Ozone treatment effectively eliminates SARS-CoV-2 from infected face masks.

The current COVID-19 pandemic is causing profound health, economic, and social problems worldwide. The global shortage of medical and personal protective equipment (PPE) in specialized centers during the outbreak demonstrated the need for efficient methods to disinfect and recycle them in times of emergency. We have previously described that high ozone concentrations destroyed viral RNA in an inactivated SARS-CoV-2 strain within a few minutes. However, the efficient ozone dosages for active SARS-CoV-2 are still unknown. The present study aimed to evaluate the systematic effects of ozone exposure on face masks from hospitalized patients infected with SARS-CoV-2. Face masks from COVID-19 patients were collected and treated with a clinical ozone generator at high ozone concentrations in small volumes for short periods. The study focused on SARS-CoV-2 gene detection (assessed by real-time quantitative polymerase chain reaction (RT-qPCR)) and on the virus inactivation by in vitro studies. We assessed the effects of different high ozone concentrations and exposure times on decontamination efficiency. We showed that high ozone concentrations (10,000, 2,000, and 4,000 ppm) and short exposure times (10, 10, and 2 minutes, respectively), inactivated both the original strain and the B.1.1.7 strain of SARS-CoV-2 from 24 contaminated face masks from COVID-19 patients. The validation results showed that the best condition for SARS-CoV-2 inactivation was a treatment of 4,000 ppm of ozone for 2 minutes. Further studies are in progress to advance the potential applications of these findings.

A transistor-based label-free immunosensor for rapid detection of tau protein.

Tau protein in cerebrospinal fluid (CSF) is a central and relevant biomarker of Alzheimer's disease (AD) that correlates with the severity of dementia. Unfortunately, so far, direct label-free detection of tau remains a challenge. Here, we present a transistor-based biosensor that detects the net charge of tau protein directly under physiological conditions. To achieve this, readily available whole anti-tau IgG antibodies are co-immobilized on the sensor surface with polyethylene glycol (PEG) molecules of different molecular weight. We show that by increasing the PEG size from 10 kDa to 20 kDa, the electrical response upon binding of tau improves significantly. These results support recent theoretical work that predicted larger PEGs to form a thicker surface layer with a higher detectable analyte charge. With 20 kDa PEG, we demonstrate label-free tau detection in a wide concentration range with detection limits <1 pM in 150 mM buffer and cell culture media, as well as < 10 pM in artificial CSF. This purely electrical method allows fast and simple tau detection within 30 min without sample processing, washing steps, or labeled detection antibodies. By exchanging the capture antibody, the platform is also amenable to different biomarkers and may enable future diagnostic tools for AD and other diseases.

Analytical Model To Describe the Effect of Polyethylene Glycol on Ionic Screening of Analyte Charges in Transistor-Based Immunosensing.

Recently, the co-immobilization of polyethylene glycol has improved sensor responses of transistor-based immunosensing by approximately three times. However, there is currently no analytical model available to explain this empirical effect. The key parameters thought to affect the potential are the receptor density, the capacitance, the analyte charge, and the dissociation constant. Based on our experimental data, only the analyte charge can account for the signal enhancement. To capture the effect of PEG on the analyte charge, we introduce a prefactor, the detectable charge qdet, which represents the portion of analyte charges effectively detected by the sensor. This parameter can quantitatively describe the PEG-induced signal enhancement and can be used to recommend the choice of PEG size for immuno-field-effect transistors. Additionally, we include the competition between electrolyte ions and the analyte for binding to the recognition molecule to more accurately describe the concentration-dependent sensor responses than the traditional Langmuir binding model does.

Catalytic Activity and Proton Translocation of Reconstituted Respiratory Complex I Monitored by Surface-Enhanced Infrared Absorption Spectroscopy.

Respiratory complex I (CpI) is a key player in the way organisms obtain energy, being an energy transducer, which couples nicotinamide adenine dinucleotide (NADH)/quinone oxidoreduction with proton translocation by a mechanism that remains elusive so far. In this work, we monitored the function of CpI in a biomimetic, supported lipid membrane system assembled on a 4-aminothiophenol (4-ATP) self-assembled monolayer by surface-enhanced infrared absorption spectroscopy. 4-ATP serves not only as a linker molecule to a nanostructured gold surface but also as pH sensor, as indicated by concomitant density functional theory calculations. In this way, we were able to monitor NADH/quinone oxidoreduction-induced transmembrane proton translocation via the protonation state of 4-ATP, depending on the net orientation of CpI molecules induced by two complementary approaches. An associated change of the amide I/amide II band intensity ratio indicates conformational modifications upon catalysis which may involve movements of transmembrane helices or other secondary structural elements, as suggested in the literature [ Di Luca , Proc. Natl. Acad. Sci. U.S.A. , 2017 , 114 , E6314 - E6321 ].

Regulation of Hormonal Control, Cell Reprogramming, and Patterning during De Novo Root Organogenesis.

Body regeneration through formation of new organs is a major question in developmental biology. We investigated de novo root formation using whole leaves of Arabidopsis (Arabidopsis thaliana). Our results show that local cytokinin biosynthesis and auxin biosynthesis in the leaf blade followed by auxin long-distance transport to the petiole leads to proliferation of J0121-marked xylem-associated tissues and others through signaling of INDOLE-3-ACETIC ACID INDUCIBLE28 (IAA28), CRANE (IAA18), WOODEN LEG, and ARABIDOPSIS RESPONSE REGULATORS1 (ARR1), ARR10, and ARR12. Vasculature proliferation also involves the cell cycle regulator KIP-RELATED PROTEIN2 and ABERRANT LATERAL ROOT FORMATION4, resulting in a mass of cells with rooting competence that resembles callus formation. Endogenous callus formation precedes specification of postembryonic root founder cells, from which roots are initiated through the activity of SHORT-ROOT, PLETHORA1 (PLT1), and PLT2. Primordia initiation is blocked in shr plt1 plt2 mutant. Stem cell regulators SCHIZORIZA, JACKDAW, BLUEJAY, and SCARECROW also participate in root initiation and are required to pattern the new organ, as mutants show disorganized and reduced number of layers and tissue initials resulting in reduced rooting. Our work provides an organ regeneration model through de novo root formation, stating key stages and the primary pathways involved.

Direct, Label-Free, and Rapid Transistor-Based Immunodetection in Whole Serum.

Transistor-based biosensors fulfill many requirements posed upon transducers for future point-of-care diagnostic devices such as scalable fabrication and label-free and real-time quantification of chemical and biological species with high sensitivity. However, the short Debye screening length in physiological samples (<1 nm) has been a major drawback so far, preventing direct measurements in serum. In this work, we demonstrate how tailoring the sensing surface with short specific biological receptors and a polymer polyethylene glycol (PEG) can strongly enhance the sensor response. In addition, the sensor performance can be dramatically improved if the measurements are performed at elevated temperatures (37 °C instead of 21 °C). With this novel approach, highly sensitive and selective detection of a representative immunosensing parameter-human thyroid-stimulating hormone-is shown over a wide measuring range with subpicomolar detection limits in whole serum. To the best of our knowledge, this is the first demonstration of direct immunodetection in whole serum using transistor-based biosensors, without the need for sample pretreatment, labeling, or washing steps. The presented sensor is low-cost, can be easily integrated into portable diagnostics devices, and offers a competitive performance compared to state-of-the-art central laboratory analyzers.

Venous thromboembolism in radiation therapy cancer patients: Findings from the RIETE registry.

BACKGROUND: Cancer patients are at high risk of venous thromboembolism, particularly during cancer treatment. Conversely to chemotherapy, data on the epidemiology and clinical features of venous thromboembolism during radiation therapy are scarce. There is lack of evidence on the influence of radiation therapy (RT) on outcome in cancer patients with acute venous thromboembolism (VTE). METHODS: We used the RIETE (Registro Informatizado de Enfermedad ThromboEmbolica) database to assess the clinical characteristics and outcome of prospectively-collected consecutive patients with cancer-associated thrombosis occurred during the course of radiation therapy for cancer. Death, venous thromboembolism recurrences and major bleeding rates during long-term follow-up according to cancer site and treatment were compared RESULTS: 9284 Patients with active cancer and VTE were enrolled in RIETE: 4605 with pulmonary embolism (PE) and 4679 with deep vein thrombosis (DVT). In all, 1202 (13%) were receiving RT. This last sub-population had a higher rate of PE recurrences and a similar rate of DVT recurrences or major bleeding than those not receiving RT. Patients on RT had a higher rate of cerebral bleeding. CONCLUSIONS: In this cohort of cancer patients with VTE, a significant proportion of them received RT before VTE, the latter experienced a higher risk of cerebral bleeding.

The direct role of selenocysteine in [NiFeSe] hydrogenase maturation and catalysis.

Hydrogenases are highly active enzymes for hydrogen production and oxidation. [NiFeSe] hydrogenases, in which selenocysteine is a ligand to the active site Ni, have high catalytic activity and a bias for H2 production. In contrast to [NiFe] hydrogenases, they display reduced H2 inhibition and are rapidly reactivated after contact with oxygen. Here we report an expression system for production of recombinant [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough and study of a selenocysteine-to-cysteine variant (Sec489Cys) in which, for the first time, a [NiFeSe] hydrogenase was converted to a [NiFe] type. This modification led to severely reduced Ni incorporation, revealing the direct involvement of this residue in the maturation process. The Ni-depleted protein could be partly reconstituted to generate an enzyme showing much lower activity and inactive states characteristic of [NiFe] hydrogenases. The Ni-Sec489Cys variant shows that selenium has a crucial role in protection against oxidative damage and the high catalytic activities of the [NiFeSe] hydrogenases.

H2 -Fueled ATP Synthesis on an Electrode: Mimicking Cellular Respiration.

ATP, the molecule used by living organisms to supply energy to many different metabolic processes, is synthesized mostly by the ATPase synthase using a proton or sodium gradient generated across a lipid membrane. We present evidence that a modified electrode surface integrating a NiFeSe hydrogenase and a F1 F0 -ATPase in a lipid membrane can couple the electrochemical oxidation of H2 to the synthesis of ATP. This electrode-assisted conversion of H2 gas into ATP could serve to generate this biochemical fuel locally when required in biomedical devices or enzymatic synthesis of valuable products.

A threonine stabilizes the NiC and NiR catalytic intermediates of [NiFe]-hydrogenase.

The heterodimeric [NiFe] hydrogenase from Desulfovibrio fructosovorans catalyzes the reversible oxidation of H2 into protons and electrons. The catalytic intermediates have been attributed to forms of the active site (NiSI, NiR, and NiC) detected using spectroscopic methods under potentiometric but non-catalytic conditions. Here, we produced variants by replacing the conserved Thr-18 residue in the small subunit with Ser, Val, Gln, Gly, or Asp, and we analyzed the effects of these mutations on the kinetic (H2 oxidation, H2 production, and H/D exchange), spectroscopic (IR, EPR), and structural properties of the enzyme. The mutations disrupt the H-bond network in the crystals and have a strong effect on H2 oxidation and H2 production turnover rates. However, the absence of correlation between activity and rate of H/D exchange in the series of variants suggests that the alcoholic group of Thr-18 is not necessarily a proton relay. Instead, the correlation between H2 oxidation and production activity and the detection of the NiC species in reduced samples confirms that NiC is a catalytic intermediate and suggests that Thr-18 is important to stabilize the local protein structure of the active site ensuring fast NiSI-NiC-NiR interconversions during H2 oxidation/production.

Induction of a proton gradient across a gold-supported biomimetic membrane by electroenzymatic H2 oxidation.

Energy-transduction mechanisms in living organisms, such as photosynthesis and respiration, store light and chemical energy in the form of an electrochemical gradient created across a lipid bilayer. Herein we show that the proton concentration at an electrode/phospholipid-bilayer interface can be controlled and monitored electrochemically by immobilizing a membrane-bound hydrogenase. Thus, the energy derived from the electroenzymatic oxidation of H2 can be used to generate a proton gradient across the supported biomimetic membrane.

Crystallographic studies of [NiFe]-hydrogenase mutants: towards consensus structures for the elusive unready oxidized states.

Catalytically inactive oxidized O2-sensitive [NiFe]-hydrogenases are characterized by a mixture of the paramagnetic Ni-A and Ni-B states. Upon O2 exposure, enzymes in a partially reduced state preferentially form the unready Ni-A state. Because partial O2 reduction should generate a peroxide intermediate, this species was previously assigned to the elongated Ni-Fe bridging electron density observed for preparations of [NiFe]-hydrogenases known to contain the Ni-A state. However, this proposition has been challenged based on the stability of this state to UV light exposure and the possibility of generating it anaerobically under either chemical or electrochemical oxidizing conditions. Consequently, we have considered alternative structures for the Ni-A species including oxidation of thiolate ligands to either sulfenate or sulfenic acid. Here, we report both new and revised [NiFe]-hydrogenases structures and conclude, taking into account corresponding characterizations by Fourier transform infrared spectroscopy (FTIR), that the Ni-A species contains oxidized cysteine and bridging hydroxide ligands instead of the peroxide ligand we proposed earlier. Our analysis was rendered difficult by the typical formation of mixtures of unready oxidized states that, furthermore, can be reduced by X-ray induced photoelectrons. The present study could be carried out thanks to the use of Desulfovibrio fructosovorans [NiFe]-hydrogenase mutants with special properties. In addition to the Ni-A state, crystallographic results are also reported for two diamagnetic unready states, allowing the proposal of a revised oxidized inactive Ni-SU model and a new structure characterized by a persulfide ion that is assigned to an Ni-'Sox' species.

Reconstitution of respiratory complex I on a biomimetic membrane supported on gold electrodes.

For the first time, respiratory complex I has been reconstituted on an electrode preserving its structure and activity. Respiratory complex I is a membrane-bound enzyme that has an essential function in cellular energy production. It couples NADH:quinone oxidoreduction to translocation of ions across the cellular (in prokaryotes) or mitochondrial membranes. Therefore, complex I contributes to the establishment and maintenance of the transmembrane difference of electrochemical potential required for adenosine triphosphate synthesis, transport, and motility. Our new strategy has been applied for reconstituting the bacterial complex I from Rhodothermus marinus onto a biomimetic membrane supported on gold electrodes modified with a thiol self-assembled monolayer (SAM). Atomic force microscopy and faradaic impedance measurements give evidence of the biomimetic construction, whereas electrochemical measurements show its functionality. Both electron transfer and proton translocation by respiratory complex I were monitored, simulating in vivo conditions.

FTIR spectroscopy of metalloproteins.

Absorption of infrared radiation by proteins gives important information about their structure and function. The most intense infrared bands correspond to the overlap of all the peptide bond absorption. Additionally, in many metalloproteins their prosthetic groups have intrinsic ligands or bind substrates/inhibitors that absorb intensively in the infrared. Here, we describe thoroughly several Fourier transform infrared methods for studying structure-function relationships in metalloproteins, using hydrogenases as an example.

Safety and efficacy of splenectomy in over 65-yrs-old patients with immune thrombocytopenia.

AIM: Few studies specifically focus on elderly splenectomized immune thrombocytopenia (ITP) patients. Older patients with ITP and excellent health are often excluded from surgery splenectomy. We aimed to compare the safety and efficacy of splenectomy in elderly and non-elderly ITP patients and to examine the effect of age on therapeutic response. MATERIAL AND METHODS: We carried out a retrospective analysis of a series of 218 patients who had undergone splenectomy for ITP. We compared the data from the elderly group (≥65 yrs, 57 patients) with the young group (<65 yrs, 162 patients). RESULTS: Surgical technique (laparoscopy or open laparotomy splenectomy) was comparable between the two age groups. The adjusted risk of major bleeding following splenectomy for elderly patients was three times that for young patients (OR 3.05, 95% CI: 1.44-6.52). The median duration of postoperative hospital stay was longer for elderly than for young patients (8 d vs. 4 d, P < 0.001). However, we identified a subgroup of elderly ITP patients, those aged between 65 and 70 yrs who had undergone laparoscopic splenectomy, with a low risk of postoperative complications. Of the 218 patients, 89% achieved a favorable response to splenectomy. A favorable response was significantly less common in elderly than in young people (79% vs. 92%, P = 0.005). However, we observed an acceptable long-term control of ITP in the elderly group, in which the probability of maintaining response for 14 yrs after splenectomy was 56%. CONCLUSIONS: Patients aged ≥65 yrs experienced negative effects on safety and efficacy outcomes of splenectomy for ITP, but further studies are needed to identify predictors of postsplenectomy outcomes in this group.

Influence of the protein structure surrounding the active site on the catalytic activity of [NiFeSe] hydrogenases.

A combined experimental and theoretical study of the catalytic activity of a [NiFeSe] hydrogenase has been performed by H/D exchange mass spectrometry and molecular dynamics simulations. Hydrogenases are enzymes that catalyze the heterolytic cleavage or production of H2. The [NiFeSe] hydrogenases belong to a subgroup of the [NiFe] enzymes in which a selenocysteine is a ligand of the nickel atom in the active site instead of cysteine. The aim of this research is to determine how much the specific catalytic properties of this hydrogenase are influenced by the replacement of a sulfur by selenium in the coordination of the bimetallic active site versus the changes in the protein structure surrounding the active site. The pH dependence of the D2/H(+) exchange activity and the high isotope effect observed in the Michaelis constant for the dihydrogen substrate and in the single exchange/double exchange ratio suggest that a "cage effect" due to the protein structure surrounding the active site is modulating the enzymatic catalysis. This "cage effect" is supported by molecular dynamics simulations of the diffusion of H2 and D2 from the outside to the inside of the protein, which show different accumulation of these substrates in a cavity next to the active site.

O2-independent formation of the inactive states of NiFe hydrogenase.

We studied the mechanism of aerobic inactivation of Desulfovibrio fructosovorans nickel-iron (NiFe) hydrogenase by quantitatively examining the results of electrochemistry, EPR and FTIR experiments. They suggest that, contrary to the commonly accepted mechanism, the attacking O(2) is not incorporated as an active site ligand but, rather, acts as an electron acceptor. Our findings offer new ways toward the understanding of O(2) inactivation and O(2) tolerance in NiFe hydrogenases.

Relation between anaerobic inactivation and oxygen tolerance in a large series of NiFe hydrogenase mutants.

Nickel-containing hydrogenases, the biological catalysts of oxidation and production, reversibly inactivate under anaerobic, oxidizing conditions. We aim at understanding the mechanism of (in)activation and what determines its kinetics, because there is a correlation between fast reductive reactivation and oxygen tolerance, a property of some hydrogenases that is very desirable from the point of view of biotechnology. Direct electrochemistry is potentially very useful for learning about the redox-dependent conversions between active and inactive forms of hydrogenase, but the voltammetric signals are complex and often misread. Here we describe simple analytical models that we used to characterize and compare 16 mutants, obtained by substituting the position-74 valine of the -sensitive NiFe hydrogenase from Desulfovibrio fructosovorans. We observed that this substitution can accelerate reactivation up to 1,000-fold, depending on the polarity of the position 74 amino acid side chain. In terms of kinetics of anaerobic (in)activation and oxygen tolerance, the valine-to-histidine mutation has the most spectacular effect: The V74H mutant compares favorably with the -tolerant hydrogenase from Aquifex aeolicus, which we use here as a benchmark.

Understanding and tuning the catalytic bias of hydrogenase.

When enzymes are optimized for biotechnological purposes, the goal often is to increase stability or catalytic efficiency. However, many enzymes reversibly convert their substrate and product, and if one is interested in catalysis in only one direction, it may be necessary to prevent the reverse reaction. In other cases, reversibility may be advantageous because only an enzyme that can operate in both directions can turnover at a high rate even under conditions of low thermodynamic driving force. Therefore, understanding the basic mechanisms of reversibility in complex enzymes should help the rational engineering of these proteins. Here, we focus on NiFe hydrogenase, an enzyme that catalyzes H(2) oxidation and production, and we elucidate the mechanism that governs the catalytic bias (the ratio of maximal rates in the two directions). Unexpectedly, we found that this bias is not mainly determined by redox properties of the active site, but rather by steps which occur on sites of the proteins that are remote from the active site. We evidence a novel strategy for tuning the catalytic bias of an oxidoreductase, which consists in modulating the rate of a step that is limiting only in one direction of the reaction, without modifying the properties of the active site.