KrioBlastTM as a new technology of hyper-fast cryopreservation of cells and tissues. Part 2. Kinetic vitrification of human pluripotent stem cells and spermatozoa

Pilot experiments on kinetic vitrification of human pluripotent stem cells and spermatozoa using a KrioBlastTM-2 without penetrating cryoprotectants have shown high survival of cells (75-85% in both cases

KrioBlastTM as a new technology of hyper-fast cryopreservation of cells and tissues. Part 1. Thermodynamic aspects and potential applications in reproductive and regenerative medicine

Kinetic (dynamic) vitrification is a promising trend in cryopreservation of biological materials because it allows avoiding the formation of lethal intracellular ice and minimizes harmful effects of highly toxic penetrating cryoprotectants. A uniform cooling protocol and the same instruments can be used for practically all types of cell

Two novel mutations associated with ataxia-telangiectasia identified using an ion ampliSeq inherited disease panel

© 2017 Kuznetsova, Trofimov, Shubina, Kochetkova, Karetnikova, Barkov, Bakharev, Gusev and Sukhikh. Ataxia-telangiectasia (A-T), or Louis-Bar syndrome, is a rare neurodegenerative disorder associated with immunodeficiency. For families with at least one affected child, timely A-T genotyping during any subsequent pregnancy allows the parents to make an informed decision about whether to continue to term when the fetus is affected. Mutations in the ATM gene, which is 150 kb long, give rise to A-T; more than 600 pathogenic variants in ATM have been characterized since 1990 and new mutations continue to be discovered annually. Therefore, limiting genetic screening to previously known SNPs by PCR or hybridization with microarrays may not identify the specific pathog enic genotype in ATM for a given A-T family. However, recent developments in next-generation sequencing technology offer prompt high-throughput full-length sequencing of genomic fragments of interest. This allows the identification of the whole spectrum of mutations in a gene, including any novel ones. We report two A-T families with affected children and current pregnancies. Both families are consanguineous and originate from Caucasian regions of Russia and Azerbaijan. Before our study, no ATM mutations had been identified in the older children of these families. We used ion semiconductor sequencing and an Ion AmpliSeq ™ Inherited Disease Panel to perform complete ATM gene sequencing in a single member of each family. Then we compared the experimentally determined genotype with the affected/normal phenotype distribution in the whole family to provide unambiguous evidence of pathogenic mutations responsible for A-T. A single novel SNP was allocated to each family. In the first case, we found a mononucleotide deletion, and in the second, a mononucleotide insertion. Both mutations lead to truncation of the ATM protein product. Identification of the pathogenic mutation in each family was performed in a timely fashion, allowing the fetuses to be tested and diagnosed. The parents chose to continue with both pregnancies as both fetuses had a healthy genotype and thus were not at risk of A-T

Effect of fluorosubstitution on the structure of single crystals, thin films and spectral properties of palladium phthalocyanines

In this work, the crystalline structure of single crystals grown by vacuum sublimation of unsubstituted palladium phthalocyanines (PdPc), its tetrafluorinated (PdPcF4) and hexadecafluorinated (PdPcF16) derivatives have been investigated using X-ray diffraction measurements. Two crystalline phases have been identified for PdPc; the molecules in both phases crystallize in stacks with herringbone arrangement in the monoclinic space groups (C2/c for -PdPc; P21/n for -PdPc). Both PdPcF4 and PdPcF16 crystallize in the triclinic P-1 space group, forming stacks of molecules in columnar arrangement with molecules in adjacent columns are aligned parallel to one another. X-ray diffraction measurements have also been used to elucidate the structural features and molecular orientation of thin films of PdPc, PdPcF4 and PdPcF16, grown by organic molecular beam deposition at different substrate temperatures. The effect of fluorosubstitution on UV-visible optical absorption and vibrational spectra of palladium phthalocyanine derivatives is also discussed

Effect of COVID-19 vaccination on the immune status and autoantibody profile in women of reproductive age

In the context of the COVID-19 pandemic, scientific interest is growing in studying the impact of the proposed vaccination on women’s reproductive health. As is known, alterations in the state of the immune system and activation of an autoimmune response can lead to reproductive failure in women and potential complications of subsequent pregnancy. Objective: to evaluate the effect of the “Gam-COVID-Vac” on the immune status parameters, the relationship of their changes and the specific immune response to vaccination with the dynamics of the level of autoantibodies in women of reproductive age.The prospective study included 120 women who were vaccinated against COVID-19 with the “Gam-COVIDVac”. The criteria for inclusion in the study were: the age from 18 to 49 years, the absence of COVID-19 in the anamnesis, a negative result of a study on SARS-CoV-2 by PCR and negative results of tests for antibodies of classes G and M to SARS-CoV-2 before vaccination, the absence of pregnancy and serious somatic diseases. The patients were examined twice: immediately before vaccination and 90-100 days after the introduction of the 1st component of the vaccine. The level of IgG antibodies to SARS-CoV-2 after vaccination was assessed using ELISA. Before and after vaccination, the levels of antiphospholipid, anti-nuclear, organ-specific and antihormonal autoantibodies were determined, peripheral blood lymphocytes were immunophenotyped to determine the main subpopulations (CD3, CD4, CD8, CD19, CD5, CD16, CD56), as well as the expression of activation markers of lymphocytes (HLA-DR, CD25, CD147) using monoclonal antibodies.The effectiveness and safety of the combined vector vaccine against COVID-19 were high. Specific IgG antibodies to SARS-CoV-2 were produced in 98.3% of vaccinated women, no serious adverse reactions were observed. After vaccination, there was an increase in the level of some autoantibodies within the reference ranges, only IgM antibodies to phosphatidylethanolamine (PE) and IgG antibodies to DNA increased above the reference values. However, this increase was transient. After vaccination, the following changes in the parameters of the immunogram were observed: an increase in the content of cells with CD3+CD25+, CD19+ phenotype in peripheral blood and a decrease in the content of cells with CD56+CD16+ phenotype within the reference ranges, a decrease in CD147+/CD3+. Weak correlations were noted between these changes in immunogram parameters and the levels of some autoantibodies. The specific antiviral immune response to vaccination did not correlate with the autoimmune response.Vaccination with “Gam-COVID-Vac” is effective and safe and does not lead to disorders in the immune system. The observed increase in the level of autoantibodies to PE and DNA is transient. Changes in the parameters of the immune status within the reference ranges may be due to vaccination and the development of a specific antiviral immune response

Risk Factors Associated with Adverse Fetal Outcomes in Pregnancies Affected by Coronavirus Disease 2019 (COVID-19): A Secondary Analysis of the WAPM study on COVID-19

To evaluate the strength of association between maternal and pregnancy characteristics and the risk of adverse perinatal outcomes in pregnancies with laboratory confirmed COVID-19. Secondary analysis of a multinational, cohort study on all consecutive pregnant women with laboratory-confirmed COVID-19 from February 1, 2020 to April 30, 2020 from 73 centers from 22 different countries. A confirmed case of COVID-19 was defined as a positive result on real-time reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assay of nasal and pharyngeal swab specimens. The primary outcome was a composite adverse fetal outcome, defined as the presence of either abortion (pregnancy loss before 22 weeks of gestations), stillbirth (intrauterine fetal death after 22 weeks of gestation), neonatal death (death of a live-born infant within the first 28 days of life), and perinatal death (either stillbirth or neonatal death). Logistic regression analysis was performed to evaluate parameters independently associated with the primary outcome. Logistic regression was reported as odds ratio (OR) with 95% confidence interval (CI). Mean gestational age at diagnosis was 30.6\ub19.5 weeks, with 8.0% of women being diagnosed in the first, 22.2% in the second and 69.8% in the third trimester of pregnancy. There were six miscarriage (2.3%), six intrauterine device (IUD) (2.3) and 5 (2.0%) neonatal deaths, with an overall rate of perinatal death of 4.2% (11/265), thus resulting into 17 cases experiencing and 226 not experiencing composite adverse fetal outcome. Neither stillbirths nor neonatal deaths had congenital anomalies found at antenatal or postnatal evaluation. Furthermore, none of the cases experiencing IUD had signs of impending demise at arterial or venous Doppler. Neonatal deaths were all considered as prematurity-related adverse events. Of the 250 live-born neonates, one (0.4%) was found positive at RT-PCR pharyngeal swabs performed after delivery. The mother was tested positive during the third trimester of pregnancy. The newborn was asymptomatic and had negative RT-PCR test after 14 days of life. At logistic regression analysis, gestational age at diagnosis (OR: 0.85, 95% CI 0.8-0.9 per week increase; p<0.001), birthweight (OR: 1.17, 95% CI 1.09-1.12.7 per 100 g decrease; p=0.012) and maternal ventilatory support, including either need for oxygen or CPAP (OR: 4.12, 95% CI 2.3-7.9; p=0.001) were independently associated with composite adverse fetal outcome. Early gestational age at infection, maternal ventilatory supports and low birthweight are the main determinants of adverse perinatal outcomes in fetuses with maternal COVID-19 infection. Conversely, the risk of vertical transmission seems negligible

Intrauterine Growth Restriction Is a Direct Consequence of Localized Maternal Uropathogenic Escherichia coli Cystitis

Despite the continually increasing rates of adverse perinatal outcomes across the globe, the molecular mechanisms that underlie adverse perinatal outcomes are not completely understood. Clinical studies report that 10% of pregnant women will experience a urinary tract infection (UTI) and there is an association of UTIs with adverse perinatal outcomes. We introduced bacterial cystitis into successfully outbred female mice at gestational day 14 to follow pregnancy outcomes and immunological responses to determine the mechanisms that underlie UTI-mediated adverse outcomes. Outbred fetuses from mothers experiencing localized cystitis displayed intrauterine growth restriction (20–80%) as early as 48 hours post-infection and throughout the remainder of normal gestation. Robust infiltration of cellular innate immune effectors was observed in the uteroplacental tissue following introduction of UTI despite absence of viable bacteria. The magnitude of serum proinflammatory cytokines is elevated in the maternal serum during UTI. This study demonstrates that a localized infection can dramatically impact the immunological status as well as the function of non-infected distal organs and tissues. This model can be used as a platform to determine the mechanism(s) by which proinflammatory changes occur between non-contiguous genitourinary organ

S-layer protein 2 of 'Lactobacillus crispatus' 2029, its structural and immunomodulatory characteristics and roles in protective potential of the whole bacteria against foodborne pathogens

We have previously demonstrated that human vaginal Lactobacillus crispatus 2029 (LC2029) strain is highly adhesive to cervicovaginal epithelial cells, exhibits antagonistic activity against genitourinary pathogens and expresses surface-layer protein (Slp). The aims of the present study were elucidation of Slp structural and immunomodulatory characteristics and its roles in protective properties of the whole vaginal LC2029 bacteria against foodborne pathogens. Enteric Caco-2 and colon HT-29 cell lines were used as the in vitro models of the human intestinal epithelial layer. LC2029 strain has two homologous surface-layer (S-layer) genes, slp1 and slp2. Whilst we found no evidence for the expression of slp1 under the growth conditions used, a very high level of expression of the slp2 gene was detected. C-terminal part of the amino sequence of Slp2 protein was found to be highly similar to that of the conserved C-terminal region of SlpA protein of L. crispatus Zj001 isolated from pig intestines and CbsA protein of L. crispatus JCM5810 isolated from chicken intestines, and was substantially variable at the N-terminal and middle regions. The amino acid sequence identity between SlpA and CbsA was as high as 84%, whilst the identity levels of these sequences with that of Slp2 were only 49% and 50% (respectively). LC2029 strain was found to be both acid and bile tolerant. Survival in simulated gastric and intestinal juices of LC2029 cells unable to produce Slp2 was reduced by 2-3 logs. Vaginal L. crispatus 1385 (LC1385) strain not expressing Slp was also very sensitive to gastric and intestinal stresses. Slp2 was found to be non-covalently bound to the surface of the bacterium, acting as an adhesin and facilitating interaction of LC2029 lactobacilli with the host immature or fully differentiated Caco-2 cells, as well as HT-29 cells. No toxicity to or damage of Caco-2 or HT-29 epithelial cells were detected after 24 h of colonization by LC2029 lactobacilli. Both Slp2 protein and LC2029 cells induced NF-kB activation in Caco-2 and HT-29 cells, but did not induce expression of innate immunity mediators Il-8, Il-1β, and TNF-α. Slp2 and LC2029 inhibited Il-8 production in Caco-2 and HT-29 cells induced by MALP-2 and increased production of anti-inflammatory cytokine Il-6. Slp2 inhibited production of CXCL1 and RANTES by Caco-2 cells during differentiation and maturation process within 15 days. Culturing Caco-2 and HT-29 cells in the presence of Slp2 increased adhesion of bifidobacteria BLI-2780 to these enterocytes. Upon binding to Caco-2 and HT-29 cells, Slp2 protein and LC2029 lactobacilli were recognized by toll-like receptors (TLR) 2/6. It was shown that LC2029 strain is a strong co-aggregator of foodborne pathogens Campylobacter jejuni, Salmonella enteritidis, and Escherichia coli O157:H used in this study. The Slp2 was responsible for the ability of LC2029 to co-aggregate these enteropathogens. Slp2 and intact LC2029 lactobacilli inhibited foodborne pathogen-induced activation of caspase-9 and caspase-3 as apoptotic biomarkers in Caco-2 and HT-29 cells. In addition, Slp2 and Slp2-positive LC2029 strain reduced adhesion of tested pathogenic bacteria to Caco-2 and HT-29 cells. Slp2-positive LC2029 strain but not Slp2 alone provided bactericidal effect on foodborne pathogens. These results suggest a range of mechanisms involved in inhibition of growth, viability, and cell adhesion properties of pathogenic Proteobacteria by the Slp2 producing LC2029, which may be useful in treatment of necrotizing enterocolitis (NEC) in newborns and foodborne infectious diseases in children and adults, increasing the colonization resistance and maintaining the intestinal homeostasis

Search for low-mass dilepton resonances in Higgs boson decays to four-lepton final states in proton–proton collisions at √s=13TeV

A search for low-mass dilepton resonances in Higgs boson decays is conducted in the four-lepton final state. The decay is assumed to proceed via a pair of beyond the standard model particles, or one such particle and a Z boson. The search uses proton–proton collision data collected with the CMS detector at the CERN LHC, corresponding to an integrated luminosity of 137 fb−1, at a center-of-mass energy √s = 13 TeV. No significant deviation from the standard model expectation is observed. Upper limits at 95% confidence level are set on model-independent Higgs boson decay branching fractions. Additionally, limits on dark photon and axion-like particle production, based on two specific models, are reported