Membrane Enzyme Research Articles

Repetitive and zonal expression profiles of absorption-related genes in the gastrointestinal tract of ascidian Ciona intestinalis type A.

Intestinal absorption is essential for heterotrophic bilaterians with a tubular gut. Although the fundamental features of the digestive system were shared among chordates with evolution, the gut morphologies of vertebrates diverged and adapted to different food habitats. The ascidian Ciona intestinalis type A, a genome-wide research model of basal chordates, is used to examine the functional morphology of the intestines because of its transparent juvenile body. In the present study, the characteristic gene expression patterns (GEP) of Ciona absorptive proteins, e.g., brush border membrane enzymes for terminal digestion (lactase, maltase, APA, and APN) and transporters (SGLT1, GLUT5, PEPT1, and B0AT1), were investigated in juveniles and young adults, with a special reference to the absorption of other nutrients by pinocytosis- and phagocytosis-related proteins (megalin, cubilin, amnionless, Dab2, Rab7, LAMP, cathepsins, and MRC1). Whole-mount in situ hybridization revealed that these GEP showed multi-regional and repetitive features along the Ciona gastrointestinal tract, mainly in the stomach and several regions of the intestines. In young adults, many absorption-related genes, including pinocytosis-/phagocytosis-related genes, were also expressed between the stomach and mid-intestine. In the gastrointestinal epithelium, absorption-related genes showed zonal GEP along the epithelial structure. Comparisons of GEP, including other intestinal functions, such as nutrient digestion and intestinal protection, indicated the repetitive assignment of a well-coordinated set of intestinal GEP in the Ciona gastrointestinal tract.

NADPH Oxidases: From Molecular Mechanisms to Current Inhibitors.

NADPH oxidases (NOXs) form a family of electron-transporting membrane enzymes whose main function is reactive oxygen species (ROS) generation. Strong evidence suggests that ROS produced by NOX enzymes are major contributors to oxidative damage under pathologic conditions. Therefore, blocking the undesirable actions of these enzymes is a therapeutic strategy for treating various pathological disorders, such as cardiovascular diseases, inflammation, and cancer. To date, identification of selective NOX inhibitors is quite challenging, precluding a pharmacologic demonstration of NOX as therapeutic targets in vivo. The aim of this Perspective is to furnish an updated outlook about the small-molecule NOX inhibitors described over the last two decades. Structures, activities, and in vitro/in vivo specificity are discussed, as well as the main biological assays used.

Enzymes in 3D: Synthesis, remodelling, and hydrolysis of cell wall (1,3;1,4)-β-glucans.

Recent breakthroughs in structural biology have provided valuable new insights into enzymes involved in plant cell wall metabolism. More specifically, the molecular mechanism of synthesis of (1,3;1,4)-β-glucans, which are widespread in cell walls of commercially important cereals and grasses, has been the topic of debate and intense research activity for decades. However, an inability to purify these integral membrane enzymes or apply transgenic approaches without interpretative problems associated with pleiotropic effects has presented barriers to attempts to define their synthetic mechanisms. Following the demonstration that some members of the CslF sub-family of GT2 family enzymes mediate (1,3;1,4)-β-glucan synthesis, the expression of the corresponding genes in a heterologous system that is free of background complications has now been achieved. Biochemical analyses of the (1,3;1,4)-β-glucan synthesized in vitro, combined with 3-dimensional (3D) cryogenic-electron microscopy and AlphaFold protein structure predictions, have demonstrated how a single CslF6 enzyme, without exogenous primers, can incorporate both (1,3)- and (1,4)-β-linkages into the nascent polysaccharide chain. Similarly, 3D structures of xyloglucan endo-transglycosylases and (1,3;1,4)-β-glucan endo- and exohydrolases have allowed the mechanisms of (1,3;1,4)-β-glucan modification and degradation to be defined. X-ray crystallography and multi-scale modeling of a broad specificity GH3 β-glucan exohydrolase recently revealed a previously unknown and remarkable molecular mechanism with reactant trajectories through which a polysaccharide exohydrolase can act with a processive action pattern. The availability of high-quality protein 3D structural predictions should prove invaluable for defining structures, dynamics, and functions of other enzymes involved in plant cell wall metabolism in the immediate future.

Adsorption of Ammonia in Wastewater Using Hyacinth (Eichornia Crassipes) Powder with The Assistance of Bio Balls

<p><span><strong>Abstract.</strong> </span>Water pollution can be caused by the discharge of domestic wastewater containing contaminants. Ammonia is a water pollutant that has a bad impact because it can cause an unpleasant pungent odor and can inhibit or stop the growth of aquatic organisms because it interferes with oxygen binding, changes pH and affects enzymatic reactions and membrane stability in aquatic organisms. This research treats domestic wastewater contaminated with ammonia by means of adsorption using water hyacinth powder. In order to reduce ammonia contaminants in domestic wastewater. This study used a quantitative method which included the acclimatization stage, preliminary test, water hyacinth powder production, adsorption process, ammonia content analysis. The results showed that the use of water hyacinth powder in the most efficient adsorption process was 8 grams of water hyacinth powder and 60 bioballs in 10 liters of wastewater with a decrease in ammonia content of 93.47% for 24 hours. With the bioremediation process ratio, the ammonia content in wastewater can be reduced from 4.810 ppm to 0.314 ppm.</p><p> </p><p><strong>Keywords:</strong></p><p>Adsorption, Domestic Wastewater, Ammonia, Water Hyacinth Powder</p><p><strong><br /></strong></p>

Quantitative flow cytometric selection of tau conformational nanobodies specific for pathological aggregates.

Single-domain antibodies, also known as nanobodies, are broadly important for studying the structure and conformational states of several classes of proteins, including membrane proteins, enzymes, and amyloidogenic proteins. Conformational nanobodies specific for aggregated conformations of amyloidogenic proteins are particularly needed to better target and study aggregates associated with a growing class of associated diseases, especially neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. However, there are few reported nanobodies with both conformational and sequence specificity for amyloid aggregates, especially for large and complex proteins such as the tau protein associated with Alzheimer's disease, due to difficulties in selecting nanobodies that bind to complex aggregated proteins. Here, we report the selection of conformational nanobodies that selectively recognize aggregated (fibrillar) tau relative to soluble (monomeric) tau. Notably, we demonstrate that these nanobodies can be directly isolated from immune libraries using quantitative flow cytometric sorting of yeast-displayed libraries against tau aggregates conjugated to quantum dots, and this process eliminates the need for secondary nanobody screening. The isolated nanobodies demonstrate conformational specificity for tau aggregates in brain samples from both a transgenic mouse model and human tauopathies. We expect that our facile approach will be broadly useful for isolating conformational nanobodies against diverse amyloid aggregates and other complex antigens.

Regulation of survival, growth performance, and intestinal health of large yellow croaker (Larimichthys crocea) larvae by sodium decanoate supplementation

A 30-day feeding trial was undertaken to elucidate the impact of diets supplemented with sodium decanoate (Sd) on survival, growth performance, and intestinal health in Larimichthys crocea larvae. A series of experimental diets consisting of graduated levels of Sd were formulated, including 0.0% (the control), 0.1%, 0.2%, 0.4%, or 0.8%. The study revealed no significant variations in survival among dietary groups. The growth performance (final body weight and specific growth rate) exhibited significant linear and quadratic trends, with the highest values observed in larvae fed a diet containing 0.2% Sd, which was significantly higher than that of the control group. Notably, Larvae fed diets with 0.2%–0.8% Sd had significantly higher intestinal fold height than the control group. A finding that was substantiated by the increased mRNA expression of pcna and odc, which are known to reflect processes of epithelial proliferation and differentiation. Meanwhile, the significant increase in the activity of the intestinal brush border membrane enzyme (Leucine-aminopeptidase), observed in diets supplemented with 0.1%–0.8% Sd, provides compelling evidence supporting the promotion of intestinal development and maturation. In terms of digestive and absorptive capacity, dietary supplementation with 0.4% Sd significantly increased the activity of trypsin and creatine kinase, while 0.1%–0.4% Sd specifically enhanced the activity of Na+-K+-Adenosinetriphosphatase than the control group. Importantly, larvae consuming Sd-supplemented diets at appropriate levels exhibited increased mRNA expression of tight junction-associated genes. Additionally, diets supplemented appropriate Sd improved the antioxidant capacity by modulating antioxidant enzymes and genes, antioxidant substances, and lipid peroxidation products. In summary, the diet incorporating 0.2% Sd could potentially boost the growth of larvae, which may be connected to Sd promoting intestinal health by regulating intestinal morphology and development, digestion and absorption capacity, intestinal barrier function, and antioxidant capacity. This study sheds light into the application of medium-chain fatty acids in marine fish larvae and contributes to the development of nutritional regulation strategies aimed at enhancing larval development.

CB1 receptor expression and signaling are required for dexamethasone-induced aversive memory consolidation

The molecular processes that underlie long-term memory formation involve signaling pathway activation by neurotransmitter release, which induces the expression of immediate early genes, such as Zif268, having a key role in memory formation. In this work, we show that the cannabinoid CB1 receptor signaling is necessary for the effects of dexamethasone on the behavioral response in an inhibitory avoidance task, on dexamethasone-induced ERK phosphorylation, and on dexamethasone-dependent Zif268 expression. Furthermore, we provide primary evidence for the mechanism responsible for this crosstalk between cannabinoid and glucocorticoid-mediated signaling pathways, showing that dexamethasone regulates endocannabinoid metabolism by inhibiting the activity of the Fatty acid amide hydrolase (FAAH), an integral membrane enzyme that hydrolyzes endocannabinoids and related amidated signaling lipids. Our results provide novel evidence regarding the role of the endocannabinoid system, and in particular of the CB1 receptor, as a mediator of the effects of glucocorticoids on the consolidation of aversive memories.

Production and Bioconversion Efficiency of Enzyme Membrane Bioreactors in the Synthesis of Valuable Products.

The demand for bioactive molecules with nutritional benefits and pharmaceutically important properties is increasing, leading researchers to develop modified production strategies with low-cost purification processes. Recent developments in bioreactor technology can aid in the production of valuable products. Enzyme membrane bioreactors (EMRs) are emerging as sustainable synthesis processes in various agro-food industries, biofuel applications, and waste management processes. EMRs are modified reactors used for chemical reactions and product separation, particularly large-molecule hydrolysis and the conversion of macromolecules. EMRs generally produce low-molecular-weight carbohydrates, such as oligosaccharides, fructooligosaccharides, and gentiooligosaccharides. In this review, we provide a comprehensive overview of the use of EMRs for the production of valuable products, such as oligosaccharides and oligodextrans, and we discuss their application in the bioconversion of inulin, lignin, and sugars. Furthermore, we critically summarize the application and limitations of EMRs. This review provides important insights that can aid in the production of valuable products by food and pharmaceutical industries, and it is intended to assist scientists in developing improved quality and environmentally friendly prebiotics using EMRs.

Detoxification and decolorization of complex textile effluent in an enzyme membrane reactor: batch and continuous studies.

There is an urgent need to look for bio-based technologies to address the pollution related to textile dyes in waterbodies. The aim of this study was to evaluate an engineered laccase variant, LCC1-62 of Cyathus bulleri, expressed in recombinant Pichia pastoris, for the decolorization and detoxification of real textile effluent. The partially purified laccase effectively (~60-100%) decolorized combined effluent from different dyeing units at a laccase concentration of 500 U/L at a 50-mL level. Decolorization and detoxification of the combined effluents, from a local textile mill, were evaluated at 0.3 L volumetric level in a ray-flow membrane reactor in batch and continuous modes of operation. In batch studies, maximum decolorization of 97% and detoxification of 96% occurred at a hydraulic retention time (HRT) of 6 h without any additional laccase requirement. In continuous studies, the reactor was operated at an HRT of 6 h with a lower enzyme dosage (~120 U/L of the effluent). Decolorization was accompanied by a loss in laccase activity which was restored to ~120 U/L by the addition of laccase in two regimes. The addition of laccase, when the residual laccase activity decreased to 40% (~50 U/L), resulted in high decolorization (~5 ppm residual dye concentration) and low variance (σ2) of 2.77, while laccase addition, when the residual dye concentration decreased to ~8% (~10 U/L), resulted in an average dye concentration of 13 ppm with a high variance of 62.08. The first regime was implemented, and the continuous reactor was operated for over 80 h at an HRT of 3 and 6 h, with the latter resulting in ~95% decolorization and 96% reduction in the mutagenicity of the effluent. Less than 10% membrane fouling was observed over long operations of the reactor. The findings strongly suggest the feasibility of using LCC1-62 in an enzyme membrane reactor for large-scale treatment of textile effluents.

Effect of Microplastics on the Activity of Digestive and Oxidative-Stress-Related Enzymes in Peled Whitefish (Coregonus peled Gmelin) Larvae.

Microplastics (MPs) are emergent pollutants in freshwater environments and may impact aquatic organisms, including those of nutritional value. The specific activities of digestive and antioxidant enzymes can be used as good bioindicators of the potential effects of MPs on fish in case of waterborne MP contamination. In this study, we used fluorescent polystyrene microplastics (PS-MPs) to analyze the alterations in enzyme activities in larvae of Coregonus peled Gmelin (peled or Northern whitefish), one of the most valuable commercial fish species of Siberia. Our results indicate that peled larvae can ingest 2 µm PS microspheres in a waterborne exposure model. A positive correlation (rs = 0.956; p < 0.01) was found between MP concentration in water and the number of PS microspheres in fish guts, with no significant differences between 24 h and 6-day exposure groups. The ingestion of MPs caused alterations in digestive enzyme activity and antioxidant responses at the whole-body level. The presence of PS-MPs significantly stimulated (p < 0.05) the specific activity of α-Amylase and non-specific esterases in peled larvae after 24 h. However, a pronounced positive effect (p < 0.05) of MPs on the activity of pancreatic trypsine and bile salt-activated lipase was only found after 6 days of exposure compared to after 24 h. Intestinal membrane enzyme aminopeptidase N was also stimulated in the presence of PS-MPs after 6-day exposure. We also observed a significant increase in the specific activity of catalase in peled larvae after 6 days of exposure, which indicates the MP-induced modulation of oxidative stress. Taken together, these results highlight the potential impact of environmental MPs on northern commercial fish, their importance for estimating fish stocks, and the sustainability of freshwater ecosystems.

Reduction of the Renal Radioactivity of 111In-DOTA-Labeled Antibody Fragments with a Linkage Cleaved by the Renal Brush Border Membrane Enzymes.

The interposition of a cleavable linkage by enzymes on the renal brush border membrane constitutes a promising approach for reducing the renal radioactivity levels of radiolabeled low-molecular-weight antibody fragments and constructs (LMW Abs). Herein, we applied the molecular design to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-based reagents for radiotheranostic applications with trivalent radiometals. DOTA or a derivative thereof was conjugated to a Fab through an FGK linkage ([111In]In-DO3AiBu-Bn-FGK-Fab or [111In]In-DOTA-Bn-FGK-Fab). When injected into mice, both generated radiometabolites, [111In]In-DO3AiBu-Bn-F and [111In]In-DOTA-Bn-F, by the angiotensin-converting enzyme at similar rates. Both exhibited significantly lower renal radioactivity levels than a 111In-labeled Fab prepared by the conventional procedure ([111In]In-DOTA-Bn-SCN-Fab). The different elimination rates of each radiometabolite from the kidney significantly affected the renal radioactivity levels. [111In]In-DO3AiBu-Bn-FGK-Fab preferentially reduced the renal localization without impairing tumor accumulation. These findings would pave the way for developing a DOTA-based radiotheranostic platform for LMW Abs bearing cleavable linkers for renal brush border enzymes.

Evaluation of transmembrane domain deletions on hyaluronic acid polymerization of hyaluronan synthase isolated from Streptococcus equisimilis group G.

The membrane enzyme of hyaluronan synthase (HAS) is the key enzyme in hyaluronic acid (HA) biosynthesis by coupling UDP-sugars. Prior studies proposed the C-terminus region of HAS enzyme mediates the production rate and molecular weight of HA. The current study describes the isolation and characterizations of a transmembrane HAS enzyme isolated from Streptococcus equisimilis Group G (GGS-HAS) in vitro. The effect of transmembrane domains (TMDs) on HA productivity was determined and the shortest active variant was also identified by recombinant expression of full-length and five truncated forms of GGS-HAS in Escherichia coli. We found that the GGS-HAS enzyme is longer than that of S. equisimilis group C (GCS-HAS) which includes three more residues (LER) at the C-terminus region (positions 418-420) and also one-point mutation at position 120 (E120D). Amino acid sequence alignment demonstrated 98% and 71% identity of GGS-HAS with that of S. equisimilis Group C and S. pyogenes Group A, respectively. The in vitro productivity of the full-length enzyme was 35.57µg/nmol, however, extended TMD deletions led to a reduction in the HA productivity. The HAS-123 variant showed the highest activity among the truncated forms, indicating the essential role of first, second, and third TMDs for the full activity. Despite a decline in activity, the intracellular variant can still mediate the binding and polymerization of HA without any need for TMDs. This significant finding suggests that the intracellular domain is the core for HA biosynthesis in the enzyme and other domains are probably involved in other attributes including the enzyme kinetics that affect the size distribution of the polymer. However, more investigations on the recombinant forms are still needed to confirm clearly the role of each transmembrane domain on these properties.

Oral administration of Nigella sativa oil attenuates arsenic-induced redox imbalance, DNA damage, metabolic distress, and histopathological alterations in rat intestine

BackgroundExposure to arsenic, a widespread environmental toxin, produces multiple organ toxicity, including gastrointestinal toxicity. Nigella sativa (NS) has long been revered for its numerous health benefits under normal and pathological states. In view of this, the present study attempts to evaluate the protective efficacy of orally administered Nigella sativa oil (NSO) against arsenic-induced cytotoxic and genotoxic alterations in rat intestine and elucidate the underlying mechanism of its action. MethodsRats were categorized into the control, NaAs, NSO, and NaAs+NSO groups. After pre-treatment of rats in the NaAs+NSO and NSO groups daily with NSO (2 ml/kg bwt, orally) for 14 days, NSO treatment was further continued for 30 days, with and without NaAs treatment (5 mg/kg bwt, orally), respectively. Various biochemical parameters, such as enzymatic and non-enzymatic antioxidants, carbohydrate metabolic and brush border membrane marker enzyme activities were evaluated in the mucosal homogenates of all the groups. Intestinal brush border membrane vesicles (BBMV) were isolated, and the activities of membrane marker enzyme viz. ALP, GGTase, LAP, and sucrase were determined. Further, the effect on kinetic parameters viz KM (Michaelis-Menten constant) and Vmax of these enzymes was assessed. Integrity of enterocyte DNA was examined using the comet assay. Histopathology of the intestines was performed to evaluate the histoarchitectural alterations induced by chronic arsenic exposure and/or NSO supplementation. Arsenic accumulation in the intestine was studied by inductively coupled plasma-mass spectroscopy (ICP-MS). ResultsNaAs treatment caused substantial changes in the activities of brush border membrane (BBM), carbohydrate metabolism, and antioxidant defense enzymes in the intestinal mucosal homogenates. The isolated BBM vesicles (BBMV) also showed marked suppression in the marker enzyme activities. Severe DNA damage and mucosal arsenic accumulation were observed in rats treated with NaAs alone. In contrast, oral NSO supplementation significantly alleviated all the adverse alterations induced by NaAs treatment. Histopathological examination supported the biochemical findings. ConclusionNSO, by improving the antioxidant status and energy metabolism, could significantly alter the ability of the intestine to protect against free radical-mediated arsenic toxicity in intestine. Thus, NSO may have an excellent scope in managing gastrointestinal distress in arsenic intoxication.

Molecular diagnosis of hereditary hemolytic anemias: Recent updates

AbstractHereditary hemolytic anemia (HHA) is a heterogeneous group of disorders due to genetically caused defects in red blood cell membrane structure, enzymes, heme and globin synthesis, erythroid proliferation, and differentiation. Traditionally, the diagnostic process is complex and includes a plethora of tests from routine to highly specialized ones. The inclusion of molecular testing has significantly improved the diagnostic yield. The value of molecular testing is broader than just rendering the correct diagnosis, as it may also guide therapeutic decisions. As more molecular modalities become available for clinical use, it is imperative to understand their benefits and disadvantages pertaining to the HHA diagnostics. Re‐evaluation of the traditional diagnostic workflow may also bring forth additional benefits. This review focuses on the current state of molecular testing for HHA.

Characterization of a Model Bioreactive Membrane for the Simultaneous Separation and Hydrolysis of Lipopolysaccharides

The design of functionalized selective layers to develop novel membranes is essential to provide potential solutions that meet the continuously growing demand of providing safe water. Herein, we present the interfacial response of an enzymatic thin-film composite (E-TFC) membrane displaying dual functionality and fabricated as a proof-of-concept for both efficient lipopolysaccharide (LPS) separation and ester bond hydrolysis. The enzymatic membrane model was constructed by employing lipase b from Candida antarctica (CALB) covalently coupled via chemically activated bisepoxide groups onto the surface of the di-block copolymer polystyrene-b-poly(4-vinyl pyridine) (PS-b-P4VP) layer. Our results show a complete rejection of size-excluded LPS molecules when using the fabricated E-TFC membrane in a forward osmosis (FO) application. Moreover, the immobilized enzyme was able to retain 97% of its enzymatic activity when using 4-nitrophenyl acetate (pNPA) and up to 74% to liberate free fatty acids from LPS molecules within the feed side of the FO system. This work provides fundamental insights into new emerging functional biomaterials that find applications in hybrid catalytic filtration processes that also selectively remove LPS molecules from water sources.

Synthesis of butyl cinnamate using enzymatic membrane reactor in a free solvent system

Cinnamic acid is a phenolic compound that has the potential to act as a natural antioxidant. Cinnamic acid esterification can be performed by adding alcohol as an alkyl group donor. This esterification is carried out to increase the antioxidant capacity of cinnamic acid. Esterification of cinnamic acid with butanol as the alkyl group donor was the best condition to modify cinnamic acid. In this study, the synthesis of butyl cinnamate using a enzymatic membrane reactor (EMR) is carried out continuously with Lipozyme TL IM as the catalyst. This study aimed to determine the optimal conditions for the synthesis of butyl cinnamate using various concentrations of cinnamic acid, biocatalyst, residence time, and the presence of molecular sieves. The synthesis of butyl cinnamate at 40°C was optimum when molecular sieves were present with 0.01 M cinnamic acid concentration, 1% (w/v) biocatalyst, and 12 h of residence time.

Oral administration of curcumin and gallic acid alleviates pentachlorophenol-induced oxidative damage in rat intestine.

Pentachlorophenol (PCP) is used as pesticide and wood preservative. We have previously shown that PCP causes oxidative damage in rat intestine. This study aimed to delineate the possible therapeutic potential of curcumin (CUR) and gallic acid (GA) against PCP-induced damage in rat intestine. PCP alone group received 125mg PCP/kg body weight/day orally for 4 days. Animals in combination groups received CUR or GA (100mg/kg bw) for 18 days and PCP (125mg/kg bw) for the last four days. Rats were sacrificed and intestinal preparations were analyzed for various parameters. Administration of PCP alone altered the activities of metabolic, antioxidant and brush border membrane enzymes. It also increased DNA-protein crosslinking and DNA-strand scission. Animals in combinations groups showed significant amelioration against PCP-induced oxidative damage. Histological abrasions were seen in PCP alone group which were reduced in the intestines of combination groups. CUR was more effective protectant than GA. CUR and GA protected rat intestine from PCP-mediated changes in the activities of metabolic, antioxidant and brush border membrane enzymes. They also prevented DNA damage and histological abrasions. The antioxidant character of CUR and GA may be responsible for the diminution of PCP-mediated oxidative damage.

Alteration of circulating ACE2-network related miRNAs in patients with COVID-19

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Medical University of Warsaw and São Paulo Research Foundation (FAPESP). Introduction Coronavirus disease 2019 (COVID-19) is associated with an increased risk of mortality and adverse cardiovascular (CV) occurrences, especially in a group of patients with CV diseases (CVD). SARS-CoV-2 virus enters cells through angiotensin converting enzyme 2 (ACE2) cell membrane enzymes, leading to the downregulation in the ACE2-related pathways. Based on our previous in silico [1] results we hypothesized that alteration in the ACE2 interaction network caused by virus infection can affect the expression of miRNAs and influence severity of the disease. Purpose In this study we aimed to analyse the diagnostic and predictive utility of ACE2-related miRNAs, which were identified in our previous in silico analysis (miR-10b-5p, miR-124-3p, miR-200b-3p, miR-26b-5p, miR-302c-5p) in patients with COVID-19. We also aimed to unravel the functions of analyzed miRNAs, by using machine learning based tools, including SHAP for clinical data analysis, and bioinformatic tools for data mining, target predictions and enrichment analyses. Methods Blood samples and clinical data of 79 COVID-19 patients were collected at three different time points, including the day of admission, 7-days and 21-days after admission, as well as one time-point of 32 healthy volunteers. The composite endpoint was hospitalisation length of stay (&amp;gt;21 days) and/or death in follow-up. Results Delta low miR-200b-3p expression (after 7-days of admission) presents predictive utility in assessment of the hospital length of stay and/or death in ROC curve analysis (AUC:0.730, p=0.002) (Fig. 1). Delta low miR-200b-3p expression, together with diabetes mellitus (DM) are independent predictors of increased hospital length of stay and/or death (OR= 5.775; CI= 1.572- 21.214; p= 0.008, OR= 4.888; CI= 1.001- 23.858; p= 0.050, respectively). The expression levels of miR-26b-5p and miR-10b-5p in COVID-19 patients were found lower at the baseline, 7 and 21-days after admission compared to the healthy controls (p&amp;lt;0.0001 for all time points). SHAP analysis indicated levels of miR 200b-3p (day 7th), miR-302c-5p (day 7th), CRP (day 7th), neutrophils (day 0), and D-Dimer (day 0) as the most promising predictors of long hospitalisation for the COVID-19 patients. Pathway enrichment analysis showed that among top shared pathways between targets of analyzed miRNAs were IL-2 signaling pathway, and Pathways in cancer. miR-200b-3p showed regulation of COVID-19-related targets associated with T cell protein tyrosine phosphatase and HIF-1 transcriptional activity in hypoxia, key pathways in COVID-19. Bioinformatics analysis pointed out the role of those miRNAs in multiple CVD phenotypes associated with COVID-19 (Fig. 2). Conclusions In this study we validated and characterized miRNAs which could serve as novel, predictive biomarkers of the COVID-19 long term hospitalisation and can be used for early stratification of patients and prediction of severity of infection development in an individual.

Nanoarchitectonic approaches for measuring the catalytic behavior of a membrane anchored enzyme. From Langmuir-Blodgett to a novel Langmuir-Schaefer based nanofilm building device

Self-organized lipid monolayers at the air-water interface (Langmuir films, LF) are commonly used for measuring the catalytic properties of membrane-bound enzymes. This methodology allows to provide a consistent flat topography molecular density, packing defects and thickness. The aim of the present work was to show the methodological advantages of using the horizontal transfer method (Langmuir-Schaefer) with respect to the vertical transfer method (Langmuir-Blodgett) when mounting a device to measure catalytic activity of membrane enzymes. Based on the results obtained we can conclude that it is possible to prepare stable Langmuir-Blodgett (LB) and Langmuir-Schaefer (LS) films from Bovine Erythrocyte Membranes (BEM) preserving the catalytic activity of its native Acetylcholinesterase (BEA). In comparison, the LS films showed Vmax values more similar to the enzyme present in the vesicles of natural membranes. In addition, it was much easier to produce large amounts of transferred areas with the horizontal transfer methodology. It was possible to decrease the time required to mount an assay with numerous activity points, such as building activity curves as a function of substrate concentration. The present results show that LSBEM provides a proof of concept for the development of biosensors based on transferred purified membrane for the screening of new products acting on an enzyme embedded on its natural milieu. In the case of BEA, the application of these enzymatic sensors could have medical interest, providing drug screening tools for the treatment of Alzheimer's disease.

Treatment of the Neutropenia Associated with GSD1b and G6PC3 Deficiency with SGLT2 Inhibitors.

Glycogen storage disease type Ib (GSD1b) is due to a defect in the glucose-6-phosphate transporter (G6PT) of the endoplasmic reticulum, which is encoded by the SLC37A4 gene. This transporter allows the glucose-6-phosphate that is made in the cytosol to cross the endoplasmic reticulum (ER) membrane and be hydrolyzed by glucose-6-phosphatase (G6PC1), a membrane enzyme whose catalytic site faces the lumen of the ER. Logically, G6PT deficiency causes the same metabolic symptoms (hepatorenal glycogenosis, lactic acidosis, hypoglycemia) as deficiency in G6PC1 (GSD1a). Unlike GSD1a, GSD1b is accompanied by low neutrophil counts and impaired neutrophil function, which is also observed, independently of any metabolic problem, in G6PC3 deficiency. Neutrophil dysfunction is, in both diseases, due to the accumulation of 1,5-anhydroglucitol-6-phosphate (1,5-AG6P), a potent inhibitor of hexokinases, which is slowly formed in the cells from 1,5-anhydroglucitol (1,5-AG), a glucose analog that is normally present in blood. Healthy neutrophils prevent the accumulation of 1,5-AG6P due to its hydrolysis by G6PC3 following transport into the ER by G6PT. An understanding of this mechanism has led to a treatment aimed at lowering the concentration of 1,5-AG in blood by treating patients with inhibitors of SGLT2, which inhibits renal glucose reabsorption. The enhanced urinary excretion of glucose inhibits the 1,5-AG transporter, SGLT5, causing a substantial decrease in the concentration of this polyol in blood, an increase in neutrophil counts and function and a remarkable improvement in neutropenia-associated clinical signs and symptoms.