The Caenorhabditis elegans CPI-2a cystatin-like inhibitor has an essential regulatory role during oogenesis and fertilization

In the present study, we characterized a sterile cpi-2a(ok1256) deletion mutant in Caenorhabditis elegans and showed that CPI-2a has an essential regulatory role during oogenesis and fertilization. We have also shown that the CPI2a inhibitor and both Ce-CPL-1 and Ce-CPZ-1 enzymes are present in the myoepithelial sheath surrounding germ cells, oocytes, and embryos as well as in the yolk granules within normal oocytes. Staining of mutant worms with anti-yolk protein antibodies has indicted that the proteins are not present in the mature oocytes. Moreover, green fluorescent protein expression was absence or reduced in cpi-2a/yp170:gfp mutant oocytes, although it was expressed in one of the successfully developed embryos. Based on these results, we hypothesize that the sterility in cpi-2a(ok1256) mutant worms is potentially caused by two possible mechanisms: 1) defects in the uptake and/or processing of yolk proteins by the growing oocytes and 2) indirect induction of defects in cell-cell signaling that is critical for promoting germ line development, oocyte maturation, ovulation, and fertilization. A defect in any of these processes would have detrimental effects on the development of normal embryos and consequently normal production of progenies as we observed in cpi-2a mutant worms. This is the first study that demonstrates the expression of cysteine proteases and their endogenous inhibitor in the gonadal sheath cells surrounding germ cells and oocytes, which indirectly have established their potential involvement in proteolytic processing of molecules within the gonadal sheath cells, such as components of the extracellular matrix or the cytoskeletal proteins, which are essential for proper cell-cell signaling activities of the gonadal sheath cells during normal maturation and ovulation processes.     

The C terminus of the Vps34p phosphoinositide 3-kinase is necessary and sufficient for the interaction with the Vps15p protein kinase.

Vps34p is a phosphatidylinositol 3-kinase that is part of a membrane-associated complex with the Vps15p protein kinase. This kinase complex is required for the delivery of soluble proteins to the lysosomal/vacuolar compartment of eukaryotic cells. This study examined the Vps34p-Vps15p association and identified the domains within each protein that were important for this interaction. Using several different approaches, the interaction domain within Vps34p was mapped to a 28-amino acid element near its C terminus. This Vps34p motif was both necessary and sufficient for the interaction with Vps15p. Two-hybrid mapping experiments indicated that two separate regions of Vps15p were required for the association with Vps34p; they are the N-terminal protein kinase domain and a set of three tandem repeats of about 39 amino acids each. Neither domain alone was sufficient for the interaction. These Vps15p repeat elements are similar in sequence to the HEAT motifs that have been implicated in protein interactions in other proteins, including the Huntingtin protein. Finally, these studies identified a novel motif at the very C terminus of Vps34p that was required for phosphatidylinositol 3-kinase activity. This domain is highly conserved specifically in all Vps34p-like phosphatidylinositol 3-kinases but is not required for the interaction with Vps15p. This study thus represents a first step toward a better understanding of how this Vps15p.Vps34p kinase complex is assembled and regulated in vivo.     

Silicon nanotechnologies of pigmented heterokonts

Many pigmented heterokonts are able to synthesize elements of their cell walls (the frustules) of dense biogenic silica. These include diatom algae, which occupy a significant place in the biosphere. The siliceous frustules of diatoms have species-specific patterns of surface structures between 10 and a few hundred nanometers. The present review considers possible mechanisms of uptake of silicic acid from the aquatic environment, its transport across the plasmalemma, and intracellular transport and deposition of silica inside the specialized Silica Deposition Vesicle (SDV) where elements of the new frustule are formed. It is proposed that a complex of silicic acid with positively charged proteins silaffins and polypropylamines remains a homogeneous solution during the intracellular transport to SDV, where biogenic silica precipitates. The high density of the deposited biogenic silica may be due to removal of water from the SDV by aquaporins followed by syneresis--a process during which pore water is expelled from the network of the contracting gel. The pattern of aquaporins in the silicalemma, the membrane embracing the SDV, can determine the pattern of species-specific siliceous nanostructures.     

Sulfinylated azadecalins act as functional mimics of a pollen germination stimulant in Arabidopsis pistils

Polarized cell elongation is triggered by small molecule cues during development of diverse organisms. During plant reproduction, pollen interactions with the stigma result in the polar outgrowth of a pollen tube, which delivers sperm cells to the female gametophyte to effect double fertilization. In many plants, pistils stimulate pollen germination. However, in Arabidopsis, the effect of pistils on pollen germination and the pistil factors that stimulate pollen germination remain poorly characterized. Here, we demonstrate that stigma, style, and ovules in Arabidopsis pistils stimulate pollen germination. We isolated an Arabidopsis pistil extract fraction that stimulates Arabidopsis pollen germination, and employed ultra‐high resolution electrospray ionization (ESI), Fourier‐transform ion cyclotron resonance (FT‐ICR) and MS/MS techniques to accurately determine the mass (202.126 Da) of a compound that is specifically present in this pistil extract fraction. Using the molecular formula (C₁₀H₁₉NOS) and tandem mass spectral fragmentation patterns of the m/z (mass to charge ratio) 202.126 ion, we postulated chemical structures, devised protocols, synthesized N‐methanesulfinyl 1‐ and 2‐azadecalins that are close structural mimics of the m/z 202.126 ion, and showed that they are sufficient to stimulate Arabidopsis pollen germination in vitro (30 μm stimulated approximately 50% germination) and elicit accession‐specific response. Although N‐methanesulfinyl 2‐azadecalin stimulated pollen germination in three species of Lineage I of Brassicaceae, it did not induce a germination response in Sisymbrium irio (Lineage II of Brassicaceae) and tobacco, indicating that activity of the compound is not random. Our results show that Arabidopsis pistils promote germination by producing azadecalin‐like molecules to ensure rapid fertilization by the appropriate pollen.     

Paradoxical hotspots for guanine oxidation by a chemical mediator of inflammation

Guanine in DNA is a major oxidation target owing to its low ionization potential (IP), and there is often an inverse correlation between damage frequency and sequence-dependent variation in guanine IP. We report that the biological oxidant nitrosoperoxycarbonate (ONOOCO2(-)) paradoxically selects guanines with the highest IP in GC-containing contexts. Along with sequence-dependent variation in damage chemistry, this behavior points to factors other than charge migration as determinants of genomic DNA oxidation.     

Quantitation and pharmacokinetic modeling of therapeutic antibody quality attributes in human studies

A thorough understanding of drug metabolism and disposition can aid in the assessment of efficacy and safety. However, analytical methods used in pharmacokinetics (PK) studies of protein therapeutics are usually based on ELISA, and therefore can provide a limited perspective on the quality of the drug in concentration measurements. Individual post-translational modifications (PTMs) of protein therapeutics are rarely considered for PK analysis, partly because it is technically difficult to recover and quantify individual protein variants from biological fluids. Meanwhile, PTMs may be directly linked to variations in drug efficacy and safety, and therefore understanding of clearance and metabolism of biopharmaceutical protein variants during clinical studies is an important consideration. To address such challenges, we developed an affinity-purification procedure followed by peptide mapping with mass spectrometric detection, which can profile multiple quality attributes of therapeutic antibodies recovered from patient sera. The obtained data enable quantitative modeling, which allows for simulation of the PK of different individual PTMs or attribute levels in vivo and thus facilitate the assessment of quality attributes impact in vivo. Such information can contribute to the product quality attribute risk assessment during manufacturing process development and inform appropriate process control strategy.     

Biparatopic sybodies neutralize SARS‐CoV‐2 variants of concern and mitigate drug resistance

The ongoing COVID‐19 pandemic represents an unprecedented global health crisis. Here, we report the identification of a synthetic nanobody (sybody) pair, Sb#15 and Sb#68, that can bind simultaneously to the SARS‐CoV‐2 spike RBD and efficiently neutralize pseudotyped and live viruses by interfering with ACE2 interaction. Cryo‐EM confirms that Sb#15 and Sb#68 engage two spatially discrete epitopes, influencing rational design of bispecific and tri‐bispecific fusion constructs that exhibit up to 100‐ and 1,000‐fold increase in neutralization potency, respectively. Cryo‐EM of the sybody‐spike complex additionally reveals a novel up‐out RBD conformation. While resistant viruses emerge rapidly in the presence of single binders, no escape variants are observed in the presence of the bispecific sybody. The multivalent bispecific constructs further increase the neutralization potency against globally circulating SARS‐CoV‐2 variants of concern. Our study illustrates the power of multivalency and biparatopic nanobody fusions for the potential development of therapeutic strategies that mitigate the emergence of new SARS‐CoV‐2 escape mutants.     

Delta-9-tetrahydrocannabinol protects cardiac cells from hypoxia via CB2 receptor activation and nitric oxide production

Delta-9-tetrahydrocannabinol (THC), the major active component of marijuana, has a beneficial effect on the cardiovascular system during stress conditions, but the defence mechanism is still unclear. The present study was designed to investigate the central (CB1) and the peripheral (CB2) cannabinoid receptor expression in neonatal cardiomyoctes and possible function in the cardioprotection of THC from hypoxia. Pre-treatment of cardiomyocytes that were grown in vitro with 0.1 – 10 μM THC for 24 h prevented hypoxia-induced lactate dehydrogenase (LDH) leakage and preserved the morphological distribution of α-sarcomeric actin. The antagonist for the CB2 (10 μM), but not CB1 receptor antagonist (10 μM) abolished the protective effect of THC. In agreement with these results using RT-PCR, it was shown that neonatal cardiac cells express CB2, but not CB1 receptors. Involvement of NO in the signal transduction pathway activated by THC through CB2 was examined. It was found that THC induces nitric oxide (NO) production by induction of NO synthase (iNOS) via CB2 receptors. L-NAME (NOS inhibitor, 100 μM) prevented the cardioprotection provided by THC. Taken together, our findings suggest that THC protects cardiac cells against hypoxia via CB2 receptor activation by induction of NO production. An NO mechanism occurs also in the classical pre-conditioning process; therefore, THC probably pre-trains the cardiomyocytes to hypoxic conditions.