, M. In vivo and in vitro analysis of starmaker activity in zebrafish otolith biomineralization.

Since the birth of the first baby conceived by in vitro fertilization (IVF), assisted reproductive technologies (ART) have been constantly evolving to accomodate needs of a growing number of infertile couples. Rapidly developing ART procedures are directly applied for human infertility treatment without prior long-term safety evaluation. Although the majority of ART babies are healthy at birth, a comprehensive assessment of the long-term risks associated with ART is still lacking. An increased risk of epigenetic errors has been associated with the use of ART, which may contribute to the onset of civilization disease later in adolescence/adulthood and/or in subsequent generations. Therefore, our investigations should not focus on (or be limited to) the occurrence of a few very rare imprinting disorders in ART children, which might be associated with parental age and/or the use of ART, but on the possibly increased disease susceptibilities later in life and their potential transmission to the subsequent generations. Retrospective studies do not offer exhaustive information on long-term consequences of ART. Animal models are useful tools to study long-term effects including transgenerational ones and the epigenetic risk of a given ART procedure, which could then be translated to the human context. The final goal is the establishment of common guidelines for assessing the epigenetic risk of ART in humans, which will contribute to two key objectives of the Horizon2020 programme, i.e. to improve our understanding of the causes and mechanisms underlying health and disease, and to improve our ability to monitor health and prevent/manage disease.

Recombinant antigens exhibit targeted protectiveproperties and offer important opportunities in the development of therapeutic technologies. Biophysical and structural methods have become important tools for the rational design and engineering of improved antigen-based vaccines. Vaccines containing Leptospira immunoglobulin-like (Lig) protein-derived antigens are currently the most promising candidates for protective immunity against the globally prevalent bacterial pathogen, Leptospira interrogans; however, vaccine trials using these domains have produced inconsistent results. Here, we compare the thermostability of domains from the main immunogenic regions from major leptospiral antigens, LigA and LigB. By measuring temperature-dependent fluorescence decay of the hydrophobic core tryptophan, 17 individual Lig protein immunoglobulin-like (Ig-like) domains were shown to display a broad range of unfolding temperatures. For a majority of the domains, stability issues begin to occur at physiologically relevant temperatures. A set of chimeric Ig-like domains was used to establish the ability of transplanted domain regions to enhance thermostability. Further insights into the determinants for domain stabilization were explored with nuclear magnetic resonance dynamics and mutational analysis. The current study has yielded a set of thermostable Ig-like domain scaffolds for use in engineering antigen-based vaccines and demonstrates the importance of incorporating thermostability screening as a design parameter.

Disruption of granulosa cells (GCs), the main functional cells in the ovary, is associated with impaired female fertility. Epidemiological studies demonstrated that women have detectable levels of organic pollutants (e.g., perfluorooctanoate, perfluorooctane sulfonate, 2,2-dichlorodiphenyldichloroethylene, polychlorinated biphenyl 153, and hexachlorobenzene) in their follicular fluid (FF), and thus these compounds may directly affect the function of GCs in the ovary. Considering that humans are exposed to multiple pollutants simultaneously, we elucidated the effects of a mixture of endocrine-disrupting chemicals (EDCs) on human granulosa HGrC1 cells. The EDC mixture directly increased progesterone secretion by upregulating 3β-hydroxysteroid dehydrogenase (3βHSD) expression. Furthermore, the EDC mixture increased activity of mitochondria, which are the central sites for steroid hormone biosynthesis, and the ATP content. Unexpectedly, the EDC mixture reduced glucose transporter 4 (GLUT4) expression and perturbed glucose uptake; however, this did not affect the glycolytic rate. Moreover, inhibition of GLUT1 by STF-31 did not alter the effects of the EDC mixture on steroid secretion but decreased basal estradiol secretion. Taken together, our results demonstrate that the mixture of EDCs present in FF can alter the functions of human GCs by disrupting steroidogenesis and may thus adversely affect female reproductive health. This study highlights that the EDC mixture elicits its effects by targeting mitochondria and increases mitochondrial network formation, mitochondrial activity, and expression of 3βHSD, which is associated with the inner mitochondrial membrane.

Leishmaniasis, malaria, toxoplasmosis, and acanthamoebiasis are protozoan parasitic infections. They remain important contributors to the development of kidney disease, which is associated with increased patients’ morbidity and mortality. Kidney injury mechanisms are not fully understood in protozoan parasitic diseases, bringing major difficulties to specific therapeutic interventions. The aim of this review is to present the biochemical and molecular mechanisms in kidneys infected with Leishmania spp., Plasmodium spp., Toxoplasma gondii, and Acanthamoeba spp. We present available mechanisms of an immune response, oxidative stress, apoptosis process, hypoxia, biomarkers of renal injury in the serum or urine, and the histopathological changes of kidneys infected with the selected parasites. Pathomechanisms of Leishmania spp. and Plasmodium spp. infections have been deeply investigated, while Toxoplasma gondii and Acanthamoeba spp. infections in the kidneys are not well known yet. Deeper knowledge of kidney involvement in leishmaniasis and malaria by presenting their mechanisms provides insight into how to create novel and effective treatments. Additionally, the presented work shows gaps in the pathophysiology of renal toxoplasmosis and acanthamoebiasis, which need further research.

Fucoidans, sulfated polysaccharides extracted from brown algae, are marine products with the potential to modulate bone formation and vascularization processes. The bioactivity and safety of fucoidans are highly associated with their chemical structure, which may vary with algae species and extraction method. Thus, in depth evaluation of fucoidan extracts in terms of endotoxin content, cytotoxicity, and their detailed molecular biological impact on the individual cell types in bone is needed. In this study, we characterized fucoidan extracts from three different Fucus species including Fucus vesiculosus (Fv), Fucus serratus (Fs), and Fucus distichus subsp. evanescens (Fe) for their chemical features, endotoxin content, cytotoxicity, and bioactive effects on human outgrowth endothelial cells (OEC) and human mesenchymal stem cells (MSC) as in vitro models for bone function and vascularization. Extracts contained mainly high molecular weight (HMW) fucoidans and were free of endotoxins that may cause inflammation or influence vascularization. OEC tolerated fucoidan concentrations up to 200 µg/mL, and no indication of cytotoxicity was observed. The inflammatory response, however, investigated by real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) and endothelial barrier assessed by impedance measurement differed for the individual extracts. MSC in comparison with endothelial cells were more sensitive to fucoidans and showed partly reduced metabolic activity and proliferation at higher doses of fucoidans. Further results for MSC indicated impaired osteogenic functions in alkaline phosphatase and calcification assays. All tested extracts consistently lowered important molecular mediators involved in angiogenesis, such a VEGF (vascular endothelial growth factor), ANG-1 (angiopoietin 1), and ANG-2 (angiopoietin 2), as indicated by RT-PCR and ELISA. This was associated with antiangiogenic effects at the functional level using selected extracts in co-culture models to mimic bone vascularization processes during bone regeneration or osteosarcoma.

Significance Lipid droplets (LDs) are stored in the embryo throughout the preimplantation development. Yet, the role of LDs in the embryo remains unknown. Embryonic diapause (ED) is a temporary arrest of an embryo when it waits for the uterine receptivity signal to implant. We provide evidence that LDs play a crucial role in maintaining ED. Diapausing embryos exhibit increased release of exosomes reflecting elevated embryonic signaling to the mother. During ED, the decrease in lipid is caused by a switch from carbohydrate metabolism to lipid catabolism. We have also shown that presence of LDs in the oocytes of various mammals positively corelates with their species-specific length of diapause. Our results reveal the functional role of LDs in embryonic development., Embryonic diapause (ED) is a temporary arrest of an embryo at the blastocyst stage when it waits for the uterine receptivity signal to implant. ED used by over 100 species may also occur in normally “nondiapausing” mammals when the uterine receptivity signal is blocked or delayed. A large number of lipid droplets (LDs) are stored throughout the preimplantation embryo development, but the amount of lipids varies greatly across different mammalian species. Yet, the role of LDs in the mammalian egg and embryo remains unknown. Here, using a mouse model, we provide evidence that LDs play a crucial role in maintaining ED. By mechanical removal of LDs from zygotes, we demonstrated that delipidated embryos are unable to survive during ED. LDs are not essential for normal prompt implantation, without ED. We further demonstrated that with the progression of ED, the amount of intracellular lipid reduces, and composition changes. This decrease in lipid is caused by a switch from carbohydrate metabolism to lipid catabolism in diapausing blastocysts, which also exhibit increased release of exosomes reflecting elevated embryonic signaling to the mother. We have also shown that presence of LDs in the oocytes of various mammals positively corelates with their species-specific length of diapause. Our results reveal the functional role of LDs in embryonic development. These results can help to develop diagnostic techniques and treatment of recurrent implantation failure and will likely ignite further studies in developmental biology and reproductive medicine fields.

SARS-CoV-2 Nsp1 protein binds the mRNA export receptor NXF1-NXT1 to inhibit nuclear export of mRNAs and promote infection., The ongoing unprecedented severe acute respiratory syndrome caused by the SARS-CoV-2 outbreak worldwide has highlighted the need for understanding viral-host interactions involved in mechanisms of virulence. Here, we show that the virulence factor Nsp1 protein of SARS-CoV-2 interacts with the host messenger RNA (mRNA) export receptor heterodimer NXF1-NXT1, which is responsible for nuclear export of cellular mRNAs. Nsp1 prevents proper binding of NXF1 to mRNA export adaptors and NXF1 docking at the nuclear pore complex. As a result, a significant number of cellular mRNAs are retained in the nucleus during infection. Increased levels of NXF1 rescues the Nsp1-mediated mRNA export block and inhibits SARS-CoV-2 infection. Thus, antagonizing the Nsp1 inhibitory function on mRNA export may represent a strategy to restoring proper antiviral host gene expression in infected cells.

Fucoidans are algal polysaccharides that exhibit protective properties against oxidative stress. The aim of this study was to investigate different fucoidans from brown seaweeds for their ability to protect against iron-dependent oxidative stress (ferroptosis), a main hallmark of retinal and brain diseases, including hemorrhage. We investigated five new high-molecular weight fucoidan extracts from Fucus vesiculosus, F. serratus, and F. distichus subsp. evanescens, a previously published Laminaria hyperborean extract, and commercially available extracts from F. vesiculosus and Undaria pinnatifida. We induced oxidative stress by glutathione depletion (erastin) and H2O2 in four retinal and neuronal cell lines as well as primary cortical neurons. Only extracts from F. serratus, F. distichus subsp. evanescens, and Laminaria hyperborea were partially protective against erastin-induced cell death in ARPE-19 and OMM-1 cells, while none of the extracts showed beneficial effects in neuronal cells. Protective fucoidans also attenuated the decrease in protein levels of the antioxidant enzyme GPX4, a key regulator of ferroptosis. This comprehensive analysis demonstrates that the antioxidant abilities of fucoidans may be cell type-specific, besides depending on the algal species and extraction method. Future studies are needed to further characterize the health-benefiting effects of fucoidans and to determine the exact mechanism underlying their antioxidative abilities.

Assisted Reproductive Technologies (ART) allowed the births of >8 million babies worldwide. Even if ART children are healthy at birth, several studies reported that ART may cause changes in foetal programming, leading to an increased predisposition to metabolic disorders in adulthood. Previous studies on mouse model showed obesity, glucose intolerance, and hepatic lipid accumulation in ART offspring. A cumulative effect of the different components of ART protocol has been previously described, for example, in the occurrence of epigenetic defects. Here, we investigated whether there is a cumulative effect of embryo transfer (ET), in vitro culture (IVC) and blastomere biopsy (BB) in the onset of metabolic disorders in mouse offspring vs those naturally conceived (Control - CTR). To this aim, proteomic analysis was performed on the livers from adult mouse offspring developed following ET, IVC and BB vs CTR. We observed deregulated expression of proteins involved in lipid, carbohydrate, energy metabolisms and cellular processes in ART offspring. Moreover, we found increased body weight in all ART offspring while i) insulin resistance in BB male, ii) females glucose intolerance and high level of triglycerides and cholesterol in BB females and iii) low levels of interleukin-6 in BB, IVC and ET males. In conclusion, our study suggests that the use of various embryo manipulations influences the metabolic health of adult offspring, resulting in an increased predisposition to hepatic diseases and metabolic syndrome in a sex-specific manner.

Postponement of fatherhood is growing worldwide due to socio-economic factors. The choice to conceive the first child above the age of 35 years is often associated with reduced fertility and poor pregnancy outcome. As widely known, several factors (e.g., lifestyle, environment, health problems) can affect spermatogenesis leading to poor reproductive outcome. Currently, the debate on the influence of aging on male gametes and safety/risk of conception at advanced age is still ongoing. Controversial results have been published so far on the changes in semen features of aging men and other mammalian species (mainly rodents). In this study, we aimed to assess how aging affects sperm quality in an inbreed mouse model, without underlying infertility, using a flow cytometry approach. Our data showed that aging is associated with increased sperm chromatin condensation, but not changes in the DNA integrity, metabolic activity or viability. These data suggest a mild effect of aging on sperm quality in a mouse model without underlying infertility. This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska Curie grant agreement No 834621

Tetrameric transthyretin (TTR) transports thyroid hormones and retinol in plasma and cerebrospinal fluid and performs protective functions under stress conditions. Ageing and mutations result in TTR destabilisation and the formation of the amyloid deposits that dysregulate Ca2+ homeostasis. Our aim was to determine whether Ca2+ affects the structural stability of TTR. We show, using multiple techniques, that Ca2+ does not induce prevalent TTR dissociation and/or oligomerisation. However, in the presence of Ca2+, TTR exhibits altered conformational flexibility and different interactions with the solvent molecules. These structural changes lead to the formation of the sub-populations of non-native TTR conformers and to the destabilisation of the structure of TTR. Moreover, the sub-population of TTR molecules undergoes fragmentation that is augmented by Ca2+. We postulate that Ca2+ constitutes the structural and functional switch between the native and non-native forms of TTR, and therefore tip the balance towards age-dependent pathological calcification.

The ongoing pandemic spread of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) demands skillful strategies for novel drug development, drug repurposing and cotreatments, in particular focusing on existing candidates of host-directed antivirals (HDAs). The developmental drug IMU-838, currently being investigated in a phase 2b trial in patients suffering from autoimmune diseases, represents an inhibitor of human dihydroorotate dehydrogenase (DHODH) with a recently proven antiviral activity in vitro and in vivo. Here, we established an analysis system for assessing the antiviral potency of IMU-838 and DHODH-directed back-up drugs in cultured cell-based infection models. By the use of SARS-CoV-2-specific immunofluorescence, Western blot, in-cell ELISA, viral yield reduction and RT-qPCR methods, we demonstrated the following: (i) IMU-838 and back-ups show anti-SARS-CoV-2 activity at several levels of viral replication, i.e., protein production, double-strand RNA synthesis, and release of infectious virus, (ii) antiviral efficacy in Vero cells was demonstrated in a micromolar range (IMU-838 half-maximal effective concentration, EC50, of 7.6 &plusmn, 5.8 &micro, M), (iii) anti-SARS-CoV-2 activity was distinct from cytotoxic effects (half-cytotoxic concentration, CC50, &gt, 100 &micro, (iv) the drug in vitro potency was confirmed using several Vero lineages and human cells, (v) combination with remdesivir showed enhanced anti-SARS-CoV-2 activity, (vi) vidofludimus, the active determinant of IMU-838, exerted a broad-spectrum activity against a selection of major human pathogenic viruses. These findings strongly suggest that developmental DHODH inhibitors represent promising candidates for use as anti-SARS-CoV-2 therapeutics.

Waste solid residue from the hydrothermal extraction of pectin derived from sugar beet pulp was used as feedstock in the production of 5-hydroxymethylfurfural (5-HMF). The depolymerization of pectin-free sugar beet pulp (PF-SBP) to monosaccharides and their dehydration to 5-HMF were conducted in subcritical water using a batch reactor. The experimental design methodology was used in order to model the hydrothermal process and to optimize the operational parameters of the reaction, namely temperature and holding time. These parameters are required to achieve the highest yield of 5-HMF. The model predicts, in good agreement with experimental results (R2 = 0.935), an optimal yield of 5-HMF (of approximately 38% in relation to the cellulosic fraction content in the PF-SBP) at a temperature of 192.5 °C and a holding time of about 51.2 min. 5-HMF was successfully isolated from the reaction mixture using the liquid–liquid extraction method. The results are suitable for industrial upscaling and may become an incentive to introduce a new, environmentally friendly, uncomplicated, and efficient waste treatment method. The method would be used to treat products from the sugar refining industry, the treatment of which has proven to be problematic until now.

The human head is a complex multi-layered structure of hard and soft tissues, governed by complex materials laws and interactions. Computational models of the human head have been developed over the years, reaching high levels of detail, complexity, and precision. However, most of the attention has been devoted to the brain and other intracranial structures. The skull, despite playing a major role in direct head impacts, is often overlooked and simplified. In this work, a new skull model is developed for the authors&rsquo, head model, the YEAHM, based on the original outer geometry, but segmenting it with sutures, diplo&euml, and cortical bone, having variable thickness across different head sections and based on medical craniometric data. These structures are modeled with constitutive models that consider the non-linear behavior of skull bones and also the nature of their failure. Several validations are performed, comparing the simulation results with experimental results available in the literature at several levels: (i) local material validation, (ii) blunt trauma from direct impact against stationary skull, (iii) three impacts at different velocities simulating falls, (iv) blunt ballistic temporoparietal head impacts. Accelerations, impact forces, and fracture patterns are used to validate the skull model.

We previously discovered suppressor T cell-derived, antigen (Ag)-specific exosomes inhibiting mouse hapten-induced contact sensitivity effector T cells by targeting antigen-presenting cells (APCs). These suppressive exosomes acted Ag-specifically due to a coating of antibody free light chains (FLC) from Ag-activated B1a cells. Current studies are aimed at determining if similar immune tolerance could be induced in cutaneous delayed-type hypersensitivity (DTH) to the protein Ag (ovalbumin, OVA). Intravenous administration of a high dose of OVA-coupled, syngeneic erythrocytes similarly induced CD3+CD8+ suppressor T cells producing suppressive, miRNA-150-carrying exosomes, also coated with B1a cell-derived, OVA-specific FLC. Simultaneously, OVA-immunized B1a cells produced an exosome subpopulation, originally coated with Ag-specific FLC, that could be rendered suppressive by in vitro association with miRNA-150. Importantly, miRNA-150-carrying exosomes from both suppressor T cells and B1a cells efficiently induced prolonged DTH suppression after single systemic administration into actively immunized mice, with the strongest effect observed after oral treatment. Current studies also showed that OVA-specific FLC on suppressive exosomes bind OVA peptides suggesting that exosome-coating FLC target APCs by binding to peptide-Ag-major histocompatibility complexes. This renders APCs capable of inhibiting DTH effector T cells. Thus, our studies describe a novel immune tolerance mechanism mediated by FLC-coated, Ag-specific, miRNA-150-carrying exosomes that act on the APC and are particularly effective after oral administration.

We previously discovered suppressor T cell-derived, antigen (Ag)-specific exosomes inhibiting mouse hapten-induced contact sensitivity effector T cells by targeting antigen-presenting cells (APCs). These suppressive exosomes acted Ag-specifically due to a coating of antibody free light chains (FLC) from Ag-activated B1a cells. Current studies aimed at determining if similar immune tolerance could be induced in cutaneous delayed-type hypersensitivity (DTH) to the protein Ag (ovalbumin, OVA). Intravenous administration of a high dose of OVA-coupled, syngeneic erythrocytes induced CD3+CD8+ suppressor T cells producing suppressive, miRNA-150-carrying exosomes, also coated with B1a cell-derived, OVA-specific FLC. Simultaneously, OVA-immunized B1a cells produced exosome subpopulation, originally coated with Ag-specific FLC, that could be rendered suppressive by in vitro association with miRNA-150. Importantly, miRNA-150-carrying exosomes from both suppressor T cells and B1a cells efficiently induced prolonged DTH suppression after single systemic administration into actively immunized mice, with the strongest effect observed after oral administration. Current studies also showed that OVA-specific FLC on suppressive exosomes bind OVA peptides, suggesting that exosome-coating FLC target APCs by binding to Ag-major histocompatibility complexes. This renders APCs able to inhibit DTH effector T cells. Thus, our studies described a novel immune tolerance mechanism mediated by FLC-coated, Ag-specific, miRNA-150-carrying exosomes that are particularly effective after oral administration.

&times, Amarine hybrids are attractive ornamental geophytes grown for cut flower production. Their cultivation is limited due to lesser flowering percentages and lesser bulb weight gain. To optimize the growth and propagation of geophytes, plant growth regulators (PGRs) are used, but so far none have been tested in &times, Amarine. We investigated the effect of gibberellic acid (GA3, 50, 100, and 200 mg dm&minus, 3) and methyl jasmonate (MeJA, 100, 500, and 1000 &micro, mol dm&minus, 3) on growth, flowering, bulb yield, and select physiological parameters of &times, A. tubergenii &ldquo, Zwanenburg&rdquo, PGRs were applied as foliar sprays on the 70th and 77th day after planting. GA3 treatment at 200 mg dm&minus, 3 exhibited the greatest leaf number, leaf length, bulb weight, daughter bulb number, CO2 assimilation intensity, greenness index, total sugars, and total protein content in bulbs. GA3 application at 100 and 200 mg dm&minus, 3 accelerated flowering and at 50 and 100 mg dm&minus, 3 significantly increased the bulb flowering percentage. MeJA at all tested concentrations prolonged anthesis time and reduced the bulb flowering percentage. GA3 at all concentrations and MeJA at 500 and 1000 &micro, 3 stimulated daughter bulbs formation. GA3, especially at 200 mg dm&minus, 3 can improve anthesis and increase &times, bulb yield.

In somatic cells/tissues, methylation of ribosomal DNA (rDNA) increases with age and age‐related pathologies, which has a direct impact on the regulation of nucleolar activity and cellular metabolism. Here, we used bisulfite pyrosequencing and show that methylation of the rDNA transcription unit including upstream control element (UCE), core promoter, 18S rDNA, and 28S rDNA in human sperm also significantly increases with donor's age. This positive correlation between sperm rDNA methylation and biological age is evolutionarily conserved among mammals with widely different life spans such as humans, marmoset, bovine, and mouse. Similar to the tandemly repeated rDNA, methylation of human α‐satellite and interspersed LINE1 repeats, marmoset α‐satellite, bovine alpha‐ and testis satellite I, mouse minor and major satellite, and LINE1‐T repeats increases in the aging male germline, probably related to their sperm histone packaging. Deep bisulfite sequencing of single rDNA molecules in human sperm revealed that methylation does not only depend on donor's age, but also depend on the region and sequence context (A vs. G alleles). Both average rDNA methylation of all analyzed DNA molecules and the number of fully (>50%) methylated alleles, which are thought to be epigenetically silenced, increase with donor's age. All analyzed CpGs in the sperm rDNA transcription unit show comparable age‐related methylation changes. Unlike other epigenetic aging markers, the rDNA clock appears to operate in similar ways in germline and soma in different mammalian species. We propose that sperm rDNA methylation, directly or indirectly, influences nucleolar formation and developmental potential in the early embryo., Sperm methylation of the rDNA transcription unit increases with donor's age in mammals with widely different life spans. Scatter plots show positive correlations between donor's age (x‐axis in percentage of life span) and mean methylation (y‐axis in %) of 18S rDNA in 80 mouse (purple dots), 36 bull (yellow), 16 marmoset (red), and 295 human (green) sperm samples. An orthologous region covering 8 CpGs was analyzed by bisulfite pyrosequencing. The black regression line applies to all 427 analyzed samples.

Bovine enteric bacterial pathogens are a major cause of health decline in agricultural cattle populations. The identification of host-derived microbiota with probiotic characteristics is key for the development of treatments utilizing pathogen displacement and recolonization by commensal flora. In this study, intestinal microbiota in fecal samples from four Holstein dairy cows were analyzed using 16S ribosomal RNA gene next-generation sequencing, leading to the identification of three Lactobacillus isolates (Lactobacillus gasseri, Lactobacillus reuteri, and Lactobacillus salivarius). By taking advantage of the preferential growth in acidified culture media, bacterial characteristics examination, and restriction fragment length polymorphism analysis of 16S rRNA genes, the three lactic acid bacteria (LAB) strains were successfully isolated. The three LAB isolates possess the prerequisite growth tolerances for probiotic functionality, as well as exhibit effective antimicrobial potency against enteric bacterial pathogens of cattle, including Escherichia coli O157:H7, Mycobacterium avium subspecies paratuberculosis, and Salmonella species (Salmonella enteritidis, Salmonella typhimurium, and Salmonella Dublin). Moreover, the LAB isolates showed significant adhesion to cattle intestine, implying greater survivability potential due to their species specificity when administered in the same host species. The LAB isolates were sensitive to most antibiotics with notable resistances of L. gasseri to streptomycin and L. salivarius to kanamycin. Genes attributed to specific antibiotic resistances demonstrated a low risk of lateral transfer in a conjugation study. Our in vitro results demonstrate the promising probiotic characteristics of these newly identified Lactobacillus strains and their considerable potential to serve as probiotics feed supplements for cows.

In addition to their role in regulating transport across the nuclear envelope, increasing evidence suggests nuclear pore complexes (NPCs) function in regulating gene expression. For example, the induction of certain genes (e.g., yeast INO1) is accompanied by their movement from the nuclear interior to NPCs. As sumoylation has been linked to the regulation of chromatin spatial organization and transcriptional activity, we investigated the role of sumoylation in the expression and NPC recruitment of the INO1 gene. We observed that induction of INO1 is accompanied by both increased and decreased sumoylation of proteins associated with specific regions along the INO1 locus. Furthermore, we show that the E3 ligase Siz2/Nfi1 is required for targeting the INO1 locus to the NPC where it interacts with the SUMO isopeptidase Ulp1. Our data suggest that this interaction is required for both the association of INO1 with the NPC and for its normal expression. These results imply that sumoylation is a key regulator of INO1 targeting to the NPC, and a cycle of sumoylation and NPC-associated desumoylation events contribute to the regulation of INO1 expression.

We report a sensitive PCR-based assay called Repetitive Element AneupLoidy Sequencing System (RealSeqS) that can detect aneuploidy in samples containing as little as 3 pg of DNA. Using a single primer pair, we amplified ∼350,000 amplicons distributed throughout the genome. Aneuploidy was detected in 49% of liquid biopsies from a total of 883 nonmetastatic, clinically detected cancers of the colorectum, esophagus, liver, lung, ovary, pancreas, breast, or stomach. Combining aneuploidy with somatic mutation detection and eight standard protein biomarkers yielded a median sensitivity of 80% in these eight cancer types, while only 1% of 812 healthy controls scored positive.

The paper presents the results of chemical analysis of lake waters in Bory Tucholskie National Park (BTNP). The BTNP area is unique due to its location within a single catchment and high variability in geological structure. Moreover, the lakes have different morphometric parameters, represent different hydrological types, trophic types and thermal regimes. Another unique feature is the existence of five lobelia lakes. This name comes from the Latin name of the taxon &ndash, Lobelia dortmanna L. which has been included in the Polish Red Data Book of Plants. The chemical analysis included 55 parameters, within macro elements (MEs), trace elements (TEs) and rare earth elements (REEs). Low concentrations of MEs, TEs and REEs confirm the absence of anthropogenic pressure. High variation of ME, TE and REE contents between individual lakes is due to different geological structure. The cluster analysis enabled lakes to be divided into six groups taking into account all analyzed water quality parameters. The lobelia lakes were characterized by the lowest concentrations of MEs and REEs, which mainly result from the small catchment area and their mainly endorheic character. The highest variability of MEs, TEs and REEs occurred in endorheic lakes, where the geological structure was dominant. The lowest variability of MEs, TEs and REEs occurred in the lakes connected by the Struga Siedmiu Jezior stream. The analysis of MEs, TEs and REEs in relation to the environmental factors and trophic, hydrologic and thermal typology allowed a better understanding of their spatial distribution in the BTNP lakes. The obtained results indicate that the values of the studied elements were generally close to the average values noted in surface waters according to the Geochemical Atlas of Europe.

BackgroundLeucojum aestivumL. is an important medicinal plant which produces Amaryllidaceae alkaloids, especially galanthamine and lycorine. Research is currently exploring the possibility of producing these alkaloids using biotechnological methods, including in vitro cultures. The biosynthesis of alkaloids may be affected by the types and concentrations of carbohydrate sources used in the medium. In the present investigation we performed such studies on in vitro cultures ofL. aestivumwith a view to obtaining plant material of good quality, characterized, in particular, by a high content of valuable Amaryllidaceae alkaloids.MethodsWe examined the effects of various types of carbohydrate sources—sucrose, glucose, fructose and maltose—at different concentrations (30, 60 and 90 g/L)—on the quality ofL. aestivumplants grown in the RITA®bioreactor. The plants’ quality was assessed by their biomass increments, as well by as analysing photosynthetic pigments, endogenous sugar, phenolics and Amaryllidaceae alkaloid content. We also investigated the effect of sugars on the activity of the antioxidant enzymes catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD).ResultsThe highest biomass increments were observed in plants cultivated in the medium containing 90 g/L sucrose. The highest CAT activity was noted in cultures growing in the medium supplemented with 90 g/L maltose, while the highest POD activity was observed in the presence of 90 g/L fructose and 60 g/L maltose. No differences in SOD activity were observed. Moreover, the sugars did not affect the contents of chlorophyllaand carotenoids, whereas the highest amount of chlorophyllbwas recorded in plants growing in the medium with 60 g/L maltose. No statistically significant differences were observed in the contents of endogenous sugars and phenolics in any in vitro conditions. However, the addition of sugar had a decisive effect on the biosynthesis of the Amaryllidaceae alkaloids. The highest distribution of alkaloids occurred in plants cultured in the medium containing 60 g/L sucrose. Six Amaryllidaceae alkaloids were detected in the plant tissue. The addition of 30 g/L fructose in the medium resulted in the accumulation of five alkaloids, including ismine, which was not identified in other analysed tissues. The highest concentration of galanthamine was observed in plants cultured in the presence of 30 g/L fructose and 60 g/L sucrose (39.2 and 37.5 µg/g of dry weight (DW), respectively). The plants grown in the medium containing 60 g/L sucrose exhibited the highest lycorine content (1048 µg/g of DW).ConclusionsThe type and concentration of sugar used in the medium have an essential influence on the biosynthesis of Amaryllidaceae alkaloids inL. aestivumplants cultured in a RITA®bioreactor. The results point to an interesting approach for commercial production of galanthamine and lycorine.

Acyclovir (ACV) is an effective antiviral agent for treating lytic Herpes Simplex virus, type 1 (HSV-1) infections, and it has dramatically reduced the mortality rate of herpes simplex encephalitis. However, HSV-1 resistance to ACV and its derivatives is being increasingly documented, particularly among immunocompromised individuals. The burgeoning drug resistance compels the search for a new generation of more efficacious anti-herpetic drugs. We have previously shown that trans-dihydrolycoricidine (R430), a lycorane-type alkaloid derivative, effectively inhibits HSV-1 infections in cultured cells. We now report that R430 also inhibits ACV-resistant HSV-1 strains, accompanied by global inhibition of viral gene transcription and enrichment of H3K27me3 methylation on viral gene promoters. Furthermore, we demonstrate that R430 prevents HSV-1 reactivation from latency in an ex vivo rodent model. Finally, among a panel of DNA viruses and RNA viruses, R430 inhibited Zika virus with high therapeutic index. Its therapeutic index is comparable to standard antiviral drugs, though it has greater toxicity in non-neuronal cells than in neuronal cells. Synthesis of additional derivatives could enable more efficacious antivirals and the identification of active pharmacophores.

Kamil Kucharczyk,1,2 Marek Weiss,3 Katarzyna Jastrzebska,1,2 Magdalena Luczak,4,5 Arkadiusz Ptak,3 Maciej Kozak,6,7 Andrzej Mackiewicz,1,2 Hanna Dams-Kozlowska1,2 1Department of Medical Biotechnology, Poznan University of Medical Sciences, Poznan, Poland; 2Department of Diagnostics and Cancer Immunology, Greater Poland Cancer Centre, Poznan, Poland; 3Division of Computational Physics and Nanomechanics, Institute of Physics, Faculty of Technical Physics, Poznan University of Technology, Poznan, Poland; 4Department of Biomedical Proteomics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland; 5Department of Organic Chemistry, Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznan, Poland; 6Department of Macromolecular Physics, Adam Mickiewicz University, Poznan, Poland; 7Joint Laboratory for SAXS Studies, Adam Mickiewicz University, Poznan, Poland Background: Silk is a biocompatible and biodegradable material, able to self-assemble into different morphological structures. Silk structures may be used for many biomedical applications, including carriers for drug delivery. The authors designed a new bioengineered spider silk protein, EMS2, and examined its property as a carrier of chemotherapeutics.Materials and methods: To obtain EMS protein, the MS2 silk monomer (that was based on the MaSp2 spidroin of Nephila clavipes) was modified by the addition of a glutamic acid residue. Both bioengineered silks were produced in an Escherichia coli expression system and purified by thermal method. The silk spheres were produced by mixing with potassium phosphate buffer. The physical properties of the particles were characterized using scanning electron microscopy, atomic force microscopy, Fourier-transform infrared spectroscopy, and zeta potential measurements. The MTT assay was used to examine the cytotoxicity of spheres. The loading and release profiles of drugs were studied spectrophotometrically.Results: The bioengineered silk variant, EMS2, was constructed, produced, and purified. The EMS2 silk retained the self-assembly property and formed spheres. The spheres made of EMS2 and MS2 silks were not cytotoxic and had a similar secondary structure content but differed in morphology and zeta potential values; EMS2 particles were more negatively charged than MS2 particles. Independently of the loading method (pre- or post-loading), the loading of drugs into EMS2 spheres was more efficient than the loading into MS2 spheres. The advantageous loading efficiency and release rate made EMS2 spheres a good choice to deliver neutral etoposide (ETP). Despite the high loading efficiency of positively charged mitoxantrone (MTX) into EMS2 particles, the fast release rate made EMS2 unsuitable for the delivery of this drug. A faster release rate from EMS2 particles compared to MS2 particles was observed for positively charged doxorubicin (DOX).Conclusion: By modifying its sequence, silk affinity for drugs can be controlled. Keywords: silk, bioengineering, spheres, drug delivery, chemotherapeutics, cancer therapy

Estrogen-related receptors (ERRs) α, β and γ appear to be novel molecules implicated in estrogen signaling. We blocked and activated ERRs in mouse (C57BL/6) adrenals and adrenocortical cells (H295R) using pharmacological agents XCT 790 (ERRα antagonist) and DY131 (ERRβ/γ agonist), respectively. Mice were injected with XCT 790 or DY131 (5 μg/kg bw) while cells were exposed to XCT 790 or DY131 (0.5 μg/L). Irrespectively of the agent used, changes in adrenocortical cell morphology along with changes in lutropin, cholesterol levels and estrogen production were found. Diverse and complex ERRs regulation of multilevel-acting steroidogenic proteins (perilipin; PLIN, cytochrome P450 side-chain cleavage; P450scc, translocator protein; TSPO, steroidogenic acute regulatory protein; StAR, hormone sensitive lipase; HSL and HMG-CoA reductase; HMGCR) was revealed. Blockage of ERRα decreased P450scc, StAR and TSPO expressions. Activation of ERRβ/γ increased P450scc, StAR and HMGCR while decreased HSL expressions. PLIN expression increased either after XCT 790 or DY131 treatment. Additionally, treatment with both XCT 790 or DY131 decreased activity of Ras/Raf, Erk and Akt indicating their involvement in control of morphology and steroidogenic function of cortex cells. ERRs are important in maintaining morpho-function of cortex cells through action in specific, opposite, or common manner on steroidogenic molecules.

Syndecans are transmembrane proteoglycans that, together with integrins, control cell interactions with extracellular matrix components. Despite structural similarities between all members of the syndecan family, their specific attachment to extracellular matrix proteins is defined by heparan and chondroitin chains. We postulate various unbinding kinetics for each type of single syndecan complex. Force spectroscopy data, recorded by atomic force microscope, were analyzed using two theoretical approaches describing force-induced unbinding, authored by Bell-Evans and Dudko-Hummer-Szabo. Our results reveal distinct unbinding pathways dependent on the syndecan family member. Syndecan-1 unbinds by passing over two energy barriers, inner and outer. Syndecan-4 unbinds by crossing over only one energy barrier. It has already been reported that both syndecans bear heparan chains that are structurally indistinguishable. Our finding reveals that unbinding of single syndecan complexes is family-member-dependent. Distinct unbinding pathways can be attributed to structural differences of heparan and chondroitin chains.

The objective of the study was to assess the influence of lactation number, daily milk yield, somatic cell count and milk composition on the freezing point of milk of Polish Holstein-Friesian cows. The data comprised 3,067,343 test day milk samples collected in 2014 from 865,198 first seven lactations of 714,018 Polish Holstein-Friesian cows, made available by the Polish Federation of Cattle Breeders and Dairy Farmers. The cows calved in 20,043 herds in 2013 and 2014. Four lactation classes were created: first, second, third, and fourth to seventh (4 class). Analysis of variance using the MIXED procedure in SAS was applied to milk freezing point (MFP). The following factors were included in the analysis: lactation class, daily milk yield (16 kg, 16.1–24 kg, >24 kg), fat content (4.4%), protein content (3.5%), urea content (150 mg·L-1, 151–269 mg·L-1, 270 mg·L-1), lactose content (5%), somatic cell count (

The compounds Bi2MoO6 (m = 1), Bi2WO6 (m = 1), Bi3NbTiO9 (m = 2), Bi4Ti3O12 (m = 3) and CaBi4Ti4O15 (m = 4) (Aurivillius phases with the general formula Аm–1Bi2BmO3m+3) have been synthesized by solid state reaction method. Using the high-temperature X-ray diffraction, the phase transition and melting temperatures have been determined for these compounds and their thermal expansion coefficients have been calculated. It has been established that composition and structure of Aurivillius phases have significant influence on the Curie temperature. We have also measured room temperature Raman and infrared spectra, and all observed bands have been assigned to respective motions of atoms in the unit cell. The spectra of the synthesized Auriviliius phases are in good agreement with reported in literature results of the lattice dynamics calculations. We also compare and discuss how the spectra change with increasing thickness of the perovskite-like layer.

Tetrabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA) are bisphenol A (BPA) analogs, where the phenolic moieties are substituted with halogens (Br or Cl). Previous studies indicate that BPA has significant proliferative effects on in vitro cultured epithelial ovarian cancer (EOC) cells. Considering this, we analyzed the effects of both TBBPA and TCBPA at 1, 10, and 50nM on ovarian cancer cell proliferation. The majority of malignant ovarian tumors are epithelial in origin, but approximately 10% are classified as ovarian sex cord tumors, with the most common type being granulosa cell tumors (GCTs). OVCAR-3 and KGN cells were used as in vitro models to represent EOCs and GCTs, respectively. Here, we found that TBBPA, but not TCBPA, stimulated OVCAR-3 and KGN cell proliferation, with lower potency than BPA. The stimulatory effects of TBBPA and BPA on cell proliferation were reversed by pre-treatment with a G protein-coupled receptor 30 (GPR30) antagonist in both cell lines, which possess similar basal GPR30 expression levels. Taken together, our results show for the first time that TBBPA, which has lower potency than BPA, stimulates ovarian cancer cell proliferation through the GPR30 pathway.

Simple Summary The European bison (Bison bonasus) is an endangered species which faces a number of health threats. One potentially dangerous disease is paratuberculosis, which can cause diarrhea and cachexia in animals and is a potentially dangerous disease for humans. The aim of this study was to conduct a serological survey of paratuberculosis in Polish bison herds. Of the tested 165 European bison, three were found to be positive, indicating that paratuberculosis is not currently an urgent problem in this population. However, as the appearance of symptomatic paratuberculosis in a single European bison subpopulation could be fatal for the restitution of the species as whole, further random checks are needed. Abstract The European bison (Bison bonasus) is an endangered species which faces a range of health threats. As little is known of exposure of European bison to paratuberculosis caused by Mycobacterium avium spp. paratuberculosis, known to cause losses in cattle, the aim of the present study was to conduct serological survey in Polish bison herds. Between September 2018 and February 2021, blood samples were collected from 165 European bison from different regions of Poland. Samples were taken whenever the animals were immobilized (e.g., putting on telemetry collars) as well as from any dead animal. The serum samples were tested with ELISA. Three individuals, originating from different captive herds, were found to be seropositive. In conclusion, it was found that Mycobacterium avium subsp. paratuberculosis infections are not currently a problem in European bison, especially in free-range herds.

Epidemiological studies have shown a link between problems with offspring of couples living in a contaminated environment in comparison to those who live in an uncontaminated environment. We measured the concentrations of 16 priority polycyclic aromatic hydrocarbons (PAHs) in maternal and cord blood. To explore the mechanism of the effects of PAH mixtures on nonluteinized granulosa cells (HGrC1) and granulosa tumor cells (COV434), as well as cell proliferation and apoptosis, we investigated the effect of PAH mixtures on the expression of the aryl hydrocarbon receptor (AHR), aryl hydrocarbon receptor nuclear translocator (ARNT) and aryl hydrocarbon receptor repressor (AHRR) genes, as well as the expression and activity of target genes cytochrome P450 1A1 (CYP1A1) and catechol-O-methyltransferase (COMT). The cells were exposed to mixture 1 (M1), composed of all 16 priority PAHs, and mixture 2 (M2), composed of five PAHs which are not classified as human carcinogens, and which are observed in the highest amounts both in maternal and cord blood. All 16 priority PAHs were bioavailable in maternal and cord plasma, suggesting that perinatal exposure should be considered. In HGrC1 cells, M1 increased AHR and ARNT, but decreased AHRR expression, in parallel with increased CYP1A1 and COMT expression and activity. M2 decreased AHR and AHRR, and increased ARNT, with no effect on CYP1A1 expression and activity; however, it did increase COMT expression and activity. In tumor cells, M1 lowered AHR and up-regulated AHRR and ARNT expression, consequently decreasing CYP1A1 expression and COMT activity. M2 up-regulated AHR and ARNT, down-regulated AHRR, and had no effect on CYP1A1 and COMT expression, but decreased COMT activity. We hypothesise that, dependent on composition, mixtures of PAHs activate the AHR differently through varying transcription responses: in HGrC1, a canonical AHR mechanism of M1, with activation of CYP1A1 important for detoxication, while in COV434, a noncanonical AHR mechanism, probably by activation the nuclear factor NFkB.

To characterize polychlorinated biphenyls (PCBs) action on Leydig cells, PCBs congeners, low-chlorinated (delor 103; d103) and high-chlorinated ones (delor 106; d106) were selected. The cells were treated according to PCBs dose (d103 or d106 0.2 ng/ml in low doses:, or 2 ng/ml in high doses) and type (d103 + d106 in low doses or 103 + 106 in high doses). After 24 h treatment with PCBs, a distinct increase in estrogen-related receptors (ERRs type α, β and γ) expression was revealed. However, the dose- and type-dependent PCBs effect was mostly exerted on ERRα expression. A similar increase in ERRs expression was demonstrated by estradiol but not testosterone, which was without an effect on ERRs. PCBs caused no decrease in the membrane potential status of Leydig cells (either in dose or type schedule) but had severe effects on the mitochondria number and structure. Moreover, PCBs markedly increased calcium (Ca2+) concentration and sex steroid secretion (both androgens and estrogens were elevated). These findings suggest a similar estrogenic action of PCBs congeners (d103 and d106) on Leydig cell function. We report dose- and type-specific effects of PCBs only on Leydig cell ERRs expression. Both delors showed common effects on the mitochondria ultrastructural and functional status. Based on our results, ERRα seems to be the most sensitive to hormonal modulation. The increases in Ca2+ and sex steroid secretion may be due to the activation of ERRs by PCBs binding and/or direct effect of PCBs on ERRs mRNA/protein expression. Nevertheless, to confirm the existence of possible relationships between ERRs signaling (including PCBs as ligands) and mitochondria function in Leydig cells, further intensive studies are needed.