Several orphan solute carriers functionally identified as organic cation transporters: substrates specificity compared with known cation transporters.

Organic cations comprise a significant part of medically relevant drugs and endogenous substances. Such substances need organic cation transporters (OCT) for efficient transfer via cell membranes. However, the membrane transporters of most natural or synthetic organic cations the membrane transporters are still unknown. To identify these transporters, genes of 10 known OCTs and 18 orphan solute carriers (SLC) were overexpressed in HEK293 cells and characterized concerning their transport activities with a broad spectrum of low molecular weight substances emphasizing organic cations. Several SLC35 transporters and SLC38A10 significantly enhanced the transport of numerous relatively hydrophobic organic cations. Significant organic cation transport activities have been found in gene families classified as transporters of other substance classes. For instance, SLC35G3 and SLC38A10 significantly accelerated the uptake of several cations, such as clonidine, 3,4-methylenedioxymethamphetamine, and nicotine, which are known as substrates of a thus far genetically unidentified proton/organic cation antiporter. The transporters SLC35G4 and SLC35F5 stood out by their significantly increased choline uptake, and several other SLC transported choline together with a broader spectrum of organic cations. Overall, there are many more polyspecific organic cation transporters than previously estimated. Several transporters had one predominant substrate but accepted some other cationic substrates, and others showed no particular preference for one substrate but transported several organic cations. The role of these transporters in biology and drug therapy remains to be elucidated.

Can isometric testing substitute for the one repetition maximum squat test?

When measuring maximum strength, a high accuracy and precision is required to monitor the training adaptations. Based on available reliability parameters, the literature suggests the replacement of the one repetition maximum (1RM) by isometric testing to save testing time. However, from a statistical point of view, correlation coefficients do not provide the required information when aiming to replace one test by another. Therefore, the literature suggests the inclusion of the mean absolute error (MAE), the mean absolute percentage error (MAPE) for agreement analysis. Consequently, to check the replaceability of 1RM testing methods, the current study examined the agreement of isometric and dynamic testing methods in the squat and the isometric mid-thigh pull. While in accordance with the literature, correlations were classified high r = 0.638-0.828 and ICC = 0.630-0.828, the agreement analysis provided MAEs of 175.75-444.17 N and MAPEs of 16.16-57.71% indicating an intolerable high measurement error between isometric and dynamic testing conditions in the squat and isometric mid-thigh pull. In contrast to previous studies, using MAE, MAPE supplemented by CCC and BA analysis highlights the poor agreement between the included strength tests. The recommendation to replace 1RM testing with isometric testing routines in the squat does not provide suitable concordance and is not recommended.

Imperfect symmetry facilitated the evolution of specificity and high-order stoichiometry in vertebrate hemoglobin.

Many proteins form paralogous multimers - molecular complexes in which evolutionarily related proteins are arranged into specific quaternary structures. Little is known about the mechanisms by which they acquired their stoichiometry (the number of total subunits in the complex) and heterospecificity (the preference of subunits for their paralogs rather than other copies of the same protein). Here we use ancestral protein reconstruction and biochemical experiments to study historical increases in stoichiometry and specificity during the evolution of vertebrate hemoglobin (Hb), a α2ß2 heterotetramer that evolved from a homodimeric ancestor after a gene duplication. We show that the mechanisms for this evolutionary transition was simple. One hydrophobic substitution in subunit ß after the gene duplication was sufficient to cause the ancestral dimer to homotetramerize with high affinity across a new interface. During this same interval, a single-residue deletion in subunit α at the older interface conferred specificity for the heterotetrameric form and the trans-orientation of subunits within it. These sudden transitions in stoichiometry and specificity were possible because the interfaces in Hb are isologous - involving the same surface patch on interacting subunits, rotated 180° relative to each other - but the symmetry is slightly imperfect. This architecture amplifies the impacts of individual mutations on stoichiometry and specificity, especially in higher-order complexes, and allows single substitutions to differentially affect heteromeric vs homomeric interactions. Many multimers are isologous, and symmetry in proteins is always imperfect; our findings therefore suggest that elaborate and specific molecular complexes may often evolve via simple genetic and physical mechanisms.

Upregulation of hsa-miR-141-3p promotes uterine cervical carcinoma progression via targeting dual-specificity protein phosphatase 1.

We aimed to explore the aberrant expression status of hsa-miR-141-3p and dual-specificity protein phosphatase 1 (DUSP1) and their relative mechanisms in uterine cervical carcinoma (UCC).Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was conducted to detect the expression of hsa-miR-141-3p. Immunohistochemical (IHC) staining was performed to examine the expression of DUSP1 in UCC. Gene chips and RNA-seq datasets were also obtained to assess the expression level. Integrated standardized mean difference (SMD) was calculated to evaluate the expression status of hsa-miR-141-3p in UCC tissues comprehensively. DUSP1-overexpression and hsa-miR-141-3p-inhibition HeLa cells were established, and CCK-8, transwell, wound healing, cell cycle, and apoptosis assays were implemented. The targets of hsa-miR-141-3p were obtained with online tools, and the combination of hsa-miR-141-3p and DUSP1 was validated via dual-luciferase reporter assay. Single-cell RNA-seq data were analyzed to explore hsa-miR-141-3p and DUSP1 in different cells. An integrated SMD of 1.41 (95% CI[0.45, 2.38], p = 0.0041) with 558 samples revealed the overexpression of hsa-miR-141-3p in UCC tissues. And the pooled SMD of -1.06 (95% CI[-1.45, -0.66], p < 0.0001) with 1,268 samples indicated the downregulation of DUSP1. Inhibition of hsa-miR-141-3p could upregulate DUSP1 expression and suppress invasiveness and metastasis of HeLa cells. Overexpression of DUSP1 could hamper proliferation, invasion, and migration and boost apoptosis and distribution of G1 phase. The dual-luciferase reporter assay validated the combination of hsa-miR-141-3p and DUSP1. Moreover, the targets of hsa-miR-141-3p were mainly enriched in the MAPK signaling pathway and activated in fibroblasts and endothelial cells. The current study illustrated the upregulation of hsa-miR-141-3p and the downregulation of DUSP1 in UCC tissues. Hsa-miR-141-3p could promote UCC progression by targeting DUSP1.

Using biothesiometer, Neuropathy Symptom Score, and Neuropathy Disability Score for the early detection of peripheral neuropathy: A cross-sectional study.

Patients with peripheral neuropathy could have damaged peripheral nerves, which leads to sensory and motor dysfunction. Diabetes, infections, and trauma are the major causes of peripheral neuropathy. Vibratory perception threshold (VPT) tools are commonly used to detect peripheral neuropathy. This study aims to determine the assessment of peripheral neuropathy through the different diagnostic tools in the community in Malaysia. A total number of 1283 participants were recruited from the seven retail pharmacies located in Selangor, Malaysia. The peripheral neuropathy test was conducted based on VPT tools on both feet using the digital biothesiometer. Following that, Neurological Symptom Score (NSS) and Neurological Disability Score (NDS) were taken from the participants to assess the neurological symptoms. Participants had an average age of 40.6 ± 12.9 years and were mostly of Chinese ethnicity (54.1%). The findings show that increasing age was associated with more severe peripheral neuropathy across the various assessment tools, but gender differences were found with the biothesiometer test and ethnicity has severity in the biothesiometer and disability scores. The sensitivity and specificity of the biothesiometer test were 0.63 and 0.84, respectively. The combined tool NSS and NDS had high specificity and a high positive predictive value, suggesting that it could be a reliable indicator of peripheral neuropathy when both scores are elevated. The findings show that the biothesiometer test, NSS, and NDS are considered screening VPT tools for diagnosing peripheral neuropathy. However, further evaluation and diagnostic testing are necessary in cases of a positive test result.

Emerging biomarkers for non-invasive diagnosis and treatment of cancer: a systematic review.

Cancer remains a global health challenge, necessitating continuous advancements in diagnostic and treatment strategies. This review focuses on the utility of non-invasive biomarkers in cancer diagnosis and treatment, their role in early detection, disease monitoring, and personalized therapeutic interventions. Through a systematic review of the literature, we identified 45 relevant studies that highlight the potential of these biomarkers across various cancer types, such as breast, prostate, lung, and colorectal cancers. The non-invasive biomarkers discussed include liquid biopsies, epigenetic markers, non-coding RNAs, exosomal cargo, and metabolites. Notably, liquid biopsies, particularly those based on circulating tumour DNA (ctDNA), have emerged as the most promising method for early, non-invasive cancer detection due to their ability to provide comprehensive genetic and epigenetic information from easily accessible blood samples. This review demonstrates how non-invasive biomarkers can facilitate early cancer detection, accurate subtyping, and tailored treatment strategies, thereby improving patient outcomes. It underscores the transformative potential of non-invasive biomarkers in oncology, highlighting their application for enhancing early detection, survival rates, and treatment precision in cancer care. Systematic review registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42023474749 PROSPERO, identifier CRD42023474749.

Genital and Subjective Sexual Arousal in Androphilic Women and Gynephilic Men in Response to the Copulatory Movements of Different Animal Species.

Research has repeatedly shown marked differences in men's and women's sexual response patterns; genital response in men tends to be elicited by cues that correspond to their sexual preference (preferred gender), while women's genital response is less sensitive to gender cues and more sensitive to the presence and intensity of other sexual cues (e.g., sexual activities). We tested whether the cue of copulatory movement in a general sexual context elicited a genital response in androphilic women but not in gynephilic men. If so, women should react to stimuli depicting not only the non-preferred gender but also other animal species differing in phylogenetic distance to humans. We studied the genital and self-reported arousal of 30 gynephilic men and 28 androphilic women to two sexual videos depicting penetrative human sexual intercourse (female-male and female-female) and nine videos depicting animal copulation. Neither women nor men showed genital or subjective sexual arousal to non-human sexual stimuli. Moreover, both sexes demonstrated a highly cue-specific pattern of arousal. Our results suggest that copulatory movement displayed in non-human species is not a sexual cue that can elicit genital or subjective sexual arousal in humans.

Predictive accuracy of the ASIG algorithm in a prospective systemic sclerosis cohort undergoing annual screening for pulmonary arterial hypertension.

The Australian Scleroderma Interest Group (ASIG) algorithm for screening pulmonary arterial hypertension (PAH) in systemic sclerosis (SSc) requires only respiratory function tests and serum N-terminal pro-brain natriuretic peptide as first-tier tests, and is recommended in international guidelines. In this communication, we present the findings of the application of the ASIG screening algorithm to a Singaporean cohort undergoing prospective annual screening for PAH, which shows a high negative predictive value. The ASIG algorithm may offer an alternative to more complex and costly SSc-PAH screening algorithms.

Metagenome comparison (MC): A new framework for detecting unique/enriched OMUs (operational metagenomic units) derived from whole-genome sequencing reads.

BACKGROUND: Current methods for comparing metagenomes, derived from whole-genome sequencing reads, include top-down metrics or parametric models such as metagenome-diversity, and bottom-up, non-parametric, model-free machine learning approaches like Naïve Bayes for k-mer-profiling. However, both types are limited in their ability to effectively and comprehensively identify and catalogue unique or enriched metagenomic genes, a critical task in comparative metagenomics. This challenge is significant and complex due to its NP-hard nature, which means computational time grows exponentially, or even faster, with the problem size, rendering it impractical for even the fastest supercomputers without heuristic approximation algorithms. METHOD: In this study, we introduce a new framework, MC (Metagenome-Comparison), designed to exhaustively detect and catalogue unique or enriched metagenomic genes (MGs) and their derivatives, including metagenome functional gene clusters (MFGC), or more generally, the operational metagenomic unit (OMU) that can be considered the counterpart of the OTU (operational taxonomic unit) from amplicon sequencing reads. The MC is essentially a heuristic search algorithm guided by pairs of new metrics (termed MG-specificity or OMU-specificity, MG-specificity diversity or OMU-specificity diversity). It is further constrained by statistical significance (P-value) implemented as a pair of statistical tests. RESULTS: We evaluated the MC using large metagenomic datasets related to obesity, diabetes, and IBD, and found that the proportions of unique and enriched metagenomic genes ranged from 0.001% to 0.08 % and 0.08%-0.82 % respectively, and less than 10 % for the MFGC. CONCLUSION: The MC provides a robust method for comparing metagenomes at various scales, from baseline MGs to various function/pathway clusters of metagenomes, collectively termed OMUs.

C-reactive protein and neutrophil-to-lymphocyte ratio as key inflammatory indicators in the diagnosis of cervical necrotizing fasciitis.

BACKGROUND: Cervical necrotizing fasciitis (CNF) is a life-threatening bacterial infection with a diagnostic challenge. Currently, there is insufficient evidence on the diagnostic accuracy of inflammatory indicators in CNF. OBJECTIVE: This study aims to identify key inflammatory indicators and assess their diagnostic accuracy for CNF. METHODS: A diagnostic case-control study was conducted at a tertiary healthcare facility from January 2020 to December 2023. Laboratory data from patients with CNF and non-CNF at admission were evaluated. Key inflammatory indicators were identified through consistent outcomes from multivariable logistic regression and receiver operating characteristic curves analyses. The diagnostic accuracy of these indicators, with the results of combined tests, were calculated. RESULTS: CNF was confirmed in 21 of the 67 patients investigated. C-reactive protein (CRP) and neutrophil-to-lymphocyte ratio (NLR) were identified as key inflammatory indicators, with sensitivities of 0.905 and 0.810, and specificities of 0.870 and 0.913, respectively, at CRP threshold of 165.0 mg/L and NLR of 15.8. Combining CRP and NLR in parallel and serial tests increased sensitivity to 0.952 and specificity to 1.0, respectively. CONCLUSIONS AND SIGNIFICANCE: CRP and NLR have been verified as key inflammatory indicators with satisfactory diagnostic abilities for CNF diagnosis, providing a strong foundation for future studies.

In vitro evolution driven by epistasis reveals alternative cholesterol-specific binding motifs of perfringolysin O.

The crucial molecular factors that shape the interfaces of lipid-binding proteins with their target ligands and surfaces remain unknown due to the complex makeup of biological membranes. Cholesterol, the major modulator of bilayer structure in mammalian cell membranes, is recognised by various proteins, including the well-studied cholesterol-dependent cytolysins (CDCs). Here, we use in vitro evolution to investigate the molecular adaptations that preserve the cholesterol specificity of perfringolysin O (PFO), the prototypical CDC from Clostridium perfringens. We identify variants with altered membrane-binding interfaces whose cholesterol-specific activity exceeds that of the wild-type PFO. These novel variants represent alternative evolutionary outcomes and have mutations at conserved positions that can only accumulate when epistatic constraints are alleviated. Our results improve the current understanding of the biochemical malleability of the surface of a lipid-binding protein.

Crossmodal to unimodal transfer of temporal perceptual learning.

Subsecond temporal processing is crucial for activities requiring precise timing. Here, we investigated perceptual learning of crossmodal (auditory-visual or visual-auditory) temporal interval discrimination (TID) and its impacts on unimodal (visual or auditory) TID performance. The research purpose was to test whether learning is based on a more abstract and conceptual representation of subsecond time, which would predict crossmodal to unimodal learning transfer. The experiments revealed that learning to discriminate a 200-ms crossmodal temporal interval, defined by a pair of visual and auditory stimuli, significantly reduced crossmodal TID thresholds. Moreover, the crossmodal TID training also minimized unimodal TID thresholds with a pair of visual or auditory stimuli at the same interval, even if crossmodal TID thresholds are multiple times higher than unimodal TID thresholds. Subsequent training on unimodal TID failed to reduce unimodal TID thresholds further. These results indicate that learning of high-threshold crossmodal TID tasks can benefit low-threshold unimodal temporal processing, which may be achieved through training-induced improvement of a conceptual representation of subsecond time in the brain.

Sex-specific epigenetic signatures of circulating urate and its increase after BCG vaccination.

Background: Urate concentration and the physiological regulation of urate homeostasis exhibit clear sex differences. DNA methylation has been shown to explain a substantial proportion of serum urate variance, mediate the genetic effect on urate concentration, and co-regulate with cardiometabolic traits. However, whether urate concentration is associated with DNA methylation in a sex-dependent manner is unknown. Additionally, it is worth investigating if urate changes after perturbations, such as vaccination, are associated with DNA methylation in a sex-specific manner. Methods: We investigated the association between DNA methylation and serum urate concentrations in a Dutch cohort of 325 healthy individuals. Urate concentration and DNA methylation were measured before and after Bacillus Calmette-Guérin (BCG) vaccination, used as a perturbation associated with increased gout flares. The association analysis included united, interaction, and sex-stratified analysis. Validation of the identified CpG sites was conducted using three independent cohorts. Results: 215 CpG sites were associated with serum urate in males, while 5 CpG sites were associated with serum urate in females, indicating sex-specific associations. Circulating urate concentrations significantly increased after BCG vaccination, and baseline DNA methylation was associated with differences in urate concentration before and after vaccination in a sex-specific manner. The CpG sites associated with urate concentration in males were enriched in neuro-protection pathways, whereas in females, the urate change-associated CpG sites were related to lipid and glucose metabolism. Conclusion: Our study enhances the understanding of how epigenetic factors contribute to regulating serum urate levels in a sex-specific manner. These insights have significant implications for the diagnosis, prevention, and treatment of various urate-related diseases and highlight the importance of personalized and sex-specific approaches in medicine.

Detection and quantification of Brucella abortus DNA in water buffaloes (bubalus bubalis) using droplet digital polymerase chain reaction.

Brucellosis represents a major public health concern worldwide. Human transmission is mainly due to the consumption of unpasteurized milk and dairy products of infected animals. The gold standard for the diagnosis of Brucella spp in ruminants is the bacterial isolation, but it is time-consuming. Polymerase Chain Reaction (PCR) is a quicker and more sensitive technique than bacterial culture. Droplet digital PCR (ddPCR) is a novel molecular assay showing high sensitivity in samples with low amount of DNA and lower susceptibility to amplification inhibitors. Present study aimed to develop a ddPCR protocol for the detection of Brucella abortus in buffalo tissue samples. The protocol was validated using proficiency test samples for Brucella spp by real time qPCR. Furthermore, 599 tissue samples were examined. Among reference materials, qPCR and ddPCR demonstrated same performance and were able to detect up to 225 CFU/mL. Among field samples, ddPCR showed higher sensitivity (100%), specificity and accuracy of 93.4% and 94.15%, respectively. ddPCR could be considered a promising technique to detect B. abortus in veterinary specimens, frequently characterized by low amount of bacteria, high diversity in matrices and species and poor storage conditions.

Profiling of coronaviral Mpro and enteroviral 3Cpro specificity provides a framework for the development of broad-spectrum antiviral compounds.

The main protease from coronaviruses and the 3C protease from enteroviruses play a crucial role in processing viral polyproteins, making them attractive targets for the development of antiviral agents. In this study, we employed a combinatorial chemistry approach-HyCoSuL-to compare the substrate specificity profiles of the main and 3C proteases from alphacoronaviruses, betacoronaviruses, and enteroviruses. The obtained data demonstrate that coronavirus Mpros exhibit overlapping substrate specificity in all binding pockets, whereas the 3Cpro from enterovirus displays slightly different preferences toward natural and unnatural amino acids at the P4-P2 positions. However, chemical tools such as substrates, inhibitors, and activity-based probes developed for SARS-CoV-2 Mpro can be successfully applied to investigate the activity of the Mpro from other coronaviruses as well as the 3Cpro from enteroviruses. Our study provides a structural framework for the development of broad-spectrum antiviral compounds.

Improving stereoselectivity of phosphotriesterase (PTE) for kinetic resolution of chiral phosphates.

Specific stereoisomer is paramount as it is vital for optimizing drug efficacy and safety. The quest for the isolation of desired stereoisomer of active pharmaceutical ingredients or key intermediates drives innovation in drug synthetic and biocatalytic methods. Chiral phosphoramidate is an important building block for the synthesis of antiviral drugs such as remdesivir and sofosbuvir. Given the clinical potency of the (Sp)-diastereomer of the drugs, an enzyme capable of completely hydrolyzing the (Rp)-diastereomer is needed to achieve the purified diastereomers via biocatalytic reaction. In this study, protein engineering of phosphotriesterase (PTE) was aimed to improve the specificity. Employing rational design and site-directed mutagenesis, we generated a small library comprising 24 variants for activity screening. Notably, W131M and I106A/W131M variants demonstrated successful preparation of pure (Sp)-diastereomer of remdesivir and sofosbuvir precursors within a remarkably short hydrolysis time (<20 min). Our work unveils a promising methodology for producing pure stereoisomeric compounds, utilizing novel biocatalysts to enable the chemoenzymatic synthesis of phosphoramidate nucleoside prodrugs.

Catalytic Mode and Product Specificity of an α-Agarase Reveal Its Direct Catalysis for the Production of Agarooligosaccharides.

Many α-agarases have been characterized and are utilized for producing agarooligosaccharides through the degradation of agar and agarose, which are considered valuable for applications in the food and medicine industries. However, the catalytic mechanism and product transformation process of α-agarase remain unclear, limiting further enzyme engineering for industrial applications. In this study, an α-agarase from Catenovulum maritimus STB14 (Cm-AGA) was employed to degrade agarose oligosaccharides (AGOs) with varying degrees of polymerization (DPs) to investigate the catalytic mechanism of α-agarases. The results demonstrated that Cm-AGA could degrade agarose into agarotetraose and agarohexaose. The reducing ends of agarotetraose and agarohexaose spontaneously release unstable 3,6-anhydro-α-l-galactose molecules, which were further degraded into agarotriose and agaropentose. Cm-AGA cannot act on α-1,3-glucoside bonds in agarotriose, agarotetraose, neoagarobiose, and neoagarotetraose but can act on AGOs with a DP greater than four. The product analysis was further verified by ß-galactosidase hydrolysis, which specifically cleaves the non-reducing glycosidic bond of agarooligosaccharides. Multiple sequence alignment results showed that two conserved residues, Asp994 and Glu1129, were proposed as catalytic residues and were further identified by site-directed mutagenesis. Molecular docking of Cm-AGA with agaroheptose revealed the potential substrate binding mode of the α-agarase. These findings enhance the understanding of Cm-AGA's catalytic mode and could guide enzyme engineering for modulating the production of agarooligosaccharides.

UV-B Radiation Exhibited Tissue-Specific Regulation of Isoflavone Biosynthesis in Soybean Cell Suspension Cultures.

Isoflavones, a class of substances with high biological activity, are abundant in soybeans. This study investigated isoflavone biosynthesis in soybean cell suspension cultures under UV-B radiation. UV-B radiation enhanced the transcription level and activity of key enzymes involved in isoflavone synthesis in cell suspension cultures. As a result, the isoflavone contents significantly increased by 19.80% and 91.21% in hypocotyl and cotyledon suspension cultures compared with the control, respectively. Meanwhile, a significant difference was observed in the composition of isoflavones between soybean hypocotyl and cotyledon suspension cultures. Genistin was only detected in hypocotyl suspension cultures, whereas glycitin, daidzein, and genistein accumulated in cotyledon suspension cultures. Therefore, UV-B radiation exhibited tissue-specific regulation of isoflavone biosynthesis in soybean cell suspension cultures. The combination of suspension cultures and abiotic stress provides a novel technological approach to isoflavone accumulation.

Support Enzyme Loading Influences the Effect of Aldehyde Dextran Modification on the Specificity of Immobilized Ficin for Large Proteins.

It has been reported that the modification of immobilized glyoxyl-ficin with aldehyde dextran can promote steric hindrances that greatly reduce the activity of the immobilized protease against hemoglobin, while the protease still maintained a reasonable level of activity against casein. In this paper, we studied if this effect may be different depending on the amount of ficin loaded on the support. For this purpose, both the moderately loaded and the overloaded glyoxyl-ficin biocatalysts were prepared and modified with aldehyde dextran. While the moderately loaded biocatalyst had a significantly reduced activity, mainly against hemoglobin, the activity of the overloaded biocatalyst was almost maintained. This suggests that aldehyde dextran was able to modify areas of the moderately loaded enzyme that were not available when the enzyme was overloaded. This modification promoted a significant increase in biocatalyst stability for both biocatalysts, but the stability was higher for the overloaded biocatalyst (perhaps due to a combination of inter- and intramolecular crosslinking).