A conformational switch in collybistin determines the differentiation of inhibitory postsynapses

The formation of neuronal synapses and the dynamic regulation of their efficacy depend on the assembly of the postsynaptic neurotransmitter receptor apparatus. Receptor recruitment to inhibitory GABAergic and glycinergic synapses is controlled by the scaffold protein gephyrin and the adaptor protein collybistin. We derived new insights into the structure of collybistin and used these to design biochemical, cell biological, and genetic analyses of collybistin function. Our data define a collybistin‐based protein interaction network that controls the gephyrin content of inhibitory postsynapses. Within this network, collybistin can adopt open/active and closed/inactive conformations to act as a switchable adaptor that links gephyrin to plasma membrane phosphoinositides. This function of collybistin is regulated by binding of the adhesion protein neuroligin‐2, which stabilizes the open/active conformation of collybistin at the postsynaptic plasma membrane by competing with an intramolecular interaction in collybistin that favors the closed/inactive conformation. By linking trans‐synaptic neuroligin‐dependent adhesion and phosphoinositide signaling with gephyrin recruitment, the collybistin‐based regulatory switch mechanism represents an integrating regulatory node in the formation and function of inhibitory postsynapses.     

Preventive effects of omega-3 and omega-6 Fatty acids on peroxide mediated oxidative stress responses in primary human trabecular meshwork cells

Pathologic processes in glaucoma include increased apoptosis, accumulation of extracellular material in the trabecular meshwork and optic nerve, condensations of the cytoskeleton and precocious cellular senescence. Oxidative stress was shown to generate these alterations in primary ocular cells. Fatty acids omega-3 and -6 are alleged to constitute a prophylaxis against these deleterious effects. Here, we tested actual preventive effects omega-3 and -6 against peroxide induced stress responses in primary human trabecular meshwork cells. Changes of mitochondrial activity, proliferation, heat shock proteins, extracellular matrix components, and inflammatory markers were evaluated. Alterations of the cytoskeleton were evaluated by phalloidin labeling. Here we report a repressive effect of omega-6 on metabolic activity and proliferation, which was not detected for omega-3. Both agents were able to prevent the anti-proliferative effect of H₂O₂, but only omega-3 prevented metabolic repression. Expression of heat shock protein 27 was unaltered by both fatty acids, whereas heat shock protein 90 was significantly induced by both. Omega-6 increased fibronectin and connective tissue growth factor synthesis, as well as the amount of secreted fibronectin. Omega-3, instead, induced plasminogen activator inhibitor 1 synthesis. H₂O₂ further increased fibronectin production in omega-6 supplemented cells, which was not the case in omega-3 treated cells. H₂O₂ stimulation of plasminogen activator inhibitor 1 and connective tissue growth factor was repressed by both fatty acids. Both fatty acids appeared to abolish H₂O₂ mediated stimulation of nuclear factor κB and IL-6, but not IL-1α and IL-8. H₂O₂ induced formation of cross-linked actin networks and stress fibers, which was reduced by preemptive application of omega-3. Omega-6, in contrast, had no protective effect on that, and even seemed to promote condensation. Based on the observed side effects of omega-6, omega-3 appears to be the more beneficial fatty acid in respect of prophylactic intake for prevention of a glaucomatous disease.     

Bioinformatic analysis of fold-type III PLP-dependent enzymes discovers multimeric racemases

Pyridoxal-5′-phosphate (PLP)-dependent enzymes are ubiquitous in nature and catalyze a variety of important metabolic reactions. The fold-type III PLP-dependent enzyme family is primarily comprised of decarboxylases and alanine racemases. In the development of a multiple structural alignment database (3DM) for the enzyme family, a large subset of 5666 uncharacterized proteins with high structural, but low sequence similarity to alanine racemase and decarboxylases was found. Compared to these two classes of enzymes, the protein sequences being the object of this study completely lack the C-terminal domain, which has been reported important for the formation of the dimer interface in other fold-type III enzymes. The 5666 sequences cluster around four protein templates, which also share little sequence identity to each other. In this work, these four template proteins were solubly expressed in Escherichia coli, purified, and their substrate profiles were evaluated by HPLC analysis for racemase activity using a broader range of amino acids. They were found active only against alanine or serine, where they exhibited Michaelis constants within the range of typical bacterial alanine racemases, but with significantly lower turnover numbers. As the already described racemases were proposed to be active and appeared to be monomers as judged from their crystal structures, we also investigated this aspect for the four new enzymes. Here, size exclusion chromatography indicated the presence of oligomeric states of the enzymes and a native-PAGE in-gel assay showed that the racemase activity was present only in an oligomeric state but not as monomer. This suggests the likelihood of a different behavior of these enzymes in solution compared to the one observed in crystalline form.

     

Outbreaks of SARS-CoV-2 in naturally infected mink farms: Impact,transmission dynamics,genetic patterns,and environmental contamination

SARS-CoV-2 infection outbreaks in minks have serious implications associated with animal health and welfare, and public health. In two naturally infected mink farms (A and B) located in Greece, we investigated the outbreaks and assessed parameters associated with virus transmission, immunity, pathology, and environmental contamination. Symptoms ranged from anorexia and mild depression to respiratory signs of varying intensity. Although the farms were at different breeding stages, mortality was similarly high (8.4% and 10.0%). The viral strains belonged to lineages B.1.1.218 and B.1.1.305, possessing the mink-specific S-Y453F substitution. Lung histopathology identified necrosis of smooth muscle and connective tissue elements of vascular walls, and vasculitis as the main early key events of the acute SARS-CoV-2-induced broncho-interstitial pneumonia. Molecular investigation in two dead minks indicated a consistently higher (0.3–1.3 log₁₀ RNA copies/g) viral load in organs of the male mink compared to the female. In farm A, the infected farmers were responsible for the significant initial infection of 229 out of 1,000 handled minks, suggesting a very efficient human-to-mink transmission. Subsequent infections across the sheds wherein animals were being housed occurred due to airborne transmission. Based on a R₀ of 2.90 and a growth rate equal to 0.293, the generation time was estimated to be 3.6 days, indicative of the massive SARS-CoV-2 dispersal among minks. After the end of the outbreaks, a similar percentage of animals were immune in the two farms (93.0% and 93.3%), preventing further virus transmission whereas, viral RNA was detected in samples collected from shed surfaces and air. Consequently, strict biosecurity is imperative during the occurrence of clinical signs. Environmental viral load monitoring, in conjunction with NGS should be adopted in mink farm surveillance. The minimum proportion of minks that need to be immunized to avoid outbreaks in farms was calculated at 65.5%, which is important for future vaccination campaigns.     

Effects of whole body X-irradiation and cyclophosphamide treatment on induction of macrophage tumoricidal function in mice

The influence of whole body X-irradiation (200–800 R) and subcutaneous cyclophosphamide (CY) treatment (150–500 mg/kg) was studied on the ability of adjuvants to induce cytotoxic macrophages in vivo. Surprisingly, radiation or CY therapy alone produced growth inhibitory macrophages whose function peaked within 2 days after treatment. When adjuvants such as Bacillus Calmette Guérin (BCG), pyran copolymer, or glucan were administered ip within 2 hr after sublethal (600 R) X-irradiation, adjuvant-induced cytotoxic function was depressed but not ablated. In addition, when noninduced peritoneal macrophages were obtained 6 days after lethal (800 R) X-irradiation, their ability to be activated in vitro by lymphokine or fibroblast-derived interferon preparations was only slightly depressed at all concentrations of inducer tested. When BCG, pyran, or glucan was administered ip concurrently with sc CY treatment, only the ability of BCG to activate macrophages was markedly reduced, indicating separate mechanisms for the induction of tumoricidal macrophages. A better understanding of the interaction of chemotherapeutic and/or radiation regimens with adjuvants which affect macrophage function may be instrumental to rationalized immunotherapy protocols.     

Significant improvement of quality for long oligonucleotides by using controlled pore glass with large pores

A key factor influencing the quality of long oligonucleotides is the choice of controlled pore glass (CPG) which is used as a solid support during oligonucleotide synthesis. We studied the influence of CPG pore size on the quality of 75-mer oligonucleotides. Using electrophoresis and HPLC, we demonstrated failure modes that can occur at certain oligo lengths with 1000A pore size, and compared yield and purity of 75-mer oligos using 1000A and larger pore size CPG. We showed that oligonucleotides with much better quality are obtained using CPG with pore sizes of 1400A and larger. We also identified the key characteristics for CPG selection that lead to the best CPG performance.     

Immunohistochemical expression of superoxide dismutase (MnSOD) anti-oxidant enzyme in invasive breast carcinoma

The most important cellular protective mechanisms against oxidative stress are antioxidant enzymes. Their action is based on decomposal of reactive oxygen species (ROS) and their transformation to H2O2. Within the mitochondria manganese superoxide dismutase (MnSOD) affords the major defense against ROS. In this study we investigated tissue sections from 101 breast carcinomas for the immunohistochemical expression of MnSOD protein and these results were assessed in relation to various clinicopathological parameters, in order to clarify the prognostic value of this enzyme. The possible relationship to hormone receptor content, anti-apoptotic protein bcl-2, p53 and cell proliferation was also estimated. High expression levels were observed, as 79/101 (78,2%) cases expressed strong immunoreactivity. In this study MnSOD increased in a direct relationship with tumor grade and is therefore inversely correlated with differentiation (p=0.0004). Furthermore, there was a strong positive correlation between MnSOD expression and p53 protein immunoreactivity (p=0.0029). The prognostic impact of MnSOD expression in determining the risk of recurrence and overall survival with both univariate (long-rang test) and multivariate (Cox regression) methods of analysis was statistically not significant. These results indicate that neoplastic cells in breast carcinomas retain their capability to produce MnSOD and thus protected from the possible cellular damage provoked by reactive oxygen species. In addition, MnSOD content varies according to the degree of differentiation of breast carcinoma.