The Na(+) /Ca(2+) exchanger provides a major Ca(2+) extrusion pathway in excitable cells and plays a key role in the control of intracellular Ca(2+) concentrations. In Canis familiaris, Na(+) /Ca(2+) exchanger (NCX) activity is regulated by the binding of Ca(2+) to two cytosolic Ca(2+) -binding domains, CBD1 and CBD2, such that Ca(2+) -binding activates the exchanger. Despite its physiological importance, little is known about the exchanger's global structure, and the mechanism of allosteric Ca(2+) -regulation remains unclear. It was found previously that for NCX in the absence of Ca(2+) the two domains CBD1 and CBD2 of the cytosolic loop are flexibly linked, while after Ca(2+) -binding they adopt a rigid arrangement that is slightly tilted. A realistic model for the mechanism of the exchanger's allosteric regulation should not only address this property, but also it should explain the distinctive behavior of Drosophila melanogaster's sodium/calcium exchanger, CALX, for which Ca(2+) -binding to CBD1 inhibits Ca(2+) exchange. Here, NMR spin relaxation and residual dipolar couplings were used to show that Ca(2+) modulates CBD1 and CBD2 interdomain flexibility of CALX in an analogous way as for NCX. A mechanistic model for the allosteric Ca(2+) regulation of the Na(+) /Ca(2+) exchanger is proposed. In this model, the intracellular loop acts as an entropic spring whose strength is modulated by Ca(2+) -binding to CBD1 controlling ion transport across the plasma membrane.

The Na + /Ca 2+ exchanger of Drosophila melanogaster, CALX, is the main Ca 2+ -extrusion mechanism in olfactory sensory neurons and photoreceptor cells. Na + /Ca 2+ exchangers have two Ca 2+ sensor domains, CBD1 and CBD2. In contrast to the mammalian homologues, CALX is inhibited by Ca 2+ -binding to CALX-CBD1, while CALX-CBD2 does not bind Ca 2+ at physiological concentrations. CALX-CBD1 consists of a β-sandwich and displays four Ca 2+ binding sites at the tip of the domain. In this study, we used NMR spectroscopy and isothermal titration calorimetry (ITC) to investigate the cooperativity of Ca 2+ -binding to CALX-CBD1. We observed that this domain binds Ca 2+ in the slow exchange regime at the NMR chemical shift time scale. Ca 2+ -binding restricts the dynamics in the Ca 2+ -binding region. Experiments of 15 N CEST and 15 N R 2 dispersion allowed the determination of Ca 2+ dissociation rates (≈ 20 s −1 ). NMR titration curves of residues in the Ca 2+ -binding region were sigmoidal due to the contribution of chemical exchange to transverse magnetization relaxation rates, R 2 . Hence, a novel approach to analyze NMR titration curves was proposed. Ca 2+ -binding cooperativity was examined assuming two different stoichiometric binding models and using a Bayesian approach for data analysis. Fittings of NMR and ITC binding curves to the Hill model yielded n Hill = 2.9 − 3.1, near maximum cooperativity (n Hill = 4). By assuming a stepwise model to interpret the ITC data, we found that the probability of binding from 2 up to 4 Ca 2+ is at least three orders of magnitude higher than that of binding a single Ca 2+ . Hence, four Ca 2+ ions bind almost simultaneously to CALX-CBD1. Cooperative Ca 2+ -binding is key to enable this exchanger to efficiently respond to changes in the intracellular Ca 2+ -concentration in sensory neuronal cells. SIGNIFICANCE CALX-CBD1 is the Ca 2+ -sensor domain of the Na + /Ca 2+ exchanger of Drosophila melanogaster. It consists of a β-sandwich, and contains four Ca 2+ binding sites at the distal loops. In this study, we examined the cooperative binding of four Ca 2+ ions to CALX-CBD1 using NMR spectroscopy and isothermal titration calorimetry (ITC) experiments. NMR and ITC data were analyzed using the framework of the binding polynomial formalism and Bayesian statistics. A novel approach to analyze NMR titration data in the slow exchange regime was proposed. These results support the view that CALX-CBD1 binds four Ca 2+ with high cooperativity. The significant ligand binding cooperativity exhibited by this domain is determinant for the efficient allosteric regulation of this exchanger by intracellular Ca 2+ .

The outbreak of COVID-19 pandemics highlighted the need of sensitive, selective, and easy-to-handle biosensing devices. In the contemporary scenario, point-of-care devices technologies for mass testing and infection mapping within a population has proven itself as of primordial importance. Here, we introduce a graphene-based Electrical-Electrochemical Vertical Device (EEVD) point-of-care biosensor, strategically engineered for serologic COVID-19 diagnosis. EEVD uses serologic IgG quantifications on SARS-CoV-2 Receptor Binding Domain (RBD) bioconjugate immobilized onto device surface. EEVD combines graphene basal plane with high charge carrier mobility, high conductivity, low intrinsic resistance, and interfacial sensitivity to capacitance alterations. EEVD application was carried out in real human serum samples. Since EEVD is a miniaturized device, it requires just 40 μL of sample for a point-of-care COVID-19 infections detection. When compared to serologic assays such ELISA and others immunochromatographic methods, EEVD presents some advantages such as time of analyses (15 min), sample preparation, and a LOD of 1.0 pg mL -1 . We glimpse that EEVD meets the principles of robustness and accuracy, desirable analytic parameters for assays destined to pandemics control strategies.

Amphiphilic copolymers have a wide variety of medical and biotechnological applications, including DNA transfection in eukaryotic cells. Still, no polymer-primed transfection of prokaryotic cells has been described. The reversible additionfragmentation chain transfer (RAFT) polymer synthesis technique and the reversible deactivation radical polymerization variants allow the design of polymers with well-controlled molar mass, morphology, and hydrophilicity/hydrophobicity ratios. RAFT was used to synthesize two amphiphilic copolymers containing different ratios of the amphiphilic poly [2-(dimethyl-amino) ethyl methacrylate] and the hydrophobic poly [methyl methacrylate]. These copolymers bound to pUC-19 DNA and successfully transfected non-competent Escherichia coli DH5a, with transformation efficiency in the range of 10 3 colony-forming units per mg of plasmid DNA. These results demonstrate prokaryote transformation using polymers with controlled amphiphilic/hydrophobic ratios.

A VDAC é a proteína mais abundante na membrana mitocondrial externa. Exerce o controle da atividade desta organela através da regulação da troca de metabólitos e tem função crucial no mecanismo de apoptose. Em nosso caso, os estudos dos complexos protéicos, das interações entre a VDAC e outras proteínas presentes no interior do neurônio que auxiliam na manutenção das funções das organelas e da célula, fazem parte da chamada interactômica. O presente estudo determinou o interactoma do complexo protéico Hexoquinase-VDAC-ANT presente em cérebros murino, bovino e aviar. Nosso objetivo foi identificar se as expressões diferenciadas da VDAC1 e VDAC2 verificadas nos cérebros murino, aviar e bovino, estão associadas a diferenças nos interactomas dessas proteínas. Este estudo revelou que as espécies aviar e bovina apresentaram o maior número de complexos protéicos contendo VDACs (5) quando comparadas com os neurônios de rato (1), o que é indicativo de uma cinética diferencial de montagem ou desmontagem do complexo. Além disso, a VDAC mitocondrial neuronal aviar também interage com mais proteínas em relação à VDAC mitocondrial neuronal bovina, o que é resultado de uma composição de subunidades diferenciada. Tais resultados indicam diferenças significativas quanto ao metabolismo energético e apoptótico no cérebro aviar, bovino e murino, existindo interações diferenciais da VDAC no cérebro aviar.

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The SARS‐CoV‐2 non‐structural protein 14 (nsp14), known as exoribonuclease is encoded from the large polyprotein of viral genome and is a major constituent of the transcription replication complex (TRC) machinery of the viral RNA synthesis. This protein is highly conserved among the coronaviruses and is a potential target for the development of a therapeutic drug. Here, we report the SARS‐CoV‐2 nsp14 expression, show its structural characterization, and ss‐RNA exonuclease activity through vibrational and electronic spectroscopies. The deconvolution of amide‐I band in the FTIR spectrum of the protein revealed a composition of 35 % α‐helix and 25 % β‐sheets. The binding between protein and RNA is evidenced from the spectral changes in the amide‐I region of the nsp14, showing protein conformational changes during the binding process. A value of 20.60±3.81 mol L−1 of the binding constant (KD) is obtained for nsp14/RNA complex. The findings reported here can motivate further studies to develop structural models for better understanding the mechanism of exonuclease enzymes for correcting the viral genome and can help in the development of drugs against SARS‐CoV‐2.

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