In Silico Comparative Analysis of Ivermectin and Nirmatrelvir Inhibitors Interacting with the SARS-CoV-2 Main Protease.

Exploring therapeutic options is crucial in the ongoing COVID-19 pandemic caused by SARS-CoV-2. Nirmatrelvir, which is a potent inhibitor that targets the SARS-CoV-2 Mpro, shows promise as an antiviral treatment. Additionally, Ivermectin, which is a broad-spectrum antiparasitic drug, has demonstrated effectiveness against the virus in laboratory settings. However, its clinical implications are still debated. Using computational methods, such as molecular docking and 100 ns molecular dynamics simulations, we investigated how Nirmatrelvir and Ivermectin interacted with SARS-CoV-2 Mpro(A). Calculations using density functional theory were instrumental in elucidating the behavior of isolated molecules, primarily by analyzing the frontier molecular orbitals. Our analysis revealed distinct binding patterns: Nirmatrelvir formed strong interactions with amino acids, like MET49, MET165, HIS41, HIS163, HIS164, PHE140, CYS145, GLU166, and ASN142, showing stable binding, with a root-mean-square deviation (RMSD) of around 2.0 Å. On the other hand, Ivermectin interacted with THR237, THR239, LEU271, LEU272, and LEU287, displaying an RMSD of 1.87 Å, indicating enduring interactions. Both ligands stabilized Mpro(A), with Ivermectin showing stability and persistent interactions despite forming fewer hydrogen bonds. These findings offer detailed insights into how Nirmatrelvir and Ivermectin bind to the SARS-CoV-2 main protease, providing valuable information for potential therapeutic strategies against COVID-19.

Comparison of the precursor, amino acid oxidation, and end-product methods for the evaluation of protein turnover in senior dogs.

Stable isotope methods have been used to study protein metabolism in humans; however, there application in dogs has not been frequently explored. The present study compared the methods of precursor (13C-Leucine), end-products (15N-Glycine), and amino acid oxidation (13C-Phenylalanine) to determine the whole-body protein turnover rate in senior dogs. Six dogs (12.7 ± 2.6 years age, 13.6 ± 0.6 kg bodyweight) received a dry food diet for maintenance and were subjected to all the above-mentioned methods in succession. To establish 13C and 15N kinetics, according to different methodologies blood plasma, urine, and expired air were collected using a specifically designed mask. The volume of CO2 was determined using respirometry. The study included four methods viz. 13C-Leucine, 13C-Phenylalanine evaluated with expired air, 13C-Phenylalanine evaluated with urine, and 15N-Glycine, with six dogs (repetitions) per method. Data was subjected to variance analysis and means were compared using the Tukey test (P<0.05). In addition, the agreement between the methods was evaluated using Pearson correlation and Bland-Altman statistics. Protein synthesis (3.39 ± 0.33 g.kg-0,75. d-1), breakdown (3.26 ± 0.18 g.kg-0.75.d-1), and flux estimations were similar among the four methods of study (P>0.05). However, only 13C-Leucine and 13C-Phenylalanine (expired air) presented an elevated Pearson correlation and concordance. This suggested that caution should be applied while comparing the results with the other methodologies.

SARS-CoV-2 Spike Protein Enhances Carboxypeptidase Activity of Angiotensin-Converting Enzyme 2.

In this study, we investigated whether severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein may modify angiotensin-converting enzyme 2 (ACE2) activity in the plasma, heart, kidney, liver, lung, and six brain regions (amygdala, brain stem, cortex, hippocampus, hypothalamus, and striatum) of diabetic and hypertensive rats. We determine ACE2 activity in the plasma and lysates of heart, kidney, liver, lung, and six brain regions. MLN-4760 inhibits ACE2 activity in the plasma and all organs. On the other hand, soluble ACE2 (sACE2) activity increased in the plasma of diabetic rats, and there was no change in the plasma of hypertensive rats. ACE2 activity was augmented in the liver, brain stem, and striatum, while it decreased in the kidney, amygdala, cortex, and hippocampus of diabetic rats. ACE2 activity increased in the kidney, liver, and lung, while it decreased in the heart, amygdala, cortex, and hypothalamus of hypertensive rats. We measured the ACE2 content via enzyme-linked immunosorbent assay and found that ACE2 protein levels increased in the heart, while it decreased in the plasma, kidney, brain stem, cortex, hippocampus, hypothalamus, and striatum of diabetic rats. ACE2 protein levels decreased in the brain stem, cortex, hippocampus, and hypothalamus of hypertensive rats. Our data showed that the spike protein enhanced ACE2 activity in the liver and lungs of diabetic rats, as well as in the heart and three of the brain regions (cortex, hypothalamus, and striatum) of hypertensive rats.

Chilean Market Protein Shakes Composition.

Understanding the nutritional content of protein supplements is crucial for optimal nutritional planning among athletes and other people. Distribution of macronutrients and aminograms in the main products available in the national Chilean market remains unknown. A descriptive cross-sectional study was conducted to identify the main protein supplements available in the Chilean market. Information on macronutrients and aminograms from the nutritional labels of each product was extracted. The analysis considered the content per portion and per 100 g. Cluster analysis models and graphical representations were explored. Eighty protein shakes were assessed in the Santiago de Chile market. The median protein dosage was 32 g (range from 25 to 52), and the median energy value stood at 390 kcal (range from 312 to 514). The median protein content per 100 g of product was found to be 75 g (range from 42.5 to 97.2). The combined median concentration of amino acids was 4749.75 mg. Among these, the essential amino acid L-Tryptophan exhibited the lowest concentration at 1591.50 mg, while the conditional amino acid L-Glutamine had the highest median concentration at 17,336 mg. There was a significant prevalence of animal-derived products, placing specific emphasis on protein supplements that feature elevated levels of the amino acids L-Glutamine and L-Leucine.

Cardioprotective Effects of Leucine Supplementation against Doxorubicin-Induced Cardiotoxicit.

Doxorubicin is one of the most important antitumor drugs used in oncology; however, its cardiotoxic effect limits the therapeutic use and raises concerns regarding patient prognosis. Leucine is a branched-chain amino acid used in dietary supplementation and has been studied to attenuate the toxic effects of doxorubicin in animals, which increases oxidative stress. Oxidative stress in different organs can be estimated using several methods, including catalase expression analysis. This study aimed to analyze the effect of leucine on catalase levels in rat hearts after doxorubicin administration. Adult male Wistar rats were separated into two groups: Standard diet (SD) and 5% Leucine-Enriched Diet (LED). The animals had free access to diet from D0 to D28. At D14, the groups were subdivided in animals injected with Doxorubicin and animals injected with vehicle, until D28, and the groups were SD, SD + Dox, LED and LED + Dox. At D28, the animals were submitted do Transthoracic Echocardiography and euthanized. Despite Dox groups had impaired body weight gain, raw heart weight was not different between the groups. No substantial alterations were observed in macroscopic evaluation of the heart. Although, Doxorubicin treatment increased total interstitial collagen in the heart, which in addition to Type I collagen, is lower in LED groups. Western blot analysis showed that catalase expression in the heart of LED groups was lower than that in SD groups. In conclusion, leucine supplementation reduced both the precocious Dox-induced cardiac remodeling and catalase levels in the heart.

A Randomized Controlled Trial on the Effects of Leucine-Supplement Combined with Nutritional Counseling on Body Composition in Mix Cancer Older Men.

BACKGROUND: Malnutrition and metabolic alterations of cancer cachexia are often associated with negative weight loss and muscle mass wasting. In this sense, protein supplementation can be a strategy to help counteract the loss and/or maintenance of mass in these patients. The aim of this study was to evaluate the effect of leucine supplementation on body composition in outpatients with gastrointestinal tract cancer. METHODS: It was a randomized, blinded, controlled, parallel trial, performed in male patients with a cancer diagnosis of the gastrointestinal tract and appendix organs undergoing chemotherapy. All the patients were allocated to one of the protocol groups: L-leucine supplement or the control group, during 8 weeks of intervention. We evaluated the body composition through bioelectrical impedance analysis, the cancer cachexia classification, and the diet intake before and after the intervention protocol. The intention-to-treat approach was performed to predict the missing values for all patients who provide any observation data. RESULTS: The patients were an average age of 65.11 ± 7.50 years old. In the body composition analysis with patients who finished all the supplementation, we observed a significant gain in body weight (61.79.9 ± 9.02 versus 64.06 ± 9.45, p = 0.01), ASMM (7.64 ± 1.24 versus 7.81 ± 1.20, p = 0.02) in the Leucine group, whereas patients in the control did not present significant variation in these parameters. There was no significant intergroup difference. While in the analysis included the patients with intention-to-treat, we found a significant increase in body weight (p = 0.01), BMI (p = 0.01), FFM (p = 0.03), and ASMM (p = 0.01) in the Leucine group. No significant intergroup differences. These results also similar among cachectic patients. CONCLUSION: A balanced diet enriched with free-Leucine supplementation was able to promotes gains in body weight and lean mass in older men diagnosticated with gastrointestinal and appendix organs of digestion cancer after 8 weeks. However, the fact that most men are non-cachectic or pre-cachectic is not clear if the increase in muscle mass was due to a high intake of leucine, since no difference between groups was detected. Moreover, we know that benefits on body composition are due to adequate calorie and macronutrients consumption and that balanced feeding according to nutrition Guidelines seems crucial and must be advised during the oncological treatment.

Cost-Utility Model of Nirmatrelvir/Ritonavir in Brazil: Analysis of a Vaccinated Population.

OBJECTIVES: The aim of this study is to conduct a cost-utility analysis of the use of the antiviral nirmatrelvir/ritonavir, applied to a vaccinated Brazilian population against COVID-19, from the perspective of the Brazilian Public Health System (SUS). METHODS: A microsimulation model was created with individual-level data and daily cycles, with a 1-year time horizon, to compare the current scenario of standard care with a scenario in which nirmatrelvir/ritonavir is offered to the population. Adults of any age group that received ≥2 doses of the COVID-19 vaccine formed the investigated population. Direct medical costs of the outpatients and inpatients admitted to the ward or intensive care unit were included. The effectiveness of the model was measured in quality-adjusted life-years (QALYs). RESULTS: In all simulations, the use of nirmatrelvir/ritonavir resulted in incremental costs per patient of US dollar (USD)245.86 and incremental effectiveness of 0.009 QALY, over a year. The incremental cost-utility ratio was USD27 220.70/QALY. The relative risk of the vaccinated population was the factor that affected the outcome most, according to the univariate sensitivity analysis. The probabilistic sensitivity analysis resulted in 100% of the simulations being more costly and effective, but that only 4% of them were below the established cost-effectiveness threshold of USD24 000.00/QALY. In the scenario considering only the population over 60 years old and immunosuppressed (of any age), the incremental cost-utility ratio was USD7589.37/QALY. CONCLUSIONS: The use of nirmatrelvir/ritonavir in the treatment of COVID-19 in a vaccinated population was cost-effective only for immunosuppressed individuals and people over 60 years of age.

The branched chain amino acids (BCAAs) modulate the development of the intra-cellular stages of Trypanosoma cruzi.

The life cycle of Trypanosoma cruzi, the etiological agent of Chagas disease, involves different forms of the parasite, which alternates between insect and vertebrate hosts. One critical process in the parasite's life cycle is metacyclogenesis, in which the replicative non-infective forms present in the insect midgut differentiate into non-dividing vertebrate-infective forms. It is known that proline (Pro) is important for this process and that leucine (Leu) and isoleucine (Ile) can act as inhibitors of metacyclogenesis. In this study, we investigated further the role of branched-chain amino acids (BCAAs) as negative modulators of parasite differentiation and infection capability in vitro. We found that BCAAs can down-regulate metacyclogenesis, inhibiting Pro-dependent differentiation. Furthermore, we evaluated the ability of all three BCAAs to influence the differentiation of intracellular stages and found that they could modulate the release of trypomastigotes from infected host cells. These findings suggest that BCAAs may have an important role in the complex life cycle of T. cruzi. Thus, enzymes of their metabolism and other interacting proteins could be potential targets for the development of new therapeutic strategies for Chagas disease.

Light effects on Lasiodiplodia theobromae metabolome cultured in vitro.

INTRODUCTION: The present work identified and compared intracellular metabolites and metabolic networks in mycelial cultures of Lasiodiplodia theobromae grown under 12 natural light and 24 hours' dark using a 1 H NMR-based metabolomics approach. MATERIALS AND METHODS: Fungal cultures were grown in potato dextrose media, and metabolites were extracted by sonication with sodium phosphate-buffered saline (pH = 6.0, 10% D2O, 0.1 mM TSP) from mycelium samples collected every week over four weeks. RESULTS: Multivariate analyses revealed that the light exposure group showed a positive correlation within beta-hydroxybutyrate, acetoacetate, acetone, betaine, choline, glycerol, and phosphocholine. On the other hand, phenyl acetate, leucine, isoleucine, valine, and tyrosine were positively correlated with dark conditions. Light favored the oxidative degradation of valine, leucine, and isoleucine, leading to the accumulation of choline, phosphocholine, betaine, and ketone bodies (ketogenesis). Ketogenesis, gluconeogenesis, and the biosynthesis of choline, phosphocholine, and betaine, were considered discriminatory routes for light conditions. The light-sensing pathways were interlinked with fungal development, as verified by the increased production of mycelia biomass without fruiting bodies and stress signaling, as demonstrated by the increased production of pigments.

Treatment of maple syrup urine disease: Benefits, risks, and challenges of liver transplantation.

Maple syrup urine disease (MSUD) is caused by a deficiency in the activity of the branched-chain α-ketoacid dehydrogenase (BCKD) complex, promoting the accumulation of the branched-chain amino acids (BCAA) leucine, isoleucine, and valine, as well as their respective α-keto acids. MSUD is an autosomal recessive hereditary metabolic disorder characterized by ketoacidosis, ataxia, coma, and mental and psychomotor retardation. The mechanisms involved in the brain damage caused by MSUD are not fully understood. Early diagnosis and treatment, as well as proper control of metabolic decompensation crises, are crucial for patients' survival and for a better prognosis. The recommended treatment consists of a high-calorie diet with restricted protein intake and specific formulas containing essential amino acids, except those accumulated in MSUD. This treatment will be maintained throughout life, being adjusted according to the patients' nutritional needs and BCAA concentration. Because dietary treatment may not be sufficient to prevent neurological damage in MSUD patients, other therapeutic strategies have been studied, including liver transplantation. With transplantation, it is possible to obtain an increase of about 10% of the normal BCKD in the body, an amount sufficient to maintain amino acid homeostasis and reduce metabolic decompensation crises. However, the experience related to this practice is very limited when considering the shortage of liver for transplantation and the risks related to the surgical procedure and immunosuppression. Thus, the purpose of this review is to survey the benefits, risks, and challenges of liver transplantation in the treatment of MSUD.

A promising new target to control fasciolosis: Fasciola hepatica leucine aminopeptidase 2.

Parasite M17 leucine aminopeptidases (LAPs) have been associated with critical roles in different key functions such as the nutrition, migration, and invasion of the natural host. Native or recombinant LAP used as a vaccine antigen has proved effective to elicit protection against Fasciola hepatica infection in sheep, pointing to potential vaccine candidates against fascioliasis in ruminant species. Previously, the FhLAP1, abundantly secreted in vitro by the mature adult parasite was used as a vaccine antigen obtaining promising protection results against F. hepatica challenge in small ruminants. Here, we report the biochemical characterization of a second recombinant LAP (FhLAP2) associated with the juvenile stage of F. hepatica. FhLAP2 showed aminopeptidase activity using different synthetic substrates, including leucine, arginine, and methionine and was increased in the presence of Mn+ 2 and Mg+ 2. The activity was inhibited by bestatin, 1,10-phenanthroline, and EDTA, specific inhibitors of aminopeptidase and/or metalloproteases. Finally, the recombinant FhLAP2 functional form was tested in combination with Freund's incomplete adjuvant in an immunization trial in mice followed by an experimental challenge with F. hepatica metacercariae. The immunization with FhLAP2/FIA resulted in a significant reduction of parasite recovery compared to control groups. The immunized group elicited total specific IgG and subclasses IgG1 and IgG2 antibody responses. This study highlights the potential of a new candidate vaccine formulation with potential applications in natural ruminant hosts, especially those targeting the juvenile stage.

Melatonin improves behavioral parameters and oxidative stress in zebrafish submitted to a leucine-induced MSUD protocol.

Maple syrup urine disease (MSUD) is an inherited metabolic disorder caused by a deficiency in branched-chain alpha-ketoacid dehydrogenase complex (BCKAC). The treatment is a standard therapy based on a protein-restricted diet with low branched-chain amino acids (BCAA) content to reduce plasma levels and, consequently, the effects of accumulating their metabolites, mainly in the central nervous system. Although the benefits of dietary therapy for MSUD are undeniable, natural protein restriction may increase the risk of nutritional deficiencies, resulting in a low total antioxidant status that can predispose and contribute to oxidative stress. As MSUD is related to redox and energy imbalance, melatonin can be an important adjuvant treatment. Melatonin directly scavenges the hydroxy radical, peroxyl radical, nitrite anion, and singlet oxygen and indirectly induces antioxidant enzyme production. Therefore, this study assesses the role of melatonin treatment on oxidative stress in brain tissue and behavior parameters of zebrafish (Danio rerio) exposed to two concentrations of leucine-induced MSUD: leucine 2 mM and 5mM; and treated with 100 nM of melatonin. Oxidative stress was assessed through oxidative damage (TBARS, DCF, and sulfhydryl content) and antioxidant enzyme activity (SOD and CAT). Melatonin treatment improved redox imbalance with reduced TBARS levels, increased SOD activity, and normalized CAT activity to baseline. Behavior was analyzed with novel object recognition test. Animals exposed to leucine improved object recognition due to melatonin treatment. With the above, we can suggest that melatonin supplementation can protect neurologic oxidative stress, protecting leucine-induced behavior alterations such as memory impairment.

Branched chain amino acids catabolism as a source of new drug targets in pathogenic protists.

Leucine, isoleucine, and valine, collectively termed Branched Chain Amino Acids (BCAA), are hydrophobic amino acids (AAs) and are essential for most eukaryotes since in these organisms they cannot be biosynthesized and must be supplied by the diet. These AAs are structurally relevant for muscle cells and, of course, important for the protein synthesis process. The metabolism of BCAA and its participation in different biological processes in mammals have been relatively well described. However, for other organisms as pathogenic parasites, the literature is really scarce. Here we review the BCAA catabolism, compile evidence on their relevance for pathogenic eukaryotes with special emphasis on kinetoplastids and highlight unique aspects of this underrated pathway.

Genetic and Epigenetic Regulation of the Innate Immune Response to Gout.

Gout is a disease caused by uric acid (UA) accumulation in the joints, causing inflammation. Two UA forms - monosodium urate (MSU) and soluble uric acid (sUA) have been shown to interact physically with inflammasomes, especially with the nod-like receptor (NLR) family pyrin domain containing 3 (NLRP3), albeit the role of the immune response to UA is poorly understood, given that asymptomatic hyperuricemia does also exist. Macrophage phagocytosis of UA activate NLRP3, lead to cytokines release, and ultimately, lead to chemoattract neutrophils and lymphocytes to the gout flare joint spot. Genetic variants of inflammasome genes and of genes encoding their molecular partners may influence hyperuricemia and gout susceptibility, while also influencing other comorbidities such as metabolic syndrome and cardiovascular diseases. In this review, we summarize the inflammatory responses in acute and chronic gout, specifically focusing on innate immune cell mechanisms and genetic and epigenetic characteristics of participating molecules. Unprecedently, a novel UA binding protein - the neuronal apoptosis inhibitor protein (NAIP) - is suggested as responsible for the asymptomatic hyperuricemia paradox.Abbreviation: ß2-integrins: leukocyte-specific adhesion molecules; ABCG2: ATP-binding cassete family/breast cancer-resistant protein; ACR: American college of rheumatology; AIM2: absent in melanoma 2, type of pattern recognition receptor; ALPK1: alpha-protein kinase 1; ANGPTL2: angiopoietin-like protein 2; ASC: apoptosis-associated speck-like protein; BIR: baculovirus inhibitor of apoptosis protein repeat; BIRC1: baculovirus IAP repeat-containing protein 1; BIRC2: baculoviral IAP repeat-containing protein 2; C5a: complement anaphylatoxin; cAMP: cyclic adenosine monophosphate; CARD: caspase activation and recruitment domains; CARD8: caspase recruitment domain-containing protein 8; CASP1: caspase 1; CCL3: chemokine (C-C motif) ligand 3; CD14: cluster of differentiation 14; CD44: cluster of differentiation 44; Cg05102552: DNA-methylation site, usually cytosine followed by guanine nucleotides; contains arbitrary identification code; CIDEC: cell death-inducing DNA fragmentation factor-like effector family; CKD: chronic kidney disease; CNV: copy number variation; CPT1A: carnitine palmitoyl transferase - type 1a; CXCL1: chemokine (CXC motif) ligand 1; DAMPs: damage associated molecular patterns; DC: dendritic cells; DNMT(1): maintenance DNA methyltransferase; eQTL: expression quantitative trait loci; ERK1: extracellular signal-regulated kinase 1; ERK2: extracellular signal-regulated kinase 2; EULAR: European league against rheumatism; GMCSF: granulocyte-macrophage colony-stimulating factor; GWAS: global wide association studies; H3K27me3: tri-methylation at the 27th lysine residue of the histone h3 protein; H3K4me1: mono-methylation at the 4th lysine residue of the histone h3 protein; H3K4me3: tri-methylation at the 4th lysine residue of the histone h3 protein; HOTAIR: human gene located between hoxc11 and hoxc12 on chromosome 12; IκBα: cytoplasmatic protein/Nf-κb transcription inhibitor; IAP: inhibitory apoptosis protein; IFNγ: interferon gamma; IL-1ß: interleukin 1 beta; IL-12: interleukin 12; IL-17: interleukin 17; IL18: interleukin 18; IL1R1: interleukin-1 receptor; IL-1Ra: interleukin-1 receptor antagonist; IL-22: interleukin 22; IL-23: interleukin 23; IL23R: interleukin 23 receptor; IL-33: interleukin 33; IL-6: interleukin 6; IMP: inosine monophosphate; INSIG1: insulin-induced gene 1; JNK1: c-jun n-terminal kinase 1; lncRNA: long non-coding ribonucleic acid; LRR: leucine-rich repeats; miR: mature non-coding microRNAs measuring from 20 to 24 nucleotides, animal origin; miR-1: miR followed by arbitrary identification code; miR-145: miR followed by arbitrary identification code; miR-146a: miR followed by arbitrary identification code, "a" stands for mir family; "a" family presents similar mir sequence to "b" family, but different precursors; miR-20b: miR followed by arbitrary identification code; "b" stands for mir family; "b" family presents similar mir sequence to "a" family, but different precursors; miR-221: miR - followed by arbitrary identification code; miR-221-5p: miR followed by arbitrary identification code; "5p" indicates different mature miRNAs generated from the 5' arm of the pre-miRNA hairpin; miR-223: miR followed by arbitrary identification code; miR-223-3p: mir followed by arbitrary identification code; "3p" indicates different mature miRNAs generated from the 3' arm of the pre-miRNA hairpin; miR-22-3p: miR followed by arbitrary identification code, "3p" indicates different mature miRNAs generated from the 3' arm of the pre-miRNA hairpin; MLKL: mixed lineage kinase domain-like pseudo kinase; MM2P: inductor of m2-macrophage polarization; MSU: monosodium urate; mTOR: mammalian target of rapamycin; MyD88: myeloid differentiation primary response 88; n-3-PUFAs: n-3-polyunsaturated fatty-acids; NACHT: acronym for NAIP (neuronal apoptosis inhibitor protein), C2TA (MHC class 2 transcription activator), HET-E (incompatibility locus protein from podospora anserina) and TP1 (telomerase-associated protein); NAIP: neuronal apoptosis inhibitory protein (human); Naip1: neuronal apoptosis inhibitory protein type 1 (murine); Naip5: neuronal apoptosis inhibitory protein type 5 (murine); Naip6: neuronal apoptosis inhibitory protein type 6 (murine); NBD: nucleotide-binding domain; Nek7: smallest NIMA-related kinase; NET: neutrophil extracellular traps; Nf-κB: nuclear factor kappa-light-chain-enhancer of activated b cells; NFIL3: nuclear-factor, interleukin 3 regulated protein; NIIMA: network of immunity in infection, malignancy, and autoimmunity; NLR: nod-like receptor; NLRA: nod-like receptor NLRA containing acidic domain; NLRB: nod-like receptor NLRA containing BIR domain; NLRC: nod-like receptor NLRA containing CARD domain; NLRC4: nod-like receptor family CARD domain containing 4; NLRP: nod-like receptor NLRA containing PYD domain; NLRP1: nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain containing 1; NLRP12: nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain containing 12; NLRP3: nod-like receptor family pyrin domain containing 3; NOD2: nucleotide-binding oligomerization domain; NRBP1: nuclear receptor-binding protein; Nrf2: nuclear factor erythroid 2-related factor 2; OR: odds ratio; P2X: group of membrane ion channels activated by the binding of extracellular; P2X7: p2x purinoceptor 7 gene; p38: member of the mitogen-activated protein kinase family; PAMPs: pathogen associated molecular patters; PBMC: peripheral blood mononuclear cells; PGGT1B: geranylgeranyl transferase type-1 subunit beta; PHGDH: phosphoglycerate dehydrogenase; PI3-K: phospho-inositol; PPARγ: peroxisome proliferator-activated receptor gamma; PPARGC1B: peroxisome proliferative activated receptor, gamma, coactivator 1 beta; PR3: proteinase 3 antigen; Pro-CASP1: inactive precursor of caspase 1; Pro-IL1ß: inactive precursor of interleukin 1 beta; PRR: pattern recognition receptors; PYD: pyrin domain; RAPTOR: regulatory associated protein of mTOR complex 1; RAS: renin-angiotensin system; REDD1: regulated in DNA damage and development 1; ROS: reactive oxygen species; rs000*G: single nuclear polymorphism, "*G" is related to snp where replaced nucleotide is guanine, usually preceded by an id number; SLC2A9: solute carrier family 2, member 9; SLC7A11: solute carrier family 7, member 11; SMA: smooth muscular atrophy; Smac: second mitochondrial-derived activator of caspases; SNP: single nuclear polymorphism; Sp3: specificity protein 3; ST2: serum stimulation-2; STK11: serine/threonine kinase 11; sUA: soluble uric acid; Syk: spleen tyrosine kinase; TAK1: transforming growth factor beta activated kinase; Th1: type 1 helper T cells; Th17: type 17 helper T cells; Th2: type 2 helper T cells; Th22: type 22 helper T cells; TLR: tool-like receptor; TLR2: toll-like receptor 2; TLR4: toll-like receptor 4; TNFα: tumor necrosis factor alpha; TNFR1: tumor necrosis factor receptor 1; TNFR2: tumor necrosis factor receptor 2; UA: uric acid; UBAP1: ubiquitin associated protein; ULT: urate-lowering therapy; URAT1: urate transporter 1; VDAC1: voltage-dependent anion-selective channel 1.

Structural basis of nirmatrelvir and ensitrelvir activity against naturally occurring polymorphisms of the SARS-CoV-2 main protease.

SARS-CoV-2 is the causative agent of COVID-19. The main viral protease (Mpro) is an attractive target for antivirals. The clinically approved drug nirmatrelvir and the clinical candidate ensitrelvir have so far showed great potential for treatment of viral infection. However, the broad use of antivirals is often associated with resistance generation. Herein, we enzymatically characterized 14 naturally occurring Mpro polymorphisms that are close to the binding site of these antivirals. Nirmatrelvir retained its potency against most polymorphisms tested, while mutants G143S and Q189K were associated with diminished inhibition constants. For ensitrelvir, diminished inhibition constants were observed for polymorphisms M49I, G143S, and R188S, but not for Q189K, suggesting a distinct resistance profile between inhibitors. In addition, the crystal structures of selected polymorphisms revealed interactions that were critical for loss of potency. In conclusion, our data will assist the monitoring of potential resistant strains, support the design of combined therapy, as well as assist the development of the next generation of Mpro inhibitors.

The Aging Process: A Metabolomics Perspective.

Aging process is characterized by a progressive decline of several organic, physiological, and metabolic functions whose precise mechanism remains unclear. Metabolomics allows the identification of several metabolites and may contribute to clarifying the aging-regulated metabolic pathways. We aimed to investigate aging-related serum metabolic changes using a metabolomics approach. Fasting blood serum samples from 138 apparently healthy individuals (20−70 years old, 56% men) were analyzed by Proton Nuclear Magnetic Resonance spectroscopy (1H NMR) and Liquid Chromatography-High-Resolution Mass Spectrometry (LC-HRMS), and for clinical markers. Associations of the metabolic profile with age were explored via Correlations (r); Metabolite Set Enrichment Analysis; Multiple Linear Regression; and Aging Metabolism Breakpoint. The age increase was positively correlated (0.212 ≤ r ≤ 0.370, p < 0.05) with the clinical markers (total cholesterol, HDL, LDL, VLDL, triacylglyceride, and glucose levels); negatively correlated (−0.285 ≤ r ≤ −0.214, p < 0.05) with tryptophan, 3-hydroxyisobutyrate, asparagine, isoleucine, leucine, and valine levels, but positively (0.237 ≤ r ≤ 0.269, p < 0.05) with aspartate and ornithine levels. These metabolites resulted in three enriched pathways: valine, leucine, and isoleucine degradation, urea cycle, and ammonia recycling. Additionally, serum metabolic levels of 3-hydroxyisobutyrate, isoleucine, aspartate, and ornithine explained 27.3% of the age variation, with the aging metabolism breakpoint occurring after the third decade of life. These results indicate that the aging process is potentially associated with reduced serum branched-chain amino acid levels (especially after the third decade of life) and progressively increased levels of serum metabolites indicative of the urea cycle.

RLPredictiOme, a Machine Learning-Derived Method for High-Throughput Prediction of Plant Receptor-like Proteins, Reveals Novel Classes of Transmembrane Receptors.

Cell surface receptors play essential roles in perceiving and processing external and internal signals at the cell surface of plants and animals. The receptor-like protein kinases (RLK) and receptor-like proteins (RLPs), two major classes of proteins with membrane receptor configuration, play a crucial role in plant development and disease defense. Although RLPs and RLKs share a similar single-pass transmembrane configuration, RLPs harbor short divergent C-terminal regions instead of the conserved kinase domain of RLKs. This RLP receptor structural design precludes sequence comparison algorithms from being used for high-throughput predictions of the RLP family in plant genomes, as has been extensively performed for RLK superfamily predictions. Here, we developed the RLPredictiOme, implemented with machine learning models in combination with Bayesian inference, capable of predicting RLP subfamilies in plant genomes. The ML models were simultaneously trained using six types of features, along with three stages to distinguish RLPs from non-RLPs (NRLPs), RLPs from RLKs, and classify new subfamilies of RLPs in plants. The ML models achieved high accuracy, precision, sensitivity, and specificity for predicting RLPs with relatively high probability ranging from 0.79 to 0.99. The prediction of the method was assessed with three datasets, two of which contained leucine-rich repeats (LRR)-RLPs from Arabidopsis and rice, and the last one consisted of the complete set of previously described Arabidopsis RLPs. In these validation tests, more than 90% of known RLPs were correctly predicted via RLPredictiOme. In addition to predicting previously characterized RLPs, RLPredictiOme uncovered new RLP subfamilies in the Arabidopsis genome. These include probable lipid transfer (PLT)-RLP, plastocyanin-like-RLP, ring finger-RLP, glycosyl-hydrolase-RLP, and glycerophosphoryldiester phosphodiesterase (GDPD, GDPDL)-RLP subfamilies, yet to be characterized. Compared to the only Arabidopsis GDPDL-RLK, molecular evolution studies confirmed that the ectodomain of GDPDL-RLPs might have undergone a purifying selection with a predominance of synonymous substitutions. Expression analyses revealed that predicted GDPGL-RLPs display a basal expression level and respond to developmental and biotic signals. The results of these biological assays indicate that these subfamily members have maintained functional domains during evolution and may play relevant roles in development and plant defense. Therefore, RLPredictiOme provides a framework for genome-wide surveys of the RLP superfamily as a foundation to rationalize functional studies of surface receptors and their relationships with different biological processes.

Inflammasome activation and assembly in Huntington's disease.

Huntington's disease (HD) is a rare neurodegenerative disease characterized by motor, cognitive, and psychiatric symptoms. Inflammasomes are multiprotein complexes capable of sensing pathogen-associated and damage-associated molecular patterns, triggering innate immune pathways. Activation of inflammasomes results in a pro-inflammatory cascade involving, among other molecules, caspases and interleukins. NLRP3 (nucleotide-binding domain, leucine-rich-repeat containing family, pyrin domain-containing 3) is the most studied inflammasome complex, and its activation results in caspase-1 mediated cleavage of the pro-interleukins IL-1ß and IL-18 into their mature forms, also inducing a gasdermin D mediated form of pro-inflammatory cell death, i.e. pyroptosis. Accumulating evidence has implicated NLRP3 inflammasome complex in neurodegenerative diseases. The evidence in HD is still scant and mostly derived from pre-clinical studies. This review aims to present the available evidence on NLRP3 inflammasome activation in HD and to discuss whether targeting this innate immune system complex might be a promising therapeutic strategy to alleviate its symptoms.

Exposure to leucine alters glutamate levels and leads to memory and social impairment in zebrafish.

Maple Syrup Urine Disease (MSUD) is a metabolic disorder characterized by high levels in blood and urine of branched-chain amino acids leucine, isoleucine, and valine and their alpha-ketoacids, by a partial or total blockade in the activity of branched-chain complex alpha-keto acids dehydrogenase. The main symptoms in MSUD occur in the central nervous system, including cognitive deficits, locomotor, poor feeding, seizures, psychomotor delay, and mental retardation, but the mechanisms of neurotoxicity and behavior alteration due to this disease are poorly understood, thus this study aimed at showing the effects of leucine exposure on glutamate levels and behavior in zebrafish. For this, we analyzed the behavior using the social preference test and novel object recognition test, moreover, we analyse the glutamate levels and uptake using scintillation and high-performance liquid chromatography methods. Our results demonstrated a decrease in glutamate levels and uptake, accompanied by memory and social impairment. In conclusion, these results suggest that alterations in glutamate levels can be associated with behavior impairment, however, more studies are necessary to understand the mechanisms for brain damage in MSUD.

Pathophysiology of maple syrup urine disease: Focus on the neurotoxic role of the accumulated branched-chain amino acids and branched-chain α-keto acids.

Maple syrup urine disease (MSUD) is an autosomal recessive neurometabolic disorder caused by severe deficiency of branched-chain α-keto acid dehydrogenase complex activity, which catalyzes the oxidative decarboxylation of the branched-chain α-keto acids (BCKA). The metabolic blockage results in tissue accumulation and high urinary excretion of the branched-chain amino acids (BCAA) leucine, isoleucine and valine, as well as alloisoleucine, and their respective BCKA α-ketoisocaproic (α-KIC), α-ketoisovaleric and α-keto-ß-methylvaleric acids. Affected patients usually manifest acute episodes of encephalopathy associated with seizures, coma and life-threatening cerebral edema in the first weeks of life, which is followed by progressive neurological deterioration with motor delay, ataxia, intellectual disability and psychiatric symptoms. The pathophysiology of the brain damage in MSUD has been mainly focused on brain amino acid imbalance leading to deficient cerebral protein and neurotransmitter synthesis. However, the acute episodes of severe neurological symptoms accompanied by large increases of BCKA/BCAA levels suggest neurotoxic actions of these compounds. In this particular, mounting evidence from humans and animal models support an important role of particularly leucine and α-KIC on the pathogenesis of the brain injury in MSUD. In this review we will present the current knowledge of the major mechanisms presumably involved in MSUD neuropathology and highlight the neurotoxic properties of the BCAA and BCKA, disturbing brain bioenergetics and redox homeostasis, besides inducing neuroinflammation. We suggest that these pathomechanisms may contribute to the neurological sequelae of MSUD patients and hopefully allow the design of novel therapeutic strategies, including antioxidant and bioenergetics stimulating drugs targeting the mitochondria.