The Viral Chemokine MCK-2 of Murine Cytomegalovirus Promotes Infection as Part of a gH/gL/MCK-2 Complex

Human cytomegalovirus (HCMV) forms two gH/gL glycoprotein complexes, gH/gL/gO and gH/gL/pUL(128,130,131A), which determine the tropism, the entry pathways and the mode of spread of the virus. For murine cytomegalovirus (MCMV), which serves as a model for HCMV, a gH/gL/gO complex functionally homologous to the HCMV gH/gL/gO complex has been described. Knock-out of MCMV gO does impair, but not abolish, virus spread indicating that also MCMV might form an alternative gH/gL complex. Here, we show that the MCMV CC chemokine MCK-2 forms a complex with the glycoprotein gH, a complex which is incorporated into the virion. We could additionally show that mutants lacking both, gO and MCK-2 are not able to produce infectious virus. Trans-complementation of these double mutants with either gO or MCK-2 showed that both proteins can promote infection of host cells, although through different entry pathways. MCK-2 has been extensively studied in vivo by others. It has been shown to be involved in attracting cells for virus dissemination and in regulating antiviral host responses. We now show that MCK-2, by forming a complex with gH, strongly promotes infection of macrophages in vitro and in vivo. Thus, MCK-2 may play a dual role in MCMV infection, as a chemokine regulating the host response and attracting specific target cells and as part of a glycoprotein complex promoting entry into cells crucial for virus dissemination.     

Essential Elements as a Distinguishing Factor between Mycorrhizal Potentials of Two Cohabiting Truffle Species in Riparian Forest Habitat in Serbia

True truffles (Tuber sp.) that establish ectomycorrhizal symbiosis (ECM) with trees in the Mediterranean and temporal regions have species specific abilities to assimilate soil born elements. Suitable habitats are usually inhabited by few truffle species, while distinguishing their symbiotic potentials appeared very difficult. Two species that commonly inhabit riparian forests in Serbia are the most prized one, Tuber magnatum Pico (Piedmont white truffle) and not so highly valued Tuber brumale Vitt . In order to assess potential differences between their assimilation and accumulation abilities, the differences between contents of elements that may be the subjects of the symbiotic trade between the host plant and fungi were evaluated in accumulation target (ascocarps) and their source (the soil). Essential (K, Na, Ca, Mg, Fe, P, S, and Zn) and essential trace elements (Co, Cr, Cu, Mn, and Se) in truffles and soil samples were determined by means of inductively coupled plasma with optical emission spectrometry (ICP‐OES). Their concentrations (mg/kg) in ascocarps were in the range from 1.364±0.591 (Cr) to 10760.862±16.058 (K), while in soil ranged from 23.035±0.010 (Cr) to 20809.300±122.934 (Fe). Element accumulation potential (bioaccumulation factor) was calculated in the system truffle/soil. The statistical approaches were used for establishing the differences, while the possible differentiation between symbiotic potentials of two mycelia in the defined soil conditions was discussed.     

Craniofacial anthropometric pattern profile in hypohidrotic ectodermal dysplasia--application in detection of gene carriers

Hypohidrotic ectodermal dysplasia (HED) is characterized by clinical manifestations of severe hypodontia or anodontia, hypotrichosis, hypohidrosis, and specific facial appearance. Affected males show complete expression of clinical features of this condition. Their mothers, who are gene carriers, express only some signs, which are usually very mild. Currently available clinical methods are not sufficient for routine identification of the HED heterozygous gene carriers. The purpose of this study was to identify and describe the facial characteristics of HED patients and their mothers and to evaluate the usefulness of craniofacial pattern profile analysis (CFPP) in the diagnosis of this syndrome and the detection of gene carriers. In this study six affected males and their mothers were evaluated. Z-scores for each variable were calculated and compared with age- and sex-matched controls. Anthropometric analysis showed a specific dysmorphic pattern in CST patients that includes decreased skull base width (t-t: -1.67 Z); decreased forehead width (ft-ft: -1.8 Z), decreased midface depth (sn-t: -2.02 Z), markedly decreased total facial height (n-gn: -3.4 Z), and markedly decreased maxillary arc (t-sn-t: -2.5 Z). Gene carriers showed a similar tendency in their pattern profiles. They showed the same tendency towards lower Z-values for forehead width, facial height, and mouth width. The values for these measurements were between those of the affected and healthy controls. The most pronounced findings were increased head width (eu-eu: +2.83 Z), increased lower face width (go-go: +2.06 Z), and reduction of total facial height (n-gn: -0.95 Z). They also displayed increased nose width (al-al: +2.41 Z) and increased biocular distance (ex-ex: +2.01 Z). When used in conjunction with other methods the anthropometrics pattern profile analysis can considerably enhance detection of gene carriers for HED and increase objective assessment of the craniofacial region in HED patients.     

Synthesis of macrolones with central piperazine ring in the linker and its influence on antibacterial activity

Three macrolides, clarithromycin, azithromycin and 11-O-Me-azithromycin have been selected for the construction of a series of new macrolone derivatives. Quinolone-linker intermediates are prepared by Sonogashira-type C(6)-alkynylation of 6-iodoquinolone precursors. The final macrolones, differing by macrolide moiety and substituents at the position N-1 of the quinolone or by the presence of an ethyl ester or free acid on the quinolone unit attached via a linker. The linker comprises of a central piperazine ring bonded to the 4″-O position of cladinose by 3-carbon ester or ether functionality. Modifications of the linker did not improve antibacterial properties compared to the previously reported macrolone compounds. Linker flexibility seems to play an important role for potency against macrolide resistant respiratory pathogens.     

EGFR protein overexpression correlates with chromosome 7 polysomy and poor prognostic parameters in clear cell renal cell carcinoma

Background

The role of epidermal growth factor (EGF) and its receptor (EGFR) in the pathogenesis and progression of various malignant tumors has long been known, but there is still disagreement concerning prognostic significance of EGFR expression in clear cell renal cell carcinoma (CCRCC). The present study was designed to analyze more objectively the protein EGFR expression in CCRCC and to compare its value with EGFR gene copy number changes and clinicopathologic characteristics including patient survival.

Methods

The protein EGFR expression was analyzed immunohistochemically on 94 CCRCC, and gene copy number alterations of EGFR by FISH analysis on 41 CCRCC selected according to distinct membrane EGFR staining.

Results

Membrane EGFR expression in tumor cells was heterogeneous with respect to the proportion of positive cells and staining intensity. FISH analysis did not reveal EGFR gene amplification, while polysomy of chromosome 7 found in 41% was associated with higher EGFR membrane expression. Moreover, EGFR overexpression was associated with a higher nuclear grade, larger tumor size and shorter patient''s survival, while there was no connection with pathological stage.

Conclusion

In conclusion, the protein expression of EGFR had an impact on prognosis in patients with CCRCC, while an increased copy number of chromosome 7 could be the possible reason for EGFR protein overexpression in the absence of gene amplification.     

SARS-CoV-2 receptor binding domain fusion protein efficiently neutralizes virus infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Currently, as dangerous mutations emerge, there is an increased demand for specific treatments for SARS-CoV-2 infected patients. The spike glycoprotein on the virus envelope binds to the angiotensin converting enzyme 2 (ACE2) on host cells through its receptor binding domain (RBD) to mediate virus entry. Thus, blocking this interaction may inhibit viral entry and consequently stop infection. Here, we generated fusion proteins composed of the extracellular portions of ACE2 and RBD fused to the Fc portion of human IgG1 (ACE2-Ig and RBD-Ig, respectively). We demonstrate that ACE2-Ig is enzymatically active and that it can be recognized by the SARS-CoV-2 RBD, independently of its enzymatic activity. We further show that RBD-Ig efficiently inhibits in-vivo SARS-CoV-2 infection better than ACE2-Ig. Mechanistically, we show that anti-spike antibody generation, ACE2 enzymatic activity, and ACE2 surface expression were not affected by RBD-Ig. Finally, we show that RBD-Ig is more efficient than ACE2-Ig at neutralizing high virus titers. We thus propose that RBD-Ig physically blocks virus infection by binding to ACE2 and that RBD-Ig should be used for the treatment of SARS-CoV-2-infected patients.     

Diastolic Field Stimulation: the Role of Shock Duration in Epicardial Activation and Propagation

Detailed knowledge of tissue response to both systolic and diastolic shock is critical for understanding defibrillation. Diastolic field stimulation has been much less studied than systolic stimulation, particularly regarding transient virtual anodes. Here we investigated high-voltage-induced polarization and activation patterns in response to strong diastolic shocks of various durations and of both polarities, and tested the hypothesis that the activation versus shock duration curve contains a local minimum for moderate shock durations, and it grows for short and long durations. We found that 0.1–0.2-ms shocks produced slow and heterogeneous activation. During 0.8–1 ms shocks, the activation was very fast and homogeneous. Further shock extension to 8 ms delayed activation from 1.55 ± 0.27 ms and 1.63 ± 0.21 ms at 0.8 ms shock to 2.32 ± 0.41 ms and 2.37 ± 0.3 ms (N = 7) for normal and opposite polarities, respectively. The traces from hyperpolarized regions during 3–8 ms shocks exhibited four different phases: beginning negative polarization, fast depolarization, slow depolarization, and after-shock increase in upstroke velocity. Thus, the shocks of >3 ms in duration created strong hyperpolarization associated with significant delay (P < 0.05) in activation compared with moderate shocks of 0.8 and 1 ms. This effect appears as a dip in the activation-versus-shock-duration curve.     

Neuroprotective Potential and Chemical Profile of Alternatively Cultivated Ganoderma lucidum Basidiocarps

Various neurodegenerative diseases are the main challenges to the modern medicine and there is a great need for novel, natural, neuroprotective agents. Ganoderma lucidum is a well‐known medicinal mushroom, which health benefits have been confirmed by numerous studies. As demand for its basidiocarps is increased and traditional cultivation on hardwoods is not environmentally friendly and economically justified, finding of alternative substrates is necessary. The aim of the study was to assess the effect of alternative cultivation substrates on the chemical profile of G. lucidum basidiocarps and their capacity to inhibit acetylcholinesterase and tyrosinase, which higher activity is directly associated with neurodegenerative processes. Extracts of basidiocarps cultivated on alternative substrates, especially on clear wheat straw, showed significantly higher inhibition capacities than extracts of commercially‐grown ones. These extracts were considerably different chemically from commercial basidiocarps extracts and even nine new compounds were isolated from them. Our results suggest that cultivation substrate greatly affect the chemical profile and neuroprotective capacity of obtained basidiocarps and wheat straw is a promising cultivation substrate.     

Dental and minor physical anomalies in children with developmental disorders--a discriminant analysis

A discriminant analysis was performed in a sample of 303 children with developmental disorders (DD) and 303 healthy controls (C) in order to test whether some oro-dental and physical minor anomalies could discriminate these groups of children. DD sample comprised 176 mentally retarded (MR) children. 70 children with impaired hearing (IH) and 57 children with impaired vision (IV). The control group included 303 healthy subjects, matched for sex and age. The analysis comprised seven common oral and dental anomalies: median diastema, hypodontia, impacted teeth, microdontia, dens invaginatus, upper lip frenulum and frenulum of the tongue. Minor physical anomalies were assessed by the method proposed by Waldrop et al., as the average number of minor anomalies per individual (W1) and as the weighted score of minor anomalies (W2). Three discriminant functions were obtained by analysis of nine initial variables. Distinct discrimination and considerable distances were found between the centroids of the controls and all groups of DD children. The first two discriminant functions were significant for discrimination between the groups and they explained 98.6% of the total variance. The first function contained 90.2% of information and was defined by the number and weighted scores of minor anomalies. The second variable explained 8.4% of the total variability and was defined by three dental anomalies. The results obtained by the discriminant analysis show that application of dental and minor physical anomalies enables discrimination between the group of healthy children and the groups of children with different developmental disorders.     

Stress analysis of a centrally fractured rib fixated by an intramedullary screw

The stress on an intramedullary screw rib fixation device holding together a centrally fractured human rib under in vivo force loadings was studied using finite element analysis (FEA). Validation of the FEA modelling using pullout from porcine ribs proved FEA to be suitable for assessing the structural integrity of screw/bone systems such as rib fixated by a screw. In the human rib fixation investigation, it was found that intramedullary bioresorbable Bioretec screws can fixate centrally fractured human ribs under normal breathing conditions. However, under coughing conditions, simulation showed Bioretec fixating screws to bend substantially. High stresses in the screw are mainly the result of flexion induced by the force loading, and are restricted to thin regions on the outside of the screw shaft. Stiffer screws result in less locally intense stress concentrations in bone, indicating that bone failure in the bone/screw contact regions can be averted with improvements in screw stiffness.