High-throughput virtual search of small molecules for controlling the mechanical stability of human CD4.

Protein mechanical stability determines the function of a myriad of proteins, especially proteins from the extracellular matrix. Failure to maintain protein mechanical stability may result in diseases and disorders such as cancer, cardiomyopathies, or muscular dystrophy. Thus, developing mutation-free approaches to enhance and control the mechanical stability of proteins using pharmacology-based methods may have important implications in drug development and discovery. Here, we present the first approach that employs computational high-throughput virtual screening and molecular docking to search for small molecules in chemical libraries that function as mechano-regulators of the stability of human cluster of differentiation 4, receptor of HIV-1. Using single-molecule force spectroscopy, we prove that these small molecules can increase the mechanical stability of CD4D1D2 domains over 4-fold in addition to modifying the mechanical unfolding pathways. Our experiments demonstrate that chemical libraries are a source of mechanoactive molecules and that drug discovery approaches provide the foundation of a new type of molecular function, that is, mechano-regulation, paving the way toward mechanopharmacology.

CdS quantum dots generated in-situ for fluorometric determination of thrombin activity.

A method is presented for sensitive determination of thrombin activity. It is based on (a) the interaction between fibrinogen after activation with thrombin, and (b) an enzymatic amplification step consisting of in-situ growth of CdS quantum dots (QDs). Fibrinogen is immobilized on the surface of the wells of a microplate and then incubated with a mixture of biotinylated fibrinogen and thrombin. Thrombin activates immobilized fibrinogen and free biotinylated fibrinogen. This leads to the formation of insoluble biotinylated fibrin that remains bound on the surface of the wells. Afterwards, the samples are incubated with avidin-labeled alkaline phosphatase (ALP) which binds to biotinylated fibrin. ALP hydrolyzes the substrate p-nitrophenyl phosphate (pNPP) under formation of phosphate ions which stabilize CdS QDs that are grown in-situ from cadmium(II) and sulfide. The generation of fibrin is correlated with the activity of thrombin. Increased thrombin concentration results in increased fluorescence that can be measured at excitation/emission wavelengths of 300/510 nm. The introduction of such an amplification step (the enzyme-triggered growth of QDs) allows for the quantification of thrombin in the picomolar concentration range, with a linear response up to 2.5 pM and a detection limit of 0.05 pM. The method was applied to the determination of thrombin activity in human plasma and of the thrombin inhibitor argatroban. Graphical abstract Schematic representation of a fluorometric method for determination of thrombin activity in the picomolar concentration range based on the interaction between fibrinogen after activation with thrombin, and an enzymatic amplification step consisting of in-situ growth of CdS quantum dots (CdS QD).

Peroxidase-mimicking DNAzyme modulated growth of CdS nanocrystalline structures in situ through redox reaction: application to development of genosensors and aptasensors.

This work demonstrates the use of the peroxidase-mimicking DNAzyme (peroxidase-DNAzyme) as general and inexpensive platform for development of fluorogenic assays that do not require organic fluorophores. The system is based on the affinity interaction between the peroxidase-DNAzyme bearing hairpin sequence and the analyte (DNA or low molecular weight molecule), which changes the folding of the hairpin structure and consequently the activity of peroxidase-DNAzyme. Hence, in the presence of the analyte the peroxidase-DNAzyme structure is disrupted and does not catalyze the aerobic oxidation of l-cysteine to cystine. Thus, l-cysteine is not removed from the system and the fluorescence of the assay increases due to the in situ formation of fluorescent CdS nanocrystals. The capability of the system as a platform for fluorogenic assays was demonstrated through designing model geno- and aptasensor for the detection of a tumor marker DNA and a low molecular weight analyte, adenosine 5'triphosphate (ATP), respectively.

Thiocholine mediated stabilization of in situ produced CdS quantum dots: application for the detection of acetylcholinesterase activity and inhibitors.

The use of acetylcholinesterase (AChE) inhibitors as chemical warfare agents or pesticides represents a strong hazard against human health. The high toxicity of these compounds arises from their ability to inhibit acetylcholinesterase from degrading acetylcholine (ACh), which could affect the physiology of the nervous system with serious or fatal consequences. Here we report a simple and fluorimetric system for a highly sensitive detection of AChE activity and inhibitors. The principle of this approach is based on the hydrolysis of acetylthiocholine (ATCh) by AChE, which yields the thiol-bearing compound thiocholine (TCh) that at trace concentrations stabilized the in situ generated CdS quantum dots (QDs). The system shows a linear relationship between the fluorescence intensity and AChE activity from 1 to 10 mU mL(-1) in buffer solution. The accuracy of the proposed system was further demonstrated through the determination of AChE activity in human serum (HS) by the standard addition method. Furthermore, this novel and highly sensitive sensing system allows the detection of 80 pM of the AChE inhibitor paraoxon and 100 nM of galanthamine. The reported methodology shows potential applications for the development of a simple and inexpensive assay for the routine quantification of AChE activity and inhibitors.

The strongest protein binder is surprisingly labile.

Bacterial adhesins are cell-surface proteins that anchor to the cell wall of the host. The first stage of infection involves the specific attachment to fibrinogen (Fg), a protein found in human blood. This attachment allows bacteria to colonize tissues causing diseases such as endocarditis. The study of this family of proteins is hence essential to develop new strategies to fight bacterial infections. In the case of the Gram-positive bacterium Staphylococcus aureus, there exists a class of adhesins known as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Here, we focus on one of them, the clumping factor A (ClfA), which has been found to bind Fg through the dock-lock-latch mechanism. Interestingly, it has recently been discovered that MSCRAMM proteins employ a catch-bond to withstand forces exceeding 2 nN, making this type of interaction as mechanically strong as a covalent bond. However, it is not known whether this strength is an evolved feature characteristic of the bacterial protein or is typical only of the interaction with its partner. Here, we combine single-molecule force spectroscopy, biophysical binding assays, and molecular simulations to study the intrinsic mechanical strength of ClfA. We find that despite the extremely high forces required to break its interactions with Fg, ClfA is not by itself particularly strong. Integrating the results from both theory and experiments we dissect contributions to the mechanical stability of this protein.

Primary and secondary postpartum haemorrhage: a review for a rationale endovascular approach.

Postpartum haemorrhage (PPH) is a significant cause of maternal mortality globally, necessitating prompt and efficient management. This review provides a comprehensive exploration of endovascular treatment dimensions for both primary and secondary PPH, with a focus on uterine atony, trauma, placenta accreta spectrum (PAS), and retained products of conception (RPOC). Primary PPH, occurring within 24 h, often results from uterine atony in 70% of causes, but also from trauma, or PAS. Uterine atony involves inadequate myometrial contraction, addressed through uterine massage, oxytocin, and, if needed, mechanical modalities like balloon tamponade. Trauma-related PPH may stem from perineal injuries or pseudoaneurysm rupture, while PAS involves abnormal placental adherence. PAS demands early detection due to associated life-threatening bleeding during delivery. Secondary PPH, occurring within 24 h to 6 weeks postpartum, frequently arises from RPOC. Medical management may include uterine contraction drugs and hemostatic agents, but invasive procedures like dilation and curettage (D&C) or hysteroscopic resection may be required.Imaging assessments, particularly through ultrasound (US), play a crucial role in the diagnosis and treatment planning of postpartum haemorrhage (PPH), except for uterine atony, where imaging techniques prove to be of limited utility in its management. Computed tomography play an important role in evaluation of trauma related PPH cases and MRI is essential in diagnosing and treatment planning of PAS and RPOC.Uterine artery embolization (UAE) has become a standard intervention for refractory PPH, offering a rapid, effective, and safe alternative to surgery with a success rate exceeding 85% (Rand T. et al. CVIR Endovasc 3:1-12, 2020). The technical approach involves non-selective uterine artery embolization with resorbable gelatine sponge (GS) in semi-liquid or torpedo presentation as the most extended embolic or calibrated microspheres. Selective embolization is warranted in cases with identifiable bleeding points or RPOC with AVM-like angiographic patterns and liquid embolics could be a good option in this scenario. UAE in PAS requires a tailored approach, considering the degree of placental invasion. A thorough understanding of female pelvis vascular anatomy and collateral pathways is essential for accurate and safe UAE.In conclusion, integrating interventional radiology techniques into clinical guidelines for primary and secondary PPH management and co-working during labour is crucial.

Enzymatic product-mediated stabilization of CdS quantum dots produced in situ: application for detection of reduced glutathione, NADPH, and glutathione reductase activity.

Glutathione is the most abundant nonprotein molecule in the cell and plays an important role in many biological processes, including the maintenance of intracellular redox states, detoxification, and metabolism. Furthermore, glutathione levels have been linked to several human diseases, such as AIDS, Alzheimer disease, alcoholic liver disease, cardiovascular disease, diabetes mellitus, and cancer. A novel concept in bioanalysis is introduced and applied to the highly sensitive and inexpensive detection of reduced glutathione (GSH), over its oxidized form (GSSG), and glutathione reductase (GR) in human serum. This new fluorogenic bioanalytical system is based on the GSH-mediated stabilization of growing CdS nanoparticles. The sensitivity of this new assay is 5 pM of GR, which is 3 orders of magnitude better than other fluorogenic methods previously reported.

Assays for methionine γ-lyase and S-adenosyl-L-homocysteine hydrolase based on enzymatic formation of CdS quantum dots in situ.

S-Adenosyl-L-homocysteine hydrolase (AHCY) hydrolyzes its substrate S-adenosyl-L-homocysteine (AdoHcy) to L-homocysteine (Hcy). Methionine γ-lyase (MGL) catalyzes the decomposition of Hcy to hydrogen sulfide which forms fluorescent CdS nanoparticles in the presence of Cd(NO(3))(2). On the basis of these enzymatic reactions, two new simple and robust fluorogenic enzymatic assays for MGL and AHCY were developed and applied to detection of AHCY inhibitors.

Quantification of prothrombin in human plasma amplified by autocatalytic reaction.

By site directed mutagenesis, we have produced recombinant mutants of human and mouse prethrombin-2 which are able to convert themselves autocatalytically into α-thrombin. We also have created a new method to amplify the signal of bioanalytical assays based on the autocatalytic activation of these mutated proenzymes. The activation of the mutants by active α-thrombin triggers an autocatalytic reaction which leads to more active thrombin resulting in the amplification of the readout signal. Addition of mutated mouse prethrombin-2 into the conventional assay for prothrombin level in human plasma, employing ecarin and the fluorogenic substrate, resulted in improvement of the detection limit by 2 orders of magnitude.

Enzymatic growth of quantum dots: applications to probe glucose oxidase and horseradish peroxidase and sense glucose.

Three innovative assays are developed for the detection of enzymatic activities of glucose oxidase (GOx) and horseradish peroxidase (HRP) by the generation of CdS quantum dots (QDs) in situ using non-conventional enzymatic reactions. In the first assay, GOx catalyzes the oxidation of 1-thio-ß-D-glucose to give 1-thio-ß-D-gluconic acid. The latter is spontaneously hydrolyzed to ß-D-gluconic acid and H2 S, which in the presence of cadmium nitrate yields fluorescent CdS nanoparticles. In the second assay HRP catalyzes the oxidation of sodium thiosulfate with hydrogen peroxide generating H2 S and consequently CdS QDs. The combination of GOx with HRP, allowed quantification of glucose in plasma by following growth of fluorescent QDs.

Functional Gastrointestinal Disorders in Neonates and Toddlers According to the Rome IV Criteria: A Systematic Review and Meta-Analysis.

Functional gastrointestinal disorders (FGIDs) are classified as a combination of persistent gastrointestinal symptoms. The Rome IV criteria can elucidate several factors in the pathogenesis of FGIDs. The frequency of FGIDs can differ between clinical and nonclinical settings and between geographic regions. To determine the global prevalence of FGIDs in neonates and toddlers according to the Rome IV criteria. We included cohort and descriptive observational studies reporting the prevalence of FGIDs according to the Rome IV criteria in children aged 0-48 months. We searched the Medline, Embase, Lilacs, and CENTRAL databases from May 2016 to the present day. Furthermore, unpublished literature was searched to supplement this information. The Strengthening the Reporting of Observational Studies in Epidemiology statement was used to evaluate the risk of bias. A meta-analysis of the proportions was performed using MetaProp in R. The results are reported in forest plots. We identified and analyzed 15 studies comprising 48,325 participants. Six studies were conducted in Europe, three in Latin America, two in North America, and four in Asia. Most participants were 12-48 months old (61.0%) and were recruited from the community. The global prevalence of FGIDs was 22.0% (95% confidence interval, 15-31%). The most common disorder was functional constipation (9.0%), followed by infant regurgitation syndrome (8.0%). Its prevalence was higher in the Americas (28.0%). FGIDs, as defined by the Rome IV criteria, are present in 22% of children, and the most common primary disorder is functional constipation. A higher prevalence of FGIDs has been reported in America.

A SYSTEMATIC REVIEW AND META-ANALYSIS IN SCHOOLCHILDREN AND ADOLESCENTS WITH FUNCTIONAL GASTROINTESTINAL DISORDERS ACCORDING TO ROME IV CRITERIA.

OBJECTIVE: To determine the prevalence of functional gastrointestinal disorders (FGIDs) in children according to Rome IV criteria. METHODS: We included cohorts and observational descriptive studies, including information for the prevalence of FGIDs according to Rome IV criteria in children 4 to 18 years old. We searched the MEDLINE (Ovid), EMBASE, LILACS, and CENTRAL databases from May 2016 to nowadays. Gray literature and other databases were also consulted. The risk of bias was assessed using the STROBE Statement. The results were reported in forest plots of the estimated effects of the included studies with a 95% confidence interval (95%CI). RESULTS: We included 14 studies involving a total of 17427 participants. Three studies were conducted in Europe, two in North America, and nine in Latin America. Most studies were school-based (n=14670, 84.18%), participants were mostly female (55.49%), white (51.73%), 8 to 18 years old (77.64%), and assisted to a public school (81.53%). Thirteen studies used the Questionnaire on Pediatric Gastrointestinal Symptoms (QPGS-RIV) to assess FGIDs. We found a global prevalence for FGIDs of 23% (95%CI 21-25%, I2 99%). Main disorders were functional constipation (FC) with 12% (95%CI 11-15%) followed by functional dyspepsia (FD) (5%, 95%CI 11-15%) and irritable bowel syndrome (IBS) (3%, 95%CI 2-4%). The prevalence of FGIDs was higher in the Americas, representing 23.67% (95%CI 21.2-26.2%, I2 91.3%). CONCLUSION: FGIDs are present in one of four children and adolescents, representing a common condition in this age group the central disorders were FC, FD, and IBS.

Development of portable CdS QDs screen-printed carbon electrode platform for electrochemiluminescence measurements and bioanalytical applications.

In this work, a portable and disposable screen-printed electrode-based platform for CdS QDs electrochemiluminescence (ECL) detection is presented. CdS QDs were synthesized in aqueous media and placed on top of carbon electrodes by drop casting. The CdS QDs spherical assemblies consisted of nanoparticles about 4 nm diameters and served as ECL sensitizers to enzymatic assays. The nanoparticles were characterized by optical techniques, TEM and XPS. Besides, the electrode modification process was optimized and further studied by SEM and confocal microscopy. The ECL emission from CdS QDs was triggered with H2O2 as cofactor and enzymatic assays were employed to modulate the CdS QDs ECL signal by blocking the surface or generating H2O2 in situ. Thiol-bearing compounds such as thiocholine generated through the hydrolysis of acetylthiocholine by acetylcholinesterase (AChE) interacted with the surface of CdS QDs thus blocking the ECL. The biosensor showed a linear range up to 5 mU mL-1 and a detection limit of 0.73 mU mL-1 for AChE. Moreover, the inhibition mechanism of the enzyme was studied by using 1,5-bis-(4-allyldimethylammonium-phenyl)pentan-3-one dibromide with a detection limit of 79.22 nM. Furthermore, the natural production of H2O2 from the oxidation of methanol by the action of alcohol oxidase was utilized to carry out the ECL process. This enzymatic assay presented a linear range up to 0.5 mg L-1 and a detection limit of 61.46 µg L-1 for methanol. The reported methodology shows potential applications for the development of sensitive and easy to hand biosensors and was applied to the determination of AChE and methanol in real samples.

Enhancement of cisplatin sensitivity in human breast cancer MCF-7 cell line through BiP and 14-3-3ζ co-knockdown.

Cisplatin treatment confers the relative resistance to MCF-7 cells as compared to other breast cancer cell lines. One principal reason is that chemotherapeutic agents induce autophagy in these cells to inhibit apoptosis. Binding immunoglobulin protein (BiP), a master regulator of unfolded protein response (UPR) and 14-3-3ζ are two critical proteins upregulated in breast cancer rendering resistance to anticancer drugs. They also play pivotal roles in autophagy with crosstalk with the apoptotic pathways of UPR through certain regulators. Thus, BiP and 14-3-3ζ were selected as the candidate targets to enhance cell death and apoptosis. First, cisplatin resistance was induced and determined by MTT assay and qPCR in MCF-7 cells. Then, the apoptosis axis of UPR was activated by knocking down either BiP or 14-3-3ζ and overactivated by co-knockdown of BiP and 14-3-3ζ. Apoptosis assays were performed using flow cytometry, TUNEL assays utilized confocal microscopy followed by western blot analysis and caspase-3 and JNK activities were investigated to assess the outcomes. Finally, an autophagy assay followed by western blotting was performed to study the effects of co-knockdown genes on cell autophagy in the presence and absence of cisplatin. The present data indicated the enhancement of cisplatin sensitivity in MCF-7 cells co-knocked down in BiP and 14-3-3ζ compared with either gene knockdown. Upregulation of JNK and cleaved-PARP1 protein levels as well as caspase-3 and JNK overactivation confirmed the results. A marked attenuation of autophagy and Beclin1 as well as ATG5 downregulation were detected in co-knockdown cells compared to knockdown with either BiP or 14-3-3ζ. Cisplatin sensitization of MCF-7 cells through double-knockdown of BiP and 14-3-3ζ highlights the potential of targeting UPR and autophagy factors to increase the effect of chemotherapy.

Analytical applications of enzymatic growth of quantum dots.

We have developed an analytical assay to detect the enzymatic activity of acetylcholine esterase and alkaline phosphatase based on the generation of quantum dots by enzymatic products. Acetylcholine esterase converts acetylthiocholine into thiocholine. The latter enhances the rate of decomposition of sodium thiosulfate into H(2)S, which in the presence of cadmium sulfate yields CdS quantum dots showing a time dependent exponential growth, typical of autocatalytic processes. This assay was also applied to detect acetylcholine esterase inhibitors. Alkaline phosphatase hydrolyzes thiophosphate and yields H(2)S, which instantly reacts with Cd(2+) to give CdS quantum dots. The formation of CdS quantum dots in both reactions was followed by fluorescence spectroscopy and showed dependence on the concentration of enzyme and substrate.

Ultrasensitive optical detection of hydrogen peroxide by triggered activation of horseradish peroxidase.

Hydrogen peroxide is a very reactive byproduct of many metabolic pathways. We describe an ultra-sensitive colorimetric method to detect hydrogen peroxide based on the reconstitution of apo-horseradish peroxidase with the hemin derivative, hemin di(N,N'-acetyl-hydrazide). Oxidation of the latter by hydrogen peroxide yields hemin, which is able to reconstitute apo-horseradish peroxidase, forming an active peroxidase. We have also applied this analyte-triggered reconstitution of horseradish peroxidase to detect the activity of the enzyme glucose oxidase.

Cloning, purification and characterization of two components of phenol hydroxylase from Rhodococcus erythropolis UPV-1.

Phenol hydroxylase that catalyzes the conversion of phenol to catechol in Rhodococcus erythropolis UPV-1 was identified as a two-component flavin-dependent monooxygenase. The two proteins are encoded by the genes pheA1 and pheA2, located very closely in the genome. The sequenced pheA1 gene was composed of 1,629 bp encoding a protein of 542 amino acids, whereas the pheA2 gene consisted of 570 bp encoding a protein of 189 amino acids. The deduced amino acid sequences of both genes showed high homology with several two-component aromatic hydroxylases. The genes were cloned separately in cells of Escherichia coli M15 as hexahistidine-tagged proteins, and the recombinant proteins His(6)PheA1 and His(6)PheA2 were purified and its catalytic activity characterized. His(6)PheA1 exists as a homotetramer of four identical subunits of 62 kDa that has no phenol hydroxylase activity on its own. His(6)PheA2 is a homodimeric flavin reductase, consisting of two identical subunits of 22 kDa, that uses NAD(P)H in order to reduce flavin adenine dinucleotide (FAD), according to a random sequential kinetic mechanism. The reductase activity was strongly inhibited by thiol-blocking reagents. The hydroxylation of phenol in vitro requires the presence of both His(6)PheA1 and His(6)PheA2 components, in addition to NADH and FAD, but the physical interaction between the proteins is not necessary for the reaction.

Antibody-Directed Synthesis of Catalytic Nanoclusters for Bioanalytical Assays.

Synthesis of atomic nanoclusters (NCs) using proteins as a scaffold has attracted great attention. Usually, the synthetic conditions for the synthesis of NCs stabilized with proteins require extreme pH values or temperature. These harsh reaction conditions cause the denaturation of the proteins and end up in the loss of their biological functions. Until now, there are no examples of the use of antibodies as NC stabilizers. In this work, we present the first method for the synthesis of catalytic NCs that uses antibodies for the stabilization of NCs. Anti-BSA IgG was used as a model to demonstrate that it is possible to use an antibody as a scaffold for the synthesis of semiconductor and metallic NCs with catalytic properties. The synthesis of antibodies modified with NCs is carried out under nondenaturing conditions, which do not affect the antibody structure. The resulting antibodies still maintain the affinity for target antigens and protein G. The catalytic properties of the anti-BSA IgG modified with NCs can be used to the quantification of bovine serum albumin (BSA) in a direct sandwich enzyme-linked immunosorbent assay (ELISA).

Modulated growth of nanoparticles. Application for sensing nerve gases.

Hydrolysis of acetylthiocholine mediated by acetylcholine esterase yields the thiol-bearing compound thiocholine. At trace concentrations, thiocholine modulates the growth of Au-Ag nanoparticles on seeding gold nanoparticles in the presence of ascorbic acid. Inhibition of the enzyme by 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284c51) or by diethyl p-nitrophenyl phosphate (paraoxon) produces lower yields of thiocholine, promoting the catalytic growth of Au-Ag nanoparticles. Here, we describe the development of a simple and sensitive colorimetric assay for the detection of AChE inhibitors.

Use of an osmium complex as a universal luminescent probe for enzymatic reactions.

The water-soluble bis(bipyridine)chloro(4-picolinic acid) osmium complex, [Os(III)(bpy)2Cl(PyCOOH)]2+ (bpy=2,2'-bipyridine, Py=pyridine), is fluorescent in aqueous solution, whereas the reduced form of the complex, [Os(II)(bpy)2Cl(PyCOOH)]+, shows no significant fluorescence under the same conditions. Such reversible redox control of the fluorescence of the complex can be easily adapted to follow any enzymatic reaction to yield oxidising or reducing products that are capable of interacting with [Os(III)(bpy)2Cl(PyCOOH)]2+ or [Os(II)(bpy)2Cl(PyCOOH)]+. Based on the redox reaction between products of biocatalytic reactions and the osmium complex, we have designed a simple bioanalytical assay for the detection of nerve gases, alpha-ketoglutarate, hydrogen peroxide and glucose.