Differential responsiveness of late passage C-6 glial cells and advanced passages of astrocytes derived from aged mouse cerebral hemispheres to cytokines and growth factor: Glutamine synthetase activity

In this study, we were interested to compare the responsiveness to growth factors, NGF, b-FGF and EGF and cytokines, IL1β, and TNF-α, in late passages (74–79) C6 glial cells committed astrocytes and astrocytes of advanced passages (26–28) in cultures derived from aged mouse cerebral hemispheres (MACH). Cultures were grown in either DMEM or chemically defined medium (CDM/TIPS) in order to test the effects of growth factors or cytokines. The activity of glutamine synthetase (GS), a marker for astrocytes, was used as a test parameter. We found that treatment with growth factors increased GS activity in both glial cell culture systems with the exception of EGF in C-6 glial cells. Treatment with cytokines markedly decreased GS activity in the late passage C6 glial cells whereas only TNF-α had a similar effect on MACH astrocytes. In view of the generally opposite effects of growth factors and cytokines on GS activity, we-speculate that these molecules which are also endogenously present in glial cells may play a role in the maintenance of cellular homeostasis.     

Back to basics in COVID-19: Antigens and antibodies—Completing the puzzle

The outbreak of the coronavirus disease 2019 (COVID-19) has gathered 1 year of scientific/clinical information. This informational asset should be thoroughly and wisely used in the coming year colliding in a global task force to control this infection. Epidemiology of this infection shows that the available estimates of SARS-CoV-2 infection prevalence largely depended on the availability of molecular testing and the extent of tested population. Within molecular diagnosis, the viability and infectiousness of the virus in the tested samples should be further investigated. Moreover, SARS-CoV-2 has a genetic normal evolution that is a dynamic process. The immune system participates to the counterattack of the viral infection by pathogen elimination, cellular homoeostasis, tissue repair and generation of memory cells that would be reactivated upon a second encounter with the same virus. In all these stages, we still have knowledge to be gathered regarding antibody persistence, protective effects and immunological memory. Moreover, information regarding the intense pro-inflammatory action in severe cases still lacks and this is important in stratifying patients for difficult to treat cases. Without being exhaustive, the review will cover these important issues to be acknowledged to further advance in the battle against the current pandemia.     

Bin1 directly remodels actin dynamics through its BAR domain

Endocytic processes are facilitated by both curvature‐generating BAR‐domain proteins and the coordinated polymerization of actin filaments. Under physiological conditions, the N‐BAR protein Bin1 has been shown to sense and curve membranes in a variety of cellular processes. Recent studies have identified Bin1 as a risk factor for Alzheimer's disease, although its possible pathological function in neurodegeneration is currently unknown. Here, we report that Bin1 not only shapes membranes, but is also directly involved in actin binding through its BAR domain. We observed a moderate actin bundling activity by human Bin1 and describe its ability to stabilize actin filaments against depolymerization. Moreover, Bin1 is also involved in stabilizing tau‐induced actin bundles, which are neuropathological hallmarks of Alzheimer's disease. We also provide evidence for this effect in vivo, where we observed that downregulation of Bin1 in a Drosophila model of tauopathy significantly reduces the appearance of tau‐induced actin inclusions. Together, these findings reveal the ability of Bin1 to modify actin dynamics and provide a possible mechanistic connection between Bin1 and tau‐induced pathobiological changes of the actin cytoskeleton.     

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.     

A domain of the α-subunit of rabbit phosphorylase kinase shows homologies with regions of rabbit α-tropomyosin,human EGF receptor,and the α chain of bovine S-100 protein

Sequence comparison of the -subunit of phosphorylase kinase with -tropomyosin revealed 32% identity, and 49% similarity, between the region of -tropomyosin coded by exon 5 and a 39 amino acid segment of the kinase subunit. A subsequence of the -subunit segment and a sequence overlapping the same -subunit region are homologous with: (a) a region of the cytoplasmic domain of EGF receptor (50% identity) and (b) a Ca²⁺-binding domain of the chain of S-100 protei (50% identity) respectively. Statistical analysis shows that these homologies are significant. The biological implication of the above similarities is discussed.     

Differential maturation of μ and δ opioid receptors in the chick embryonic brain

The developmental profiles of the binding of and opiate receptors agonists was investigated using the chick embryo brain. Binding of opioids was performed at embryonic days 5, 6, 15, 18, and 20 in the developing chick embryo brain. [³H]dihyromorphine was used as a ligand and with 5×10^–7 M levorphanol for non-specific binding, and [³H](d-Ala²-d-Leu⁵)-enkephalin was used as a with 5×10^–7 M (d-Ser-Gly-Phe-Leu-Thr)-enkephalin for non-specific binding. Crude membranes were prepared from whole brain at days, 5, 6 and cerebral hemispheres at days 15, 18, and 20 of embryonic age. Both and opiate receptors were present during early embryogenesis and as early as day 5. Analysis of binding sites revealed high and low affinity sites during early embryogenesis but only one site. By 18 days of embryonic age, only one site remained. This developmental change is interpreted as a transitory state of the receptor to the adult pattern. The presence of only one site is constant throughout embryonic age; it is high during early embryogenesis reaching a lower level by 18 days. The presence of a dual binding site pattern for the receptor in early embryogenesis is implicated to have a functional significance in the pluripotential role of the endogenous opioids in early development.     

Primary Screening for Cervical Cancer Based on High-Risk Human Papillomavirus (HPV) Detection and HPV 16 and HPV 18 Genotyping,in Comparison to Cytology

Objectives

The objective of the present study is to assess the performance of a high-risk human papillomavirus (HR-HPV) DNA test with individual HPV-16/HPV-18 genotyping as a method for primary cervical cancer screening compared with liquid-based cytology (LBC) in a population of Greek women taking part in routine cervical cancer screening.

Methods

The study, conducted by the “HEllenic Real life Multicentric cErvical Screening” (HERMES) study group, involved the recruitment of 4,009 women, aged 25–55, who took part in routine cervical screening at nine Gynecology Departments in Greece. At first visit cervical specimens were collected for LBC and HPV testing using the Roche Cobas 4800 system. Women found positive for either cytology or HPV were referred for colposcopy, whereas women negative for both tests will be retested after three years. The study is ongoing and the results of the first screening round are reported herein.

Results

Valid results for cytology and HPV testing were obtained for 3,993 women. The overall prevalence of HR-HPV was 12.7%, of HPV-16 2.7% and of HPV-18 1.4%. Of those referred for colposcopy, cervical intraepithelial neoplasia grade 2 or worse (CIN2+) was detected in 41 women (1.07%). At the threshold of CIN2+, cytology [atypical squamous cells of undetermined significance (ASC-US) or worse] and HPV testing showed a sensitivity of 53.7% and 100% respectively, without change between age groups. Cytology and HPV testing showed specificity of 96.8% and 90.3% respectively, which was increased in older women (≥30) in comparison to younger ones (25–29). Genotyping for HPV16/18 had similar accuracy to cytology for the detection of CIN2+ (sensitivity: 58.5%; specificity 97.5%) as well as for triage to colposcopy (sensitivity: 58.5% vs 53.7% for cytology).

Conclusion

HPV testing has much better sensitivity than cytology to identify high-grade cervical lesions with slightly lower specificity. HPV testing with individual HPV-16/HPV-18 genotyping could represent a more accurate methodology for primary cervical cancer screening in comparison to liquid-based cytology, especially in older women.     

Solid-phase synthesis of amidine-substituted phenylbenzimidazoles and incorporation of this DNA binding and recognition motif into amino acid and peptide conjugates

Amidine-substituted phenylbenzimidazoles are well-established DNA-binding structural motifs that have contributed to the development of diverse classes of DNA-targeted agents; this ring system not only assists in increasing the overall DNA affinity of an agent, but can also influence its site selectivity. Seeking a means to conveniently exploit these attributes, a protocol for the on-resin synthesis of amino acid- and peptide-phenylbenzimidazole-amidine conjugates was developed to facilitate installation of phenylbenzimidazole-amidines into peptide chains during the course of standard solid-phase syntheses. Building from a resin-bound amino acid or peptide on Rink amide resin, 4-formyl benzoic acid was coupled to the resin-bound free amine followed by introduction of 3,4-diamino-N′-hydroxybenzimidamide (in the presence of 1,4-benzoquinone) to construct the benzimidazole heterocycle. Finally, the resin-bound N′-hydroxybenzimidamide functionality was reduced to an amidine via 1 M SnCl₂·2H₂O in DMF prior to resin cleavage to release final product. This procedure permits the straightforward synthesis of amino acids or peptides that are N-terminally capped by a phenylbenzimidazole-amidine ring system. Employing this protocol, a series of amino acid–phenylbenzimidazole-amidine (Xaa-R) conjugates was synthesized as well as dipeptide conjugates of the general form Xaa-Gly-R (where R is the phenylbenzimidazole-amidine and Xaa is any amino acid).     

Potent 2'-aminoanilide inhibitors of cFMS as potential anti-inflammatory agents

A series of 2'-aminoanilides have been identified which exhibit potent and selective inhibitory activity against the cFMS tyrosine kinase. Initial SAR studies within this series are described which examine aroyl and amino group substitutions, as well as the introduction of hydrophilic substituents on the benzene core. Compound 47 inhibits the isolated enzyme (IC(50)=0.027 microM) and blocks CSF-1-induced proliferation of bone marrow-derived macrophages (IC(50)=0.11 microM) and as such, serves as a lead candidate for further optimization studies.