Expression Patterns and Gonadotropin Regulation of the TGF-β II Receptor (Bmpr2) during Ovarian Development in the Ricefield Eel Monopterus albus

Bmpr2 plays a central role in the regulation of reproductive development in mammals, but its role during ovarian development in fish is still unclear. To ascertain the function of bmpr2 in ovarian development in the ricefield eel, we isolated and characterized the bmpr2 cDNA sequence; the localization of Bmpr2 protein was determined by immunohistochemical staining; and the expression patterns of bmpr2 in ovarian tissue incubated with FSH and hCG in vitro were analyzed. The full-length bmpr2 cDNA was 3311 bp, with 1061 amino acids encoded. Compared to other tissues, bmpr2 was abundantly expressed in the ovary and highly expressed in the early yolk accumulation (EV) stages of the ovary. In addition, a positive signal for Bmpr2 was detected in the cytoplasm of oocytes in primary growth (PG) and EV stages. In vitro, the expression level of gdf9, the ligand of bmpr2, in the 10 ng/mL FSH treatment group was significantly higher after incubation for 4 h than after incubation for different durations. However, bmpr2 expression in the 10 ng/mL FSH treatment group at 2 h, 4 h and 10 h was significantly lower. Importantly, the expression level of bmpr2 and gdf9 in the 100 IU/mL hCG group had similar changes that were significantly decreased at 4 h and 10 h. In summary, Bmpr2 might play a pivotal role in ovarian growth in the ricefield eel, and these results provide a better understanding of the function of bmpr2 in ovarian development and the basic data for further exploration of the regulatory mechanism of gdf9 in oocyte development.     

The STIM1/2-Regulated Calcium Homeostasis Is Impaired in Hippocampal Neurons of the 5xFAD Mouse Model of Alzheimer’s Disease

Alzheimer’s disease (AD) is the most common cause of age-related dementia. Neuronal calcium homeostasis impairment may contribute to AD. Here we demonstrated that voltage-gated calcium (VGC) entry and store-operated calcium (SOC) entry regulated by calcium sensors of intracellular calcium stores STIM proteins are affected in hippocampal neurons of the 5xFAD transgenic mouse model. We observed excessive SOC entry in 5xFAD mouse neurons, mediated by STIM1 and STIM2 proteins with increased STIM1 contribution. There were no significant changes in cytoplasmic calcium level, endoplasmic reticulum (ER) bulk calcium levels, or expression levels of STIM1 or STIM2 proteins. The potent inhibitor BTP-2 and the FDA-approved drug leflunomide reduced SOC entry in 5xFAD neurons. In turn, excessive voltage-gated calcium entry was sensitive to the inhibitor of L-type calcium channels nifedipine but not to the T-type channels inhibitor ML218. Interestingly, the depolarization-induced calcium entry mediated by VGC channels in 5xFAD neurons was dependent on STIM2 but not STIM1 protein in cells with replete Ca²⁺ stores. The result gives new evidence on the VGC channel modulation by STIM2. Overall, the data demonstrate the changes in calcium signaling of hippocampal neurons of the AD mouse model, which precede amyloid plaque accumulation or other signs of pathology manifestation.     

Genome-Wide Identification and Expression Analysis of Fatty Acid Desaturase (FAD) Genes in Camelina sativa (L.) Crantz

Camelina sativa (L.) Crantz is an indispensable oilseed crop, and its seeds contain many unsaturated fatty acids. FAD (fatty acid desaturase) regulates the synthesis of unsaturated fatty acids. In this research, we performed CsFAD gene family analysis and identified 24 CsFAD genes in Camelina, which were unevenly distributed on 14 of the 19 total chromosomes. Phylogenetic analysis showed that CsFAD includes four subfamilies, supported by the conserved structures and motifs of CsFAD genes. In addition, we investigated the expression patterns of the FAD family in the different tissues of Camelina. We found that CsFAD family genes were all expressed in the stem, and CsFAD2-2 was highly expressed in the early stage of seed development. Moreover, during low temperature (4 °C) stress, we identified that the expression level of CsFAD2-2 significantly changed. By observing the transient expression of CsFAD2-2 in Arabidopsis protoplasts, we found that CsFAD2-2 was located on the nucleus. Through the detection and analysis of fatty acids, we prove that CsFAD2-2 is involved in the synthesis of linolenic acid (C18:3). In conclusion, we identified CsFAD2-2 through the phylogenetic analysis of the CsFAD gene family and further determined the fatty acid content to find that CsFAD2-2 is involved in fatty acid synthesis in Camelina.     

CuO Modified by 7,7,8,8-Tetracyanoquinodimethane and Its Application to CO2 Separation

7,7,8,8-Tetracyanoquinomethane (TCNQ) was added to polyvinylpyrrolidone (PVP)/CuO composites to modify and prevent agglomeration of the particles, and thus the CuO particles were well dispersed to a small size, thereby increasing CO₂ solubility and separation performance. When the separation performance of the PVP/CuO/TCNQ composite membrane was measured for CO₂/N₂ gases, a CO₂ separation of about 174 was measured. This improvement in performance was attributed to the fact that TCNQ was applied to PVP and CuO to prevent agglomeration between particles with surface modification. Due to TCNQ, CuO could be dispersed to a small size in PVP; the bonds between chains in PVP weakened; the interaction between molecules weakened; and the free volume increased, as confirmed by FT-IR, TGA, and UV–Vis spectroscopy.     

Purinergic Signalling in the Cochlea

The mammalian cochlea is the sensory organ of hearing with a delicate, highly organised structure that supports unique operating mechanisms. ATP release from the secretory tissues of the cochlear lateral wall (stria vascularis) triggers numerous physiological responses by activating P2 receptors in sensory, supporting and neural tissues. Two families of P2 receptors, ATP-gated ion channels (P2X receptors) and G protein-coupled P2Y receptors, activate intracellular signalling pathways that regulate cochlear development, homeostasis, sensory transduction, auditory neurotransmission and response to stress. Of particular interest is a purinergic hearing adaptation, which reflects the critical role of the P2X₂ receptor in adaptive cochlear response to elevated sound levels. Other P2 receptors are involved in the maturation of neural processes and frequency selectivity refinement in the developing cochlea. Extracellular ATP signalling is regulated by a family of surface-located enzymes collectively known as “ectonucleotidases” that hydrolyse ATP to adenosine. Adenosine is a constitutive cell metabolite with an established role in tissue protection and regeneration. The differential activation of A₁ and A_2A adenosine receptors defines the cochlear response to injury caused by oxidative stress, inflammation, and activation of apoptotic pathways. A₁ receptor agonism, A_2A receptor antagonism, and increasing adenosine levels in cochlear fluids all represent promising therapeutic tools for cochlear rescue from injury and prevention of hearing loss.     

Correlation between In Vitro Neutralization Assay and Serological Tests for Protective Antibodies Detection

Serological assays are useful in investigating the development of humoral immunity against SARS-CoV-2 in the context of epidemiological studies focusing on the spread of protective immunity. The plaque reduction neutralization test (PRNT) is the gold standard method to assess the titer of protective antibodies in serum samples. However, to provide a result, the PRNT requires several days, skilled operators, and biosafety level 3 laboratories. Therefore, alternative methods are being assessed to establish a relationship between their outcomes and PRNT results. In this work, four different immunoassays (Roche Elecsys^® Anti SARS-CoV-2 S, Snibe MAGLUMI^® SARS-CoV-2 S-RBD IgG, Snibe MAGLUMI^® 2019-nCoV IgG, and EUROIMMUN^® SARS-CoV-2 NeutraLISA assays, respectively) have been performed on individuals healed after SARS-CoV-2 infection. The correlation between each assay and the reference method has been explored through linear regression modeling, as well as through the calculation of Pearson’s and Spearman’s coefficients. Furthermore, the ability of serological tests to discriminate samples with high titers of neutralizing antibodies (>160) has been assessed by ROC curve analyses, Cohen’s Kappa coefficient, and positive predictive agreement. The EUROIMMUN^® NeutraLISA assay displayed the best correlation with PRNT results (Pearson and Spearman coefficients equal to 0.660 and 0.784, respectively), as well as the ROC curve with the highest accuracy, sensitivity, and specificity (0.857, 0.889, and 0.829, respectively).     

Insights into the Structure of the Highly Glycosylated Ffase from Rhodotorula dairenensis Enhance Its Biotechnological Potential

Rhodotorula dairenensis β-fructofuranosidase is a highly glycosylated enzyme with broad substrate specificity that catalyzes the synthesis of 6-kestose and a mixture of the three series of fructooligosaccharides (FOS), fructosylating a variety of carbohydrates and other molecules as alditols. We report here its three-dimensional structure, showing the expected bimodular arrangement and also a unique long elongation at its N-terminus containing extensive O-glycosylation sites that form a peculiar arrangement with a protruding loop within the dimer. This region is not required for activity but could provide a molecular tool to target the dimeric protein to its receptor cellular compartment in the yeast. A truncated inactivated form was used to obtain complexes with fructose, sucrose and raffinose, and a Bis-Tris molecule was trapped, mimicking a putative acceptor substrate. The crystal structure of the complexes reveals the major traits of the active site, with Asn387 controlling the substrate binding mode. Relevant residues were selected for mutagenesis, the variants being biochemically characterized through their hydrolytic and transfructosylating activity. All changes decrease the hydrolytic efficiency against sucrose, proving their key role in the activity. Moreover, some of the generated variants exhibit redesigned transfructosylating specificity, which may be used for biotechnological purposes to produce novel fructosyl-derivatives.     

Allosteric Inhibition of Neutral Sphingomyelinase 2 (nSMase2) by DPTIP: From Antiflaviviral Activity to Deciphering Its Binding Site through In Silico Studies and Experimental Validation

Flavivirus comprises globally emerging and re-emerging pathogens such as Zika virus (ZIKV), Dengue virus (DENV), and West Nile virus (WNV), among others. Although some vaccines are available, there is an unmet medical need as no effective antiviral treatment has been approved for flaviviral infections. The development of host-directed antivirals (HDAs) targeting host factors that are essential for viral replication cycle offers the opportunity for the development of broad-spectrum antivirals. In the case of flaviviruses, recent studies have revealed that neutral sphingomyelinase 2, (nSMase2), involved in lipid metabolism, plays a key role in WNV and ZIKV infection. As a proof of concept, we have determined the antiviral activity of the non-competitive nSMase2 inhibitor DPTIP against WNV and ZIKV virus. DPTIP showed potent antiviral activity with EC₅₀ values of 0.26 µM and 1.56 µM for WNV and ZIKV, respectively. In order to unravel the allosteric binding site of DPTIP in nSMase2 and the details of the interaction, computational studies have been carried out. These studies have revealed that DPTIP could block the DK switch in nSMase2. Moreover, the analysis of the residues contributing to the binding identified His463 as a crucial residue. Interestingly, the inhibitory activity of DPTIP on the H463A mutant protein supported our hypothesis. Thus, an allosteric cavity in nSMase2 has been identified that can be exploited for the development of new inhibitors with anti-flaviviral activity.     

The SARS-CoV-2 Delta-Omicron Recombinant Lineage (XD) Exhibits Immune-Escape Properties Similar to the Omicron (BA.1) Variant

Recently, a recombinant SARS-CoV-2 lineage, XD, emerged that harbors a spike gene that is largely derived from the Omicron variant BA.1 in the genetic background of the Delta variant. This finding raised concerns that the recombinant virus might exhibit altered biological properties as compared to the parental viruses and might pose an elevated threat to human health. Here, using pseudotyped particles, we show that ACE2 binding and cell tropism of XD mimics that of BA.1. Further, XD and BA.1 displayed comparable sensitivity to neutralization by antibodies induced upon vaccination with BNT162b2/Comirnaty (BNT) or BNT vaccination followed by breakthrough infection. Our findings reveal important biological commonalities between XD and Omicron BA.1 host cell entry and its inhibition by antibodies.     

Modeling RTT Syndrome by iPSC-Derived Neurons from Male and Female Patients with Heterogeneously Severe Hot-Spot MECP2 Variants

Rett syndrome caused by MECP2 variants is characterized by a heterogenous clinical spectrum accounted for in 60% of cases by hot-spot variants. Focusing on the most frequent variants, we generated in vitro iPSC-neurons from the blood of RTT girls with p.Arg133Cys and p.Arg255*, associated to mild and severe phenotype, respectively, and of an RTT male harboring the close to p.Arg255*, p.Gly252Argfs*7 variant. Truncated MeCP2 proteins were revealed by Western blot and immunofluorescence analysis. We compared the mutant versus control neurons at 42 days for morphological parameters and at 120 days for electrophysiology recordings, including girls’ isogenic clones. A precocious reduced morphological complexity was evident in neurons with truncating variants, while in p.Arg133Cys neurons any significant differences were observed in comparison with the isogenic wild-type clones. Reduced nuclear size and branch number show up as the most robust biomarkers. Patch clamp recordings on mature neurons allowed the assessment of cell biophysical properties, V-gated currents, and spiking pattern in the mutant and control cells. Immature spiking, altered cell capacitance, and membrane resistance of RTT neurons, were particularly pronounced in the Arg255* and Gly252Argfs*7 mutants. The overall results indicate that the specific markers of in vitro cellular phenotype mirror the clinical severity and may be amenable to drug testing for translational purposes.