Keratinocyte cultures from involved skin in vitiligo patients show an impaired in vitro behaviour

Vitiligo depigmentation is considered a consequence of either melanocyte disappearance or loss of functioning melanocytes in the involved areas. However, it has been reported that keratinocytes in involved vitiligo skin are damaged too. Based on this evidence, we evaluated the in vitro behaviour, in life span cultures, of involved and uninvolved vitiligo keratinocytes and their expression of proliferation, differentiation and senescence markers. An additional purpose was to investigate whether vitiligo keratinocytes from depigmented skin are able to sustain survival and growth of normal melanocytes (when added in co-culture experiments), as normal human keratinocytes manage to do. Our results demonstrate that almost all involved vitiligo keratinocytes have a shorter life span in vitro than the uninvolved cells and all of them do not maintain melanocytes in culture in a physiological ratio. Modification of proliferation and senescence marker expression also occurs. Indeed, we detected low initial expression levels of the senescence marker p16 in involved vitiligo keratinocytes, despite their shorter in vitro life span, and increased expression of proliferating cell nuclear antigen and p53. This preliminary analysis of a small number of in vitro cultured vitiligo keratinocytes suggests an impaired senescence process in lesional vitiligo keratinocytes and attempts to regulate it.     

Polyphosphate and stress response in mycobacteria

Polyphosphate (poly P) is present in every living cell. Long considered a 'molecular fossil', its role in cell physiology has been neglected. However, in the last few years it has become clear that poly P plays a role in multiple physiological functions, the best characterized of which is rpoS and recA induction during the Escherichia coli stringent response. Sureka et al. in this issue of Molecular Microbiology investigate the role of poly P in mycobacterial stress response and describe its participation in a novel regulatory pathway involving the two-component system MprAB, the alternative sigma factor sigma(E) and Rel, the enzyme responsible for (p)ppGpp metabolism in mycobacteria.     

Novel carbazole derivatives as NPY Y1 antagonists

The synthesis of a series of carbazole derivatives and their SAR at the NPY Y1 receptor is described. Modulation of physicochemical properties by appropriate decoration led to the identification of a high-affinity NPY Y1 antagonist that shows high brain penetration and modest oral bioavailability.     

1,5-Biaryl pyrrole derivatives as EP1 receptor antagonists. Structure-activity relationships of 6-substituted and 5,6-disubstituted benzoic acid derivatives

Herein we describe the SAR of 1,5-biaryl pyrrole derivatives, with substituents in the 6-position of the benzoic acid moiety, as EP(1) receptor antagonists. Substitution at this position was well tolerated and led to the identification of several analogues with high affinity for the EP(1) receptor that displayed good efficacy in the established FCA model of inflammatory pain. Furthermore, several analogues were prepared which combined substitution at the 5- and 6-positions as well as derivatives with an aromatic ring fused to the 5- and 6-positions.     

Configurational entropy elucidates the role of salt-bridge networks in protein thermostability

Detailed knowledge of how networks of surface salt bridges contribute to protein thermal stability is essential not only to understand protein structure and function but also to design thermostable proteins for industrial applications. Experimental studies investigating thermodynamic stability through measurements of free energy associated with mutational alterations in proteins provide only macroscopic evidence regarding the structure of salt-bridge networks and assessment of their contribution to protein stability. Using explicit-solvent molecular dynamics simulations to provide insight on the atomic scale, we investigate here the structural stability, defined in terms of root-mean-square fluctuations, of a short polypeptide designed to fold into a stable trimeric coiled coil with a well-packed hydrophobic core and an optimal number of intra- and interhelical surface salt bridges. We find that the increase of configurational entropy of the backbone and side-chain atoms and decreased pair correlations of these with increased temperature are consistent with nearly constant atom-positional root-mean-square fluctuations, increased salt-bridge occupancies, and stronger electrostatic interactions in the coiled coil. Thus, our study of the coiled coil suggests a mechanism in which well-designed salt-bridge networks could accommodate stochastically the disorder of increased thermal motion to produce thermostability.     

Ultrasound Imaging Versus Morphopathology in Cardiovascular Diseases: The Heart Failure

Background

Exercise echocardiography (EE) is an established method to diagnose coronary artery disease (CAD). Chronotropic incompetence (CI) during the EE may be a marker of myocardial ischemia. The purpose of this investigation was to evaluate the additive value of CI during EE in CAD diagnosis.

Methods

Between 2000 and 2006, 4042 patients (1900 men with a mean age of 56 ± 11 years) were evaluated by EE. Based on the heart rate (HR) reached during the exercise test, the subjects were divided into two groups: G1 group – 490 patients who failed to achieve 85% of the maximal age-predicted HR, and G2 group – 3552 patients who were able to achieve 85% of the maximal age-predicted HR. Clinical characteristics, left ventricular wall motion abnormalities – wall motion score index (WMSI) – and coronary angiography (CA) were the parameters compared between the two groups.

Results

The left ventricular wall motion abnormalities were more frequent in G1 group than in G2 group (54% versus 26%; P < 0.00001). WMSI was higher in G1 group than in G2 group, both at rest (1.06 ± 0.17 versus 1.02 ± 0.09; P < 0.0001) and after exercise (1.12 ± 0.23 versus 1.04 ± 0.21; P < 0.0001). In G1 group, 82% of the patients with positive EE for myocardial ischemia presented obstructive coronary, compared to 71% (P = 0.03) in G2 group.

Conclusion

CI is associated with a higher frequency of myocardial ischemia during EE, reinforcing the concept that CI is a marker of the severity of myocardial ischemia.     

Dynamics, hydration, and motional averaging of a loop-gated artificial protein cavity: the W191G mutant of cytochrome c peroxidase in water as revealed by molecular dynamics simulations

Five molecular dynamics simulations of the W191G cavity mutant of cytochrome c peroxidase in explicit water reveal distinct dynamic and hydration behavior depending on the closed or open state of the flexible loop gating the cavity, the binding of (K+ or small molecule) cations, and the system temperature. The conformational spaces sampled by the loop region and by the cavity significantly reduce upon binding. The largest ordering factor on water dynamics is the presence of the K+ ion occupying the gated cavity. Considerable water exchange occurs for the open-gate cavity when no ligand or cation is bound. In all cases, good correspondence is found between the calculated (ensemble-averaged) location of water molecules and the water sites determined by X-ray crystallography experiments. However, our simulations suggest that these sites do not necessarily correspond to the presence of bound water molecules. In fact, individual water molecules may repeatedly exchange within the cavity volume yet occupy on average these water sites. Four major conclusions emerge. First, it seems misleading to interpret the conformation of protein loop regions in terms of single dominant structures. Second, our simulations support the general picture of Pro 190 cis-trans isomerization as a determinant of the loop-opening mechanism. Third, receptor flexibility is fundamental for ligand binding and molecular recognition, and our results suggest its importance for the docking of small compounds to the artificial cavity. Fourth, after validation against the available experimental data, molecular dynamics simulations can be used to characterize the dynamics and exchange of water molecules and ions, providing atomic level and time-dependent information otherwise inaccessible to experiments.     

C9orf72 ALS/FTD dipeptide repeat protein levels are reduced by small molecules that inhibit PKA or enhance protein degradation

Intronic GGGGCC (G4C2) hexanucleotide repeat expansion within the human C9orf72 gene represents the most common cause of familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9ALS/FTD). Repeat‐associated non‐AUG (RAN) translation of repeat‐containing C9orf72 RNA results in the production of neurotoxic dipeptide‐repeat proteins (DPRs). Here, we developed a high‐throughput drug screen for the identification of positive and negative modulators of DPR levels. We found that HSP90 inhibitor geldanamycin and aldosterone antagonist spironolactone reduced DPR levels by promoting protein degradation via the proteasome and autophagy pathways respectively. Surprisingly, cAMP‐elevating compounds boosting protein kinase A (PKA) activity increased DPR levels. Inhibition of PKA activity, by both pharmacological and genetic approaches, reduced DPR levels in cells and rescued pathological phenotypes in a Drosophila model of C9ALS/FTD. Moreover, knockdown of PKA‐catalytic subunits correlated with reduced translation efficiency of DPRs, while the PKA inhibitor H89 reduced endogenous DPR levels in C9ALS/FTD patient‐derived iPSC motor neurons. Together, our results suggest new and druggable pathways modulating DPR levels in C9ALS/FTD.     

Critical literature review and pooled analysis of diagnostic accuracy of Ortho VITROS SARS-CoV-2 antigen test for diagnosing acute SARS-CoV-2 infections

BackgroundThe present study is aimed at reviewing and meta-analyzing the currently published data on the diagnostic accuracy of Ortho VITROS SARS-CoV-2 antigen test for diagnosing acute SARS-CoV-2 infections.MethodsAn electronic search was conducted in Scopus and Medline with the keywords "VITROS" AND "antigen" AND "COVID-19" OR "SARS-CoV-2" AND "immunoassay" within the search fields "TITLE" AND "ABSTRACT" AND "KEYWORDS", without no date (i.e., up to January 23, 2022) or language restrictions, aimed at detecting documents reporting the diagnostic accuracy of this SARSCoV-2 immunoassay compared with reference molecular diagnostic methods.ResultsOverall, 5 studies (n=2734 samples) were finally included in our pooled analysis, four of which also provided diagnostic sensitivity in oro-and nasopharyngeal samples with high viral load. The pooled cumulative diagnostic sensitivity and specificity were 0.82 (95%CI, 0.78-0.86) and 1.00 (95%CI, 1.00-1.00), respectively, whilst the area under the curve was 0.995 (95%CI, 0.993-0.997), the cumulative agreement 97.2% (95%CI, 96.5-97.8%), with 0.89 (95%CI, 0.86-0.91) kappa statistics, thus reflecting an almost perfect concordance with reference molecular biology techniques. The pooled diagnostic sensitivity in samples with high viral load was as high as 0.98 (95%CI, 0.96-0.99).ConclusionsThese results confirm that the automated and high-throughput Ortho VITROS SARS-CoV-2 antigen test may represent a valuable surrogate of molecular testing for diagnosing acute SARS-CoV-2 infections, especially in subjects with high viral load.