From Glycolysis to Viral Defense: The Multifaceted Impact of Glycolytic Enzymes on Human Immunodeficiency Virus Type 1 Replication.

Viruses require host cells to replicate and proliferate, which indicates that viruses hijack the cellular machinery. Human immunodeficiency virus type 1 (HIV-1) primarily infects CD4-positive T cells, and efficiently uses cellular proteins to replicate. Cells already have proteins that inhibit the replication of the foreign HIV-1, but their function is suppressed by viral proteins. Intriguingly, HIV-1 infection also changes the cellular metabolism to aerobic glycolysis. This phenomenon has been interpreted as a cellular response to maintain homeostasis during viral infection, yet HIV-1 efficiently replicates even in this environment. In this review, we discuss the regulatory role of glycolytic enzymes in viral replication and the impact of aerobic glycolysis on viral infection by introducing various host proteins involved in viral replication. Furthermore, we would like to propose a "glyceraldehyde-3-phosphate dehydrogenase-induced shock (G-shock) and kill strategy" that maximizes the antiviral effect of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to eliminate latently HIV-1-infected cells.

Identification of the Most Immunoreactive Antigens of Candida auris to IgGs from Systemic Infections in Mice.

The delay in making a correct diagnosis of Candida auris causes concern in the healthcare system setting, and immunoproteomics studies are important to identify immunoreactive proteins for new diagnostic strategies. In this study, immunocompetent murine systemic infections caused by non-aggregative and aggregative phenotypes of C. auris and by Candida albicans and Candida haemulonii were carried out, and the obtained sera were used to study their immunoreactivity against C. auris proteins. The results showed higher virulence, in terms of infection signs, weight loss, and histopathological damage, of the non-aggregative isolate. Moreover, C. auris was less virulent than C. albicans but more than C. haemulonii. Regarding the immunoproteomics study, 13 spots recognized by sera from mice infected with both C. auris phenotypes and analyzed by mass spectrometry corresponded to enolase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate mutase. These four proteins were also recognized by sera obtained from human patients with disseminated C. auris infection but not by sera obtained from mice infected with C. albicans or Aspergillus fumigatus. Spot identification data are available via ProteomeXchange with the identifier PXD049077. In conclusion, this study showed that the identified proteins could be potential candidates to be studied as new diagnostic or even therapeutic targets for C. auris.

Glycolysis is reduced in dengue virus 2 infected liver cells.

Infections with dengue virus (DENV) remain a worldwide public health problem. A number of bona fide cellular targets of DENV have been identified including liver cells. Despite the many lines of evidence confirming the involvement of hepatocytes during DENV infection, only a few studies have used proteomic analysis to understand the modulation of the cellular proteome occurring upon DENV infection. We utilized a 2D-gel electrophoresis analysis to identify proteins that were differentially regulated by DENV 2 infection of liver (Hep3B) cells at 12 h post infection (hpi) and at 48 hpi. The analysis identifies 4 proteins differentially expressed at 12 hpi, and 14 differentially regulated at 48 hpi. One candidate protein identified as downregulated at 48 hpi in the proteomic analysis (GAPDH) was validated in western blotting in Hep3B cells, and subsequently in induced pluripotent stem cell (iPSC) derived human hepatocytes. The reduced expression of GAPDH was coupled with an increase in NADH, and a significantly reduced NAD + /NADH ratio, strongly suggesting that glycolysis is down regulated in response to DENV 2 infection. Metformin, a well characterized drug used in the treatment of diabetes mellitus, is an inhibitor of hepatic gluconeogenesis was shown to reduce the level of DENV 2 infection and new virus production. Collectively these results show that although glycolysis is reduced, glucose is still required, possibly for use by the pentose phosphate pathway to generate nucleosides required for viral replication.

The role of GAPDH in the selective toxicity of CNP in melanoma cells.

BACKGROUND: Malignant melanoma is the most aggressive form of skin cancer with a rather poor prognosis. Standard chemotherapy often results in severe side effects on normal (healthy) cells finally being difficult to tolerate for the patients. Shown by us earlier, cerium oxide nanoparticles (CNP, nanoceria) selectively killed A375 melanoma cells while not being cytotoxic at identical concentrations on non-cancerous cells. In conclusion, the redox-active CNP exhibited both prooxidative as well as antioxidative properties. In that context, CNP induced mitochondrial dysfunction in the studied melanoma cells via generation of reactive oxygene species (primarily hydrogen peroxide (H2O2)), but that does not account for 100% of the toxicity. AIM: Cancer cells often show an increased glycolytic rate (Warburg effect), therefore we focused on CNP mediated changes of the glucose metabolism. RESULTS: It has been shown before that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) activity is regulated via oxidation of a cysteine in the active center of the enzyme with a subsequent loss of activity. Upon CNP treatment, formation of cellular lactate and GAPDH activity were significantly lowered. The treatment of melanoma cells and melanocytes with the GAPDH inhibitor heptelidic acid (HA) decreased viability to a much higher extent in the cancer cells than in the studied normal (healthy) cells, highlighting and supporting the important role of GAPDH in cancer cells. CONCLUSION: We identified glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a target protein for CNP mediated thiol oxidation.

Intelligent decision support systems for dementia care: A scoping review.

In the context of dementia care, Artificial Intelligence (AI) powered clinical decision support systems have the potential to enhance diagnosis and management. However, the scope and challenges of applying these technologies remain unclear. This scoping review aims to investigate the current state of AI applications in the development of intelligent decision support systems for dementia care. We conducted a comprehensive scoping review of empirical studies that utilised AI-powered clinical decision support systems in dementia care. The results indicate that AI applications in dementia care primarily focus on diagnosis, with limited attention to other aspects outlined in the World Health Organization (WHO) Global Action Plan on the Public Health Response to Dementia 2017-2025 (GAPD). A trifecta of challenges, encompassing data availability, cost considerations, and AI algorithm performance, emerges as noteworthy barriers in adoption of AI applications in dementia care. To address these challenges and enhance AI reliability, we propose a novel approach: a digital twin-based patient journey model. Future research should address identified gaps in GAPD action areas, navigate data-related obstacles, and explore the implementation of digital twins. Additionally, it is imperative to emphasize that addressing trust and combating the stigma associated with AI in healthcare should be a central focus of future research directions.

Determination of internal controls for quantitative gene expression of Spodoptera litura under microbial pesticide stress.

Quantitative real-time polymerase chain reaction (qRT-PCR) has become a commonly used method for the quantification of gene expression. However, accurate qRT-PCR analysis requires a valid internal reference for data normalization. To determine the valid reference characterized with low expression variability among Spodoptera litura samples after microbial pesticide treatments, nine housekeeping genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), arginine kinase, ubiquitin C, actin-5C (ACT5C), actin, ribosomal protein S13 (RPS13), tubulin, acidic ribosomal protein P0 (RPLP0) and ubiquinol-cytochrome c reductase, were evaluated for their suitability using geNorm, Normfinder, BestKeeper, RefFinder and the comparative delta CT methods in this study. S. litura larvae after direct treatment (larvae were immersed in biopesticides), indirect treatment (larvae were fed with biopesticide immersed artificial diets) and comprehensive treatment (larvae were treated with the first two treatments in sequence), respectively with Metarhizium anisopliae, Empedobacter brevis and Bacillus thuringiensis, were investigated. The results indicated that the best sets of internal references were as follows: RPLP0 and ACT5C for direct treatment conditions; RPLP0 and RPS13 for indirect treatment conditions; RPS13 and GAPDH for comprehensive treatment conditions; RPS13 and RPLP0 for all the samples. These results provide valuable bases for further genetic researches in S. litura.

Identification of suitable reference genes for RT-qPCR studies in human parathyroid tissue glandular cells.

Identifying a proper reference gene allows us to understand fundamental changes in many biological processes. Normalization during gene expression analyses is essential for every tissue/cell type, including parathyroid tissue glandular cells. Quantitative method of gene expression analyses via qRT-PCR method provides the accurate examination of every target gene. There are limited reports to present commonly used reference genes in human parathyroid tissues rather than for glandular cell types. This study aims to determine and compare the most stable to least stable genes for parathyroid tissue cells. 43 human parathyroid tissue obtained from primary and secondary hyperparathyroidism patients and glandular cells isolated enzymatically by the removal of extracellular matrix components. After extraction of the total RNA, cDNA synthesis was performed, then qRT-PCR evaluated 14 candidate reference genes. Stability was determined by RefFinder software (Delta ct, BestKeeper, Genorm, and NormFinder algorithms), and the outcome was evaluated for five groups. Even if assessed with different groups, the most stable genes were RPLP0 and GAPDH, while the CLTC and RNA 18S were the least stable. We have confirmed the comprehensive ranking of the most stable three genes alone with the NormFinder algorithm to understand intergroup variation and found out that RPLP0>GAPDH>PGK1. Lastly, comparisons of relative target gene (GCM2) expression revealed similar expression patterns for the most stable reference genes. The most stable reference gene is recommended for the stages where stability is evaluated using the results of four different approaches using RefFinder. We aspire for this study to assist future research to conduct thorough assessments of appropriate reference genes before engaging in gene expression analyses for parathyroid tissue.

Validation of RNA Extraction Methods and Suitable Reference Genes for Gene Expression Studies in Developing Fetal Human Inner Ear Tissue.

A comprehensive gene expression investigation requires high-quality RNA extraction, in sufficient amounts for real-time quantitative polymerase chain reaction and next-generation sequencing. In this work, we compared different RNA extraction methods and evaluated different reference genes for gene expression studies in the fetal human inner ear. We compared the RNA extracted from formalin-fixed paraffin-embedded tissue with fresh tissue stored at -80 °C in RNAlater solution and validated the expression stability of 12 reference genes (from gestational week 11 to 19). The RNA from fresh tissue in RNAlater resulted in higher amounts and a better quality of RNA than that from the paraffin-embedded tissue. The reference gene evaluation exhibited four stably expressed reference genes (B2M, HPRT1, GAPDH and GUSB). The selected reference genes were then used to examine the effect on the expression outcome of target genes (OTOF and TECTA), which are known to be regulated during inner ear development. The selected reference genes displayed no differences in the expression profile of OTOF and TECTA, which was confirmed by immunostaining. The results underline the importance of the choice of the RNA extraction method and reference genes used in gene expression studies.

Glyceraldehyde-3-Phosphate Dehydrogenase Binds with Spike Protein and Inhibits the Entry of SARS-CoV-2 into Host Cells.

INTRODUCTION: Coronavirus disease 2019 caused by coronavirus-2 (SARS-CoV-2) has emerged as an aggressive viral pandemic. Health care providers confront a challenging task for rapid development of effective strategies to combat this and its long-term after effects. Virus entry into host cells involves interaction between receptor-binding domain (RBD) of spike (S) protein S1 subunit with angiotensin converting enzyme present on host cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a moonlighting enzyme involved in cellular glycolytic energy metabolism and micronutrient homeostasis. It is deployed in various cellular compartments and the extra cellular milieu. Though it is known to moonlight as a component of mammalian innate immune defense machinery, till date its role in viral restriction remains unknown. METHOD: Recombinant S protein, the RBD, and human GAPDH protein were used for solid phase binding assays and biolayer interferometry. Pseudovirus particles expressing four different strain variants of S protein all harboring ZsGreen gene as marker of infection were used for flow cytometry-based infectivity assays. RESULTS: Pseudovirus entry into target cells in culture was significantly inhibited by addition of human GAPDH into the extracellular medium. Binding assays demonstrated that human GAPDH binds to S protein and RBD of SARS-CoV-2 with nanomolar affinity. CONCLUSIONS: Our investigations suggest that this interaction of GAPDH interferes in the viral docking with hACE2 receptors, thereby affecting viral ingress into mammalian cells.

Evaluation of reference genes for gene expression analysis by real-time quantitative PCR (qPCR) in different tissues from mice infected by Ascaris suum.

Human ascariasis is the most prevalent helminth infection, affecting 445 million people worldwide. To better understand the impact of the immune system on the pathophysiology of individuals infected with Ascaris suum, mice have been used as experimental models. The RT-qPCR technique is a critical auxiliary tool of investigation used to quantify mRNA levels. However, proper normalization using reference genes is essential to ensure reliable outcomes to avoid analytical errors and false results. Despite the importance of reference genes for experimental A. suum infection studies, no specific reference genes have been identified yet. Therefore, we conducted a study to assess five potential reference genes (GAPDH, 18s, ACTB, B2M, and HPRT1) in different tissues (liver, lungs, small and large intestines) affected by A. suum larval migration in C57BL/6j mice. Tissue collection was carried out to analyze parasite burden and confirm the presence of larvae during the peak of migration in each tissue. Upon confirmation, we analyzed different genes in the tissues and found no common gene with stable expression. Our results highlight the importance of analyzing different genes and using different software programs to ensure reliable relative expression results. Based on our findings, B2M was ranked as the ideal reference gene for the liver, while 18S was the most stable gene in the lung and small intestine. ACTB, or a combination of ACTB with GAPDH, was deemed suitable as reference genes for the large intestine due to their stable expression and less variation between the control and infected groups. To further demonstrate the impact of using different reference genes, we normalized the expression of a chemokine gene (CXCL9) in all tissues. Significant differences in CXCL9 expression levels were observed between different groups in all tissues except for the large intestine. This underscores the importance of selecting appropriate reference genes to avoid overestimating target gene expression levels and encountering normalization-related issues that can lead to false results. In conclusion, our study highlights the significance of using reliable reference genes for accurate RT-qPCR analysis, especially in the context of A. suum infection studies in different tissues. Proper normalization is crucial to ensure the validity of gene expression data and avoid potential pitfalls in interpreting results.

Analyzing the interaction of Helicobacter pylori GAPDH with host molecules and hemin: Inhibition of hemin binding.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a moonlighting enzyme. Apart from its primary role in the glycolytic pathway, in many bacterial species it is found in the extracellular milieu and also on the bacterial surface. Positioning on the bacterial surface allows the GAPDH molecule to interact with many host molecules such as plasminogen, fibrinogen, fibronectin, laminin and mucin etc. This facilitates the bacterial colonization of the host. Helicobacter pylori is a major human pathogen that causes a number of gastrointestinal infections and is the main cause of gastric cancer. The binding analysis of H. pylori GAPDH (HpGAPDH) with host molecules has not been carried out. Hence, we studied the interaction of HpGAPDH with holo-transferrin, lactoferrin, haemoglobin, fibrinogen, fibronectin, catalase, plasminogen and mucin using biolayer interferometry. Highest and lowest binding affinity was observed with lactoferrin (4.83 ± 0.70 × 10-9 M) and holo-transferrin (4.27 ± 2.39 × 10-5 M). Previous studies established GAPDH as a heme chaperone involved in intracellular heme trafficking and delivery to downstream target proteins. Therefore, to get insights into heme binding, the interaction between HpGAPDH and hemin was analyzed. Hemin binds to HpGAPDH with an affinity of 2.10 µM while the hemin bound HpGAPDH does not exhibit activity. This suggests that hemin most likely binds at the active site of HpGAPDH, prohibiting substrate binding. Blind docking of hemin with HpGAPDH also supports positioning of hemin at the active site. Metal ions were found to inhibit the activity of HpGAPDH, suggesting that it also possibly occupies the substrate binding site. Furthermore, with metal-bound HpGAPDH, hemin binding was not observed, suggesting metal ions act as an inhibitor of hemin binding. Since GAPDH has been identified as a heme chaperone, it will be interesting to analyse the biological consequences of inhibition of heme binding to GAPDH by metal ions.

Identification of the Reference Genes for Relative qRT-PCR Assay in Two Experimental Models of Rabbit and Horse Subcutaneous ASCs.

Obtaining accurate and reliable gene expression results in real-time RT-PCR (qRT-PCR) data analysis requires appropriate normalization by carefully selected reference genes, either a single or a combination of multiple housekeeping genes (HKGs). The optimal reference gene/s for normalization should demonstrate stable expression across varying conditions to diminish potential influences on the results. Despite the extensive database available, research data are lacking regarding the most appropriate HKGs for qRT-PCR data analysis in rabbit and horse adipose-derived stem cells (ASCs). Therefore, in our study, we comprehensively assessed and compared the suitability of some widely used HKGs, employing RefFinder and NormFinder, two extensively acknowledged algorithms for robust data interpretation. The rabbit and horse ASCs were obtained from subcutaneous stromal vascular fraction. ASCs were induced into tri-lineage differentiation, followed by the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) treatment of the adipose-differentiated rabbit ASCs, while horse experimental groups were formed based on adipogenic, osteogenic, and chondrogenic differentiation. At the end of the experiment, the total mRNA was obtained and used for the gene expression evaluation of the observed factors. According to our findings, glyceraldehyde 3-phosphate dehydrogenase was identified as the most appropriate endogenous control gene for rabbit ASCs, while hypoxanthine phosphoribosyltransferase was deemed most suitable for horse ASCs. The obtained results underscore that these housekeeping genes exhibit robust stability across diverse experimental conditions, remaining unaltered by the treatments. In conclusion, the current research can serve as a valuable baseline reference for experiments evaluating gene expression in rabbit and horse ASCs. It highlights the critical consideration of housekeeping gene abundance and stability in qPCR experiments, emphasizing the need for an individualized approach tailored to the specific requirements of the study.

Validation of Endogenous Control Genes by Real-Time Quantitative Reverse Transcriptase Polymerase Chain Reaction for Acute Leukemia Gene Expression Studies.

Reference genes are used as internal reaction controls for gene expression analysis, and for this reason, they are considered reliable and must meet several important criteria. In view of the absence of studies regarding the best reference gene for the analysis of acute leukemia patients, a panel of genes commonly used as endogenous controls was selected from the literature for stability analysis: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Abelson murine leukemia viral oncogene human homolog 1 (ABL), Hypoxanthine phosphoribosyl-transferase 1 (HPRT1), Ribosomal protein lateral stalk subunit P0 (RPLP0), ß-actin (ACTB) and TATA box binding protein (TBP). The stability of candidate reference genes was analyzed according to three statistical methods of assessment, namely, NormFinder, GeNorm and R software (version 4.0.3). From this study's analysis, it was possible to identify that the endogenous set composed of ACTB, ABL, TBP and RPLP0 demonstrated good performances and stable expressions between the analyzed groups. In addition to that, the GAPDH and HPRT genes could not be classified as good reference genes, considering that they presented a high standard deviation and great variability between groups, indicating low stability. Given these findings, this study suggests the main endogenous gene set for use as a control/reference for the gene expression in peripheral blood and bone marrow samples from patients with acute leukemias is composed of the ACTB, ABL, TBP and RPLP0 genes. Researchers may choose two to three of these housekeeping genes to perform data normalization.

Exploration of reference genes for the development of a diagnostic kit on vascular aging in human saliva.

Identifying reliable biomarkers in saliva can be a promising approach to developing a rapid diagnostic kit for detecting vascular aging. This study investigated the most suitable reference gene for polymerase chain reaction (PCR) in saliva that is not affected by vascular aging variables. Whole saliva samples were collected to assess the expression of reference genes: actin beta (ACTB), 18S ribosomal RNA (18S rRNA), beta-2-microglobulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The most abundantly expressed gene was 18S rRNA, and the least expressed gene was GAPDH. Four genes were ranked according to their relative stability, as determined by mathematical algorithms, indicating that ACTB and 18S rRNA were stably expressed as reference genes. 18S rRNA was identified as the most promising reference gene for detecting systemic diseases using saliva from patients with vascular aging in these limited experimental conditions.

Multi-omics analysis reveals GAPDH posttranscriptional regulation of IFN-γ and PHGDH as a metabolic checkpoint of microglia polarization.

A "switch" in the metabolic pattern of microglia is considered to be required to meet the metabolic demands of cell survival and functions. However, how metabolic switches regulate microglial function remains controversial. We found here that exposure to amyloid-ß triggers microglial inflammation accompanied by increasing GAPDH levels. The increase of GAPDH, a glycolysis enzyme, leads to the reduced release of interferon-γ (IFN-γ) from inflammatory microglia. Such alternation is translational and is regulated by the binding of glycolysis enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to IFN-γ mRNA. GAPDH, by engaging/disengaging glycolysis and through influencing IFN-γ expression, regulates microglia functions, including phagocytosis and cytokine production. Phosphoglycerate dehydrogenase (PHGDH), screened from different state microglia by metabolomics combined with METARECON analysis, is a metabolic enzyme adjacent downstream of GAPDH and synthesizes serine on the collateral pathway derived from glycolysis. Polarization of microglial with PHGDH as a metabolic checkpoint can be bidirectionally regulated by adding IL-4 or giving PHGDH inhibitors. Therefore, regulation of metabolic enzymes not only reprograms metabolic patterns, but also manipulates microglia functions. Further study should be performed to explore the mechanism of metabolic checkpoints in human microglia or more in vivo animal experiments, and may expand to the effects of various metabolic substrates or enzyme, such as lipids and amino acids, on the functions of microglia.

Glyceraldehyde 3-Phosphate Dehydrogenase on the Surface of Candida albicans and Nakaseomyces glabratus Cells-A Moonlighting Protein That Binds Human Vitronectin and Plasminogen and Can Adsorb to Pathogenic Fungal Cells via Major Adhesins Als3 and Epa6.

Candida albicans and other closely related pathogenic yeast-like fungi carry on their surface numerous loosely adsorbed "moonlighting proteins"-proteins that play evolutionarily conserved intracellular functions but also appear on the cell surface and exhibit additional functions, e.g., contributing to attachment to host tissues. In the current work, we characterized this "moonlighting" role for glyceraldehyde 3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) of C. albicans and Nakaseomyces glabratus. GAPDH was directly visualized on the cell surface of both species and shown to play a significant part in the total capacity of fungal cells to bind two selected human host proteins-vitronectin and plasminogen. Using purified proteins, both host proteins were found to tightly interact with GAPDH, with dissociation constants in an order of 10-8 M, as determined by bio-layer interferometry and surface plasmon resonance measurements. It was also shown that exogenous GAPDH tightly adheres to the surface of candidal cells, suggesting that the cell surface location of this moonlighting protein may partly result from the readsorption of its soluble form, which may be present at an infection site (e.g., due to release from dying fungal cells). The major dedicated adhesins, covalently bound to the cell wall-agglutinin-like sequence protein 3 (Als3) and epithelial adhesin 6 (Epa6)-were suggested to serve as the docking platforms for GAPDH in C. albicans and N. glabratus, respectively.

Unveiling the functional significance of the 14.3.3 protein: A key player in Paracoccidioides brasiliensis biofilm formation.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidioides spp. The interaction mediated by the presence of adhesins on the fungal surface and receptors in the extracellular matrix of the host, as well as the biofilm formation, is essential in its pathogenesis. Adhesins such as gp43, enolase, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and 14-3-3 have been demonstrated in the Paracoccidioides brasiliensis (Pb18) strain and recognized as necessary in the fungus-host interaction. The Pb 18 strain silenced to 14-3-3 showed changes in morphology, virulence, and adhesion capacity. The study aimed to evaluate the role of adhesin 14-3-3 in P. brasiliensis biofilm formation and the differential expression of genes related to adhesins, comparing planktonic and biofilm forms. The presence of biofilm was also verified in sutures in vitro and in vivo. The silenced strain (Pb14-3-3 aRNA) was compared with the wild type Pb18, determining the differential metabolic activity between the strains by the XTT reduction assay; the biomass by violet crystal and the polysaccharides by safranin, even as morphological differences by microscopic techniques. Differential gene expression for adhesins was also analyzed, comparing the relative expression of these in planktonic and biofilm forms at different times. The results suggested that the silencing of 14-3-3 protein altered the ability to form biofilm and its metabolism. The quantity of biomass was similar in both strains; however, the formation of exopolymeric substances and polysaccharide material was lower in the silenced strain. Our results showed increased expression of enolase, GAPDH, and 14-3-3 genes in the first periods of biofilm formation in the Pb18 strain. In contrast, the silenced strain showed a lower expression of these genes, indicating that gene silencing can influence the expression of other genes and be involved in the biofilm formation of P. brasiliensis. In vitro and in vivo assays using sutures confirmed this yeast's ability to form biofilm and may be implicated in the pathogenesis of paracoccidioidomycosis.

Fluorogenic Hyaluronan Nanogels Track Individual Early Protein Aggregates Originated under Oxidative Stress.

Proteins are broadly versatile biochemical materials, whose functionality is tightly related to their folding state. Native folding can be lost to yield misfolded conformations, often leading to formation of protein oligomers, aggregates, and biomolecular phase condensates. The fluorogenic hyaluronan HA-RB, a nonsulfonated glycosaminoglycan with a combination of polyanionic character and of hydrophobic spots due to rhodamine B dyes, binds to early aggregates of the model protein cytoplasmic glyceraldehyde-3-phosphate dehydrogenase 1 from Arabidopsis thaliana (AtGAPC1) since the very onset of the oligomeric phase, making them brightly fluorescent. This initial step of aggregation has, until now, remained elusive with other fluorescence- or scattering-based techniques. The information gathered from nanotracking (via light-sheet fluorescence microscopy) and from FCS in a confocal microscope converges to highlight the ability of HA-RB to bind protein aggregates from the very early steps of aggregation and with high affinity. Altogether, this fluorescence-based approach allows one to monitor and track individual early AtGAPC1 aggregates in the size range from 10 to 100 nm with high time (∼10-2 s) and space (∼250 nm) resolution.

Structure-based functional analysis of a novel NADPH-producing glyceraldehyde-3-phosphate dehydrogenase from Corynebacterium glutamicum.

Corynebacterium glutamicum is an industrial workhorse applied in the production of valuable biochemicals. In the process of bio-based chemical production, improving cofactor recycling and mitigating cofactor imbalance are considered major solutions for enhancing the production yield and efficiency. Although, glyceraldehyde-3-phosphate dehydrogenase (GapDH), a glycolytic enzyme, can be a promising candidate for a sufficient NADPH cofactor supply, however, most microorganisms have only NAD-dependent GapDHs. In this study, we performed functional characterization and structure determination of novel NADPH-producing GapDH from C. glutamicum (CgGapX). Based on the crystal structure of CgGapX in complex with NADP cofactor, the unique structural features of CgGapX for NADP stabilization were elucidated. Also, N-terminal additional region (Auxiliary domain, AD) appears to have an effect on enzyme stabilization. In addition, through structure-guided enzyme engineering, we developed a CgGapX variant that exhibited 4.3-fold higher kcat, and 1.2-fold higher kcat/KM values when compared with wild-type. Furthermore, a bioinformatic analysis of 100 GapX-like enzymes from 97 microorganisms in the KEGG database revealed that the GapX-like enzymes possess a variety of AD, which seem to determine enzyme stability. Our findings are expected to provide valuable information for supplying NADPH cofactor pools in bio-based value-added chemical production.

Identification and validation of stable reference genes for expression profiling of target genes in diverse ovine tissues.

Quantitative PCR (qPCR) is a widely-used technique for quantifying the expression of target genes across various tissues, as well as under different pathological and physiological conditions. One of the challenges associated with this method is the need to identify optimal reference genes (RGs) that maintain consistent expression levels under diverse experimental settings, thereby ensuring accurate biological interpretation. In this study, we conducted a thorough analysis of 18 candidate RGs (ACTB, BACH1, B2M, GAPDH, HMBS, HPRT1, PGK1, PPIA, PPIB, RPLP0, RPL19, RPS9, RPS15, RPS28, SDHA, TBP, UXT, and YWHAZ) across 10 ovine tissues (muscle, skin, kidney, liver, intestine, rumen, lung, testis, heart, and spleen) obtained from five individual sheep. We aimed to identify genes with stable expression across these tissues. A literature-based survey helped us shortlist candidate genes representing various functional classes from multiple livestock species. We employed four algorithms: geNorm, NormFinder, BestKeeper, and Delta Ct (ΔCt), to rank these genes based on their stability. A consistent trend in the rankings was observed across these different algorithms. RefFinder was then used for a comprehensive ranking, integrating the outputs from the various methods. ACTB, PPIB, BACH1, and B2M emerged as the most stable RGs, while RPS9, RPS15, and PGK1 displayed variable expression. We validated our findings through qPCR analysis of four target genes (ACTN2, CRYAB, DLK1, and TRIM54) in the skin samples from two different sheep breeds. Based on these results, we recommend ACTB, PPIB, BACH1, and B2M as reliable internal control genes for qPCR experiments involving diverse ovine tissues.