Computational prediction of phosphorylation sites of SARS-CoV-2 infection using feature fusion and optimization strategies.

SARS-CoV-2's global spread has instigated a critical health and economic emergency, impacting countless individuals. Understanding the virus's phosphorylation sites is vital to unravel the molecular intricacies of the infection and subsequent changes in host cellular processes. Several computational methods have been proposed to identify phosphorylation sites, typically focusing on specific residue (S/T) or Y phosphorylation sites. Unfortunately, current predictive tools perform best on these specific residues and may not extend their efficacy to other residues, emphasizing the urgent need for enhanced methodologies. In this study, we developed a novel predictor that integrated all the residues (STY) phosphorylation sites information. We extracted ten different feature descriptors, primarily derived from composition, evolutionary, and position-specific information, and assessed their discriminative power through five classifiers. Our results indicated that Light Gradient Boosting (LGB) showed superior performance, and five descriptors displayed excellent discriminative capabilities. Subsequently, we identified the top two integrated features have high discriminative capability and trained with LGB to develop the final prediction model, LGB-IPs. The proposed approach shows an excellent performance on 10-fold cross-validation with an ACC, MCC, and AUC values of 0.831, 0.662, 0.907, respectively. Notably, these performances are replicated in the independent evaluation. Consequently, our approach may provide valuable insights into the phosphorylation mechanisms in SARS-CoV-2 infection for biomedical researchers.

UHRF1 poly-auto-ubiquitination induced by the anti-cancer drug, thymoquinone, is involved in the DNA repair machinery recruitment.

DNA methylation is one of the most important epigenetic mark involved in many physiologic cellular processes and pathologies. During mitosis, the transmission of DNA methylation patterns from a mother to the daughter cells is ensured through the action of the Ubiquitin-like, containing PHD and RING domains, 1/DNA methyltransferase 1 (UHRF1/DNMT1) tandem. UHRF1 is involved in the silencing of many tumor suppressor genes (TSGs) via mechanisms that remain largely to be deciphered. The present study investigated the role and the regulation of UHRF1 poly-ubiquitination induced by thymoquinone, a natural anti-cancer drug, known to enhance or re-activate the expression of TSGs. We found that the auto-ubiquitination of UHRF1, induced by TQ, is mediated by reactive oxygen species, and occurs following DNA damage. We demonstrated that the poly-ubiquitinated form of UHRF1 is K63-linked and can still silence the tumor suppressor gene p16INK4A/CDKN2A. We further showed that TQ-induced auto-ubiquitination is mediated via the activity of Tip60. Since this latter is known as a nuclear receptor co-factor, we investigated if the glucocorticoid receptor (GR) might be involved in the regulation of UHRF1 ubiquitination. Activation of the GR, with dexamethasone, did not influence auto-ubiquitination of UHRF1. However, we could observe that TQ induced a K48-linked poly-ubiquitination of GR, probably involved in the proteosomal degradation pathway. Mass-spectrometry analysis of FLAG-HA-tagged UHRF1 identified UHRF1 partners involved in DNA repair and showed that TQ increased their association with UHRF1, suggesting that poly-ubiquitination of UHRF1 is involved in the DNA repair process. We propose that poly-ubiquitination of UHRF1 serves as a scaffold to recruit the DNA repair machinery at DNA damage sites.

Synthesis of biocomposites from microalgal peptide incorporated polycaprolactone/ κ- carrageenan nanofibers and their antibacterial and wound healing property.

Antimicrobial peptides (AMPs) are promising novel agents for targeting a wide range of pathogens. In this study, microalgal peptides derived from native microalgae were incorporated into polycaprolactone (PCL) with ƙ-Carrageenan (ƙ-C) forming nanofibers using the electrospinning method. The peptides incorporated in the nanofibers were characterized by fourier infrared spectroscopy, thermogravimetric analysis, scanning electron microscopy (SEM), and contact angle measurement. The results showed that peptides with molecular weights < 10 kDa, when loaded into nanofibers, exhibited lower wettability. The SEM analysis revealed a thin, smooth, interconnected bead-like structures. The antimicrobial activity of the electrospun nanofibers was evaluated through disc diffusion, and minimum inhibitory activity against Escherichia coli (MTTC 443), and Staphylococcus aureus (MTTC 96), resulting in zones of inhibition of 24 ± 0.5 mm and 14 ± 0.5 mm, respectively. The in vitro biocompatibility of the synthesized nanofibers was confirmed using in HEK 293 cell lines with an increased cell viability. Interestingly, the fibers also exhibited a significant wound-healing properties when used in vitro scratch assays. In conclusion, algal peptides incorporated with PCL/ ƙ-C were found to exhibit antimicrobial and biocompatible biomaterials for wound healing applications.

Identification and Characterization of HIRIP3 as a Histone H2A Chaperone.

HIRIP3 is a mammalian protein homologous to the yeast H2A.Z deposition chaperone Chz1. However, the structural basis underlying Chz's binding preference for H2A.Z over H2A, as well as the mechanism through which Chz1 modulates histone deposition or replacement, remains enigmatic. In this study, we aimed to characterize the function of HIRIP3 and to identify its interacting partners in HeLa cells. Our findings reveal that HIRIP3 is specifically associated in vivo with H2A-H2B dimers and CK2 kinase. While bacterially expressed HIRIP3 exhibited a similar binding affinity towards H2A and H2A.Z, the associated CK2 kinase showed a notable preference for H2A phosphorylation at serine 1. The recombinant HIRIP3 physically interacted with the H2A αC helix through an extended CHZ domain and played a crucial role in depositing the canonical core histones onto naked DNA. Our results demonstrate that mammalian HIRIP3 acts as an H2A histone chaperone, assisting in its selective phosphorylation by Ck2 kinase at serine 1 and facilitating its deposition onto chromatin.

MBD4 loss results in global reactivation of promoters and retroelements with low methylated CpG density.

BACKGROUND: Inherited defects in the base-excision repair gene MBD4 predispose individuals to adenomatous polyposis and colorectal cancer, which is characterized by an accumulation of C > T transitions resulting from spontaneous deamination of 5'-methylcytosine. METHODS: Here, we have investigated the potential role of MBD4 in regulating DNA methylation levels using genome-wide transcriptome and methylome analyses. Additionally, we have elucidated its function through a series of in vitro experiments. RESULTS: Here we show that the protein MBD4 is required for DNA methylation maintenance and G/T mismatch repair. Transcriptome and methylome analyses reveal a genome-wide hypomethylation of promoters, gene bodies and repetitive elements in the absence of MBD4 in vivo. Methylation mark loss is accompanied by a broad transcriptional derepression phenotype affecting promoters and retroelements with low methylated CpG density. MBD4 in vivo forms a complex with the mismatch repair proteins (MMR), which exhibits high bi-functional glycosylase/AP-lyase endonuclease specific activity towards methylated DNA substrates containing a G/T mismatch. Experiments using recombinant proteins reveal that the association of MBD4 with the MMR protein MLH1 is required for this activity. CONCLUSIONS: Our data identify MBD4 as an enzyme specifically designed to repair deaminated 5-methylcytosines and underscores its critical role in safeguarding against methylation damage. Furthermore, it illustrates how MBD4 functions in normal and pathological conditions.

Assessing Spectral Analysis of Phytoconstituents and Their In Silico Interactions with Target Proteins in Plant Seed Extracts.

The pharmacological and preventive attributes of extracts from vegetable seeds have garnered widespread recognition within the scientific community. This study systematically assessed the in vitro antibacterial, antioxidant, and anti-breast cancer properties of phytochemicals present in various solvent-based vegetable seed extracts. We also conducted molecular docking simulations to ascertain their interactions with specific target proteins. Besides, nine distinct chemical constituents were identified using gas chromatography-mass spectrometry (GCMS). Remarkably, the ethyl acetate extract exhibited robust inhibitory effects against Gram-positive and Gram-negative bacterial strains. Furthermore, its capacity for 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging was found to be noteworthy, with an IC50 value of 550.82 ± 1.7 µg/mL, representing a scavenging efficiency of 64.1 ± 2.8%. Additionally, the ethyl acetate extract demonstrated significant hydrogen peroxide (H2O2) scavenging activity, with a maximal scavenging rate of 44.1 ± 1.70% (IC50) at a concentration of 761.17 ± 1.8 µg/mL. Intriguingly, in vitro cytotoxicity assays against human breast cancer (MCF-7) cells revealed varying levels of cell viability at different extract concentrations, suggesting potential anticancer properties. Importantly, these ethyl acetate extracts did not display toxicity to L929 cells across the concentration range tested. Subsequently, we conducted in-silico molecular docking experiments utilizing Discovery Studio 4.0 against the c-Met kinase protein (hepatocyte growth factor; PDB ID: 1N0W). Among the various compounds assessed, 3,4-Dihydroxy-1,6-bis-(3-methoxy-phenyl)-hexa-2,4-diene-1,6-dione exhibited a notable binding energy of -9.1 kcal/mol, warranting further investigation into its potential anticancer properties, clinical applications, and broader pharmacological characteristics.

Pr3+ Ion-Substituted Ni-Co Nano-Spinel Ferrites: Their Synthesis, Characterization, and Biocompatibility for Colorectal Cancer and Candidaemia.

Nanotherapeutics have attracted tremendous research interest in the modern pharmaceutical and biomedical industries due to their potential for drug development, targeted delivery, and therapeutic applications. Therefore, the current study underpins the synthesis of praseodymium ion (Pr3+)-substituted Ni0.5Co0.5Fe2O4 nano-spinel ferrites, (Co0.5Ni0.5PrxFe2-xO4 (0.0 ≤ x ≤ 0.10) NSFs, CoNiPr (x ≤ 0.10) NSFs) via the sonochemical route for its application as a nanotherapeutic treatment option. The synthesized nanomaterial was characterized using various analytical techniques, including scanning/transmission electron microscopy (SEM) and X-ray powder diffractometry (XRD). After substitution with Pr (x = 0.08), the particle size, polydispersity index, and zeta potential analysis indicated an increase in hydrodynamic diameter, with an average zeta potential value of -10.2 mV. The investigation of CoNiPr (x ≤ 0.10) NSFs on colorectal cancer (HCT-116) cells demonstrated a significant effect on cancer cell viability. The inhibitory concentration (IC50) of CoNiPr (x ≤ 0.10) NSFs was between 46 ± 0.91 and 288 ± 8.21 for HCT-116 cells. The effect of CoNiPr (x ≤ 0.10) NSFs on normal human embryonic kidney (HEK-293) cells showed a reduction in the HEK-293 cell viability; however, the cell viability was better than HCT-116. The NSFs treatment also showed morphological changes in cancer cell nuclei, as revealed by DAPI (4',6-diamidino-2-phenylindole), nuclear disintegration, and chromatic fragmentation, which are signs of apoptosis or programmed cell death. To examine the potential antifungal effects of CoNiPr NSFs on Candida albicans, known to cause candidemia among cancer patients, the viability of the cells was assessed post treatment with CoNiPr (x ≤ 0.10) NSFs. The increasing ratio of dopant had a moderate impact on the percentage of cell viability loss of 42, 44, and 43% with x = 0.06, 0.08, and 0.10, respectively. These results reinforce that increased dopant significantly impacts the antifungal properties of the synthesized nanomaterial. These findings support the idea that NSFs might be useful in pharmaceuticals.

Thermodynamic Insights of the Molecular Interactions of Dopamine (Neurotransmitter) with Anionic Surfactant in Non-Aqueous Media.

This study was aimed at establishing the interactions prevailing in an anionic surfactant, sodium dodecyl sulfate, and dopamine hydrochloride in an alcoholic (ethanol) media by using volumetric, conductometric, and tensiometric techniques. Various methods were utilized to estimate the critical micelle concentration (cmc) values at different temperatures. The entire methods yielded the same cmc values. The corresponding thermodynamic parameters viz. the standard free energy of micellization (Gmico), enthalpy of micellization (Hmico), and entropy of micellization (Smico) were predicted by applying the pseudo-phase separation model. The experimental density data at different temperatures (298.15 K, 303.15 K, 308.15 K, and 313.15 K) were utilized to estimate the apparent molar volumes (Vϕo) at an infinite dilution, apparent molar volumes (Vφcmc) at the critical micelle concentration, and apparent molar volumes (ΔVφm) upon micellization. Various micellar and interfacial parameters, for example, the surface excess concentration (Γmax), standard Gibbs free energy of adsorption at the interface (ΔGoad), and the minimum surface area per molecule (Amin), were appraised using the surface tension data. The results were used to interpret the intermolecular interactions prevailing in the mixed systems under the specified experimental conditions.

Nano-sized warriors: zinc chromium vanadate nanoparticles as a dual solution for eradicating waterborne enterobacteriaceae and fighting cancer.

The revolution of biomedical applications has opened new avenues for nanotechnology. Zinc Chromium vanadate nanoparticles (VCrZnO4 NPs) have emerged as an up-and-coming candidate, with their exceptional physical and chemical properties setting them apart. In this study, a one-pot solvothermal method was employed to synthesize VCrZnO4 NPs, followed by a comprehensive structural and morphological analysis using a variety of techniques, including X-Ray diffraction, scanning electron microscopy, high-resolution transmission electron microscopy, Energy-dispersive X-ray, and X-ray photoelectron spectroscopy. These techniques confirmed the crystallinity of the NPs. The VCrZnO4 NPs were tested for their antibacterial activity against primary contaminants such as Enterobacteriaceae, including Shigella flexneri, Salmonella cholerasis, and Escherichia coli, commonly found in hospital settings, using the broth dilution technique. The results indicated a stronger antibacterial activity of VCrZnO4 NPs against Shigella and Salmonella than E. coli. Electron microscopy showed that the NPs caused severe damage to the bacterial cell wall and membrane, leading to cell death. In addition, the study evaluated the anticancer activities of the metal complexes in vitro using colorectal cancer cells (HCT-116) and cervical cancer cells (HELA), along with non-cancer cells and human embryonic kidney cells (HEK-293). A vanadium complex demonstrated efficient anticancer effects with half-inhibitory concentrations (IC50) of 38.50+3.50 g/mL for HCT-116 cells and 42.25+4.15 g/mL for HELA cells. This study highlights the potential of Zinc Chromium vanadate nanoparticles as promising candidates for antibacterial and anticancer applications. Various advanced characterization techniques were used to analyze the properties of nanomaterials, which may help develop more effective and safer antibacterial and anticancer agents in the future.

Nanocellulose-Based Passivated-Carbon Quantum Dots (P-CQDs) for Antimicrobial Applications: A Practical Review.

Passivated-carbon quantum dots (P-CQDs) have been attracting great interest as an antimicrobial therapy tool due to their bright fluorescence, lack of toxicity, eco-friendly nature, simple synthetic schemes, and possession of photocatalytic functions comparable to those present in traditional nanometric semiconductors. Besides synthetic precursors, CQDs can be synthesized from a plethora of natural resources including microcrystalline cellulose (MCC) and nanocrystalline cellulose (NCC). Converting MCC into NCC is performed chemically via the top-down route, while synthesizing CODs from NCC can be performed via the bottom-up route. Due to the good surface charge status with the NCC precursor, we focused in this review on synthesizing CQDs from nanocelluloses (MCC and NCC) since they could become a potential source for fabricating carbon quantum dots that are affected by pyrolysis temperature. There are several P-CQDs synthesized with a wide spectrum of featured properties, namely functionalized carbon quantum dots (F-CQDs) and passivated carbon quantum dots (P-CQDs). There are two different important P-CQDs, namely 2,2'-ethylenedioxy-bis-ethylamine (EDA-CQDs) and 3-ethoxypropylamine (EPA-CQDs), that have achieved desirable results in the antiviral therapy field. Since NoV is the most common dangerous cause of nonbacterial, acute gastroenteritis outbreaks worldwide, this review deals with NoV in detail. The surficial charge status (SCS) of the P-CQDs plays an important role in their interactions with NoVs. The EDA-CQDs were found to be more effective than EPA-CQDs in inhibiting the NoV binding. This difference may be attributed to their SCS as well as the virus surface. EDA-CQDs with surficial terminal amino (-NH2) groups are positively charged at physiological pH (-NH3+), whereas EPA-CQDs with surficial terminal methyl groups (-CH3) are not charged. Since the NoV particles are negatively charged, they are attracted to the positively charged EDA-CQDs, resulting in enhancing the P-CQDs concentration around the virus particles. The carbon nanotubes (CNTs) were found to be comparable to the P-CQDs in the non-specific binding with NoV capsid proteins, through complementary charges, π-π stacking, and/or hydrophobic interactions.

Limosilactobacillus fermentum KAU0021 Abrogates Mono- and Polymicrobial Biofilms Formed by Candida albicans and Staphylococcus aureus.

Candida albicans and Staphylococcus aureus, representing two different kingdoms, are the most frequently isolated pathogens from invasive infections. Their pathogenic attributes, combined with drug resistance, make them a major threat and a challenge to successful treatments, mainly when involved in polymicrobial biofilm-associated infections. In the present study, we investigated the antimicrobial potential of Lactobacillus metabolite extracts (LMEs) purified from cell-free supernatant of four Lactobacillus strains (KAU007, KAU0010, KAU0021, and Pro-65). Furthermore, LME obtained from the strain KAU0021 (LMEKAU0021), being the most effective, was analyzed for its anti-biofilm property against mono- and polymicrobial biofilms formed by C. albicans and S. aureus. The impact of LMEKAU0021 on membrane integrity in single and mixed culture conditions was also evaluated using propidium iodide. The MIC values recorded for LMEKAU0021 was 406 µg/mL, 203 µg/mL, and 406 µg/mL against planktonic cells of C. albicans SC5314, S. aureus and polymicrobial culture, respectively. The LMEKAU0021 at sub-MIC values potentially abrogates both biofilm formation as well as 24 h mature mono- and polymicrobial biofilms. These results were further validated using different microscopy and viability assays. For insight mechanism, LMEKAU0021 displayed a strong impact on cell membrane integrity of both pathogens in single and mixed conditions. A hemolytic assay using horse blood cells at different concentrations of LMEKAU0021 confirmed the safety of this extract. The results from this study correlate the antimicrobial and anti-biofilm properties of lactobacilli against bacterial and fungal pathogens in different conditions. Further in vitro and in vivo studies determining these effects will support the aim of discovering an alternative strategy for combating serious polymicrobial infections caused by C. albicans and S. aureus.

Lactiplantibacillus plantarum KAU007 Extract Modulates Critical Virulence Attributes and Biofilm Formation in Sinusitis Causing Streptococcus pyogenes.

Streptococcus pyogenes is one of the most common bacteria causing sinusitis in children and adult patients. Probiotics are known to cause antagonistic effects on S. pyogenes growth and biofilm formation. In the present study, we demonstrated the anti-biofilm and anti-virulence properties of Lactiplantibacillus plantarum KAU007 against S. pyogenes ATCC 8668. The antibacterial potential of L. plantarum KAU007 metabolite extract (LME) purified from the cell-free supernatant of L. plantarum KAU007 was evaluated in terms of minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC). LME was further analyzed for its anti-biofilm potential using crystal violet assay and microscopic examination. Furthermore, the effect of LME was tested on the important virulence attributes of S. pyogenes, such as secreted protease production, hemolysis, extracellular polymeric substance production, and cell surface hydrophobicity. Additionally, the impact of LME on the expression of genes associated with biofilm formation and virulence attributes was analyzed using qPCR. The results revealed that LME significantly inhibited the growth and survival of S. pyogenes at a low concentration (MIC, 9.76 µg/mL; MBC, 39.06 µg/mL). Furthermore, LME inhibited biofilm formation and mitigated the production of extracellular polymeric substance at a concentration of 4.88 µg/mL in S. pyogenes. The results obtained from qPCR and biochemical assays advocated that LME suppresses the expression of various critical virulence-associated genes, which correspondingly affect various pathogenicity markers and were responsible for the impairment of virulence and biofilm formation in S. pyogenes. The non-hemolytic nature of LME and its anti-biofilm and anti-virulence properties against S. pyogenes invoke further investigation to study the role of LME as an antibacterial agent to combat streptococcal infections.

The Inhibition of SARS-CoV-2 and the Modulation of Inflammatory Responses by the Extract of Lactobacillus sakei Probio65.

In the three years since the first outbreak of COVID-19 in 2019, the SARS-CoV-2 virus has continued to be prevalent in our community. It is believed that the virus will remain present, and be transmitted at a predictable rate, turning endemic. A major challenge that leads to this is the constant yet rapid mutation of the virus, which has rendered vaccination and current treatments less effective. In this study, the Lactobacillus sakei Probio65 extract (P65-CFS) was tested for its safety and efficacy in inhibiting SARS-CoV-2 replication. Viral load quantification by RT-PCR showed that the P65-CFS inhibited SARS-CoV-2 replication in human embryonic kidney (HEK) 293 cells in a dose-dependent manner, with 150 mg/mL being the most effective concentration (60.16% replication inhibition) (p < 0.05). No cytotoxicity was inflicted on the HEK 293 cells, human corneal epithelial (HCE) cells, or human cervical (HeLa) cells, as confirmed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The P65-CFS (150 mg/mL) also reduced 83.40% of reactive oxidizing species (ROS) and extracellular signal-regulated kinases (ERK) phosphorylation in virus-infected cells, both of which function as important biomarkers for the pathogenesis of SARS-CoV-2. Furthermore, inflammatory markers, including interferon-α (IFN-α), IFN-ß, and interleukin-6 (IL-6), were all downregulated by P65-CFS in virus-infected cells as compared to the untreated control (p < 0.05). It was conclusively found that L. sakei Probio65 showed notable therapeutic efficacy in vitro by controlling not only viral multiplication but also pathogenicity; this finding suggests its potential to prevent severe COVID-19 and shorten the duration of infectiousness, thus proving useful as an adjuvant along with the currently available treatments.

Characterization of Defensin-like Protein 1 for Its Anti-Biofilm and Anti-Virulence Properties for the Development of Novel Antifungal Drug against Candida auris.

Candida auris has emerged as a pan-resistant pathogenic yeast among immunocompromised patients worldwide. As this pathogen is involved in biofilm-associated infections with serious medical manifestations due to the collective expression of pathogenic attributes and factors associated with drug resistance, successful treatment becomes a major concern. In the present study, we investigated the candidicidal activity of a plant defensin peptide named defensin-like protein 1 (D-lp1) against twenty-five clinical strains of C. auris. Furthermore, following the standard protocols, the D-lp1 was analyzed for its anti-biofilm and anti-virulence properties. The impact of these peptides on membrane integrity was also evaluated. For cytotoxicity determination, a hemolytic assay was conducted using horse blood. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values ranged from 0.047-0.78 mg/mL and 0.095-1.56 mg/mL, respectively. D-lp1 at sub-inhibitory concentrations potentially abrogated both biofilm formation and 24-h mature biofilms. Similarly, the peptide severely impacted virulence attributes in the clinical strain of C. auris. For the insight mechanism, D-lp1 displayed a strong impact on the cell membrane integrity of the test pathogen. It is important to note that D-lp1 at sub-inhibitory concentrations displayed minimal hemolytic activity against horse blood cells. Therefore, it is highly useful to correlate the anti-Candida property of D-lp1 along with anti-biofilm and anti-virulent properties against C. auris, with the aim of discovering an alternative strategy for combating serious biofilm-associated infections caused by C. auris.

Evaluation of Lactiplantibacillus plantarum KAU007 against Low-Pathogenic Avian Influenza Virus (H9N2).

Avian influenza A viruses (AIVs) pose a persistent threat to humans owing to their reassortment and antigenic drift properties. Among them is H9N2, a low-pathogenic avian influenza virus first discovered in the non-human host and later found infective to humans with huge pandemic potential. In recent years, antiviral resistance has become an increasing threat to public health. Additionally, vaccination against AIVs is becoming increasingly challenging with little success due to antigenic drift. This has resulted in a growing demand for products that can replace the presently in-use medications and the development of innovative antiviral therapies. In this study, we systematically investigate the antiviral potential of lactic acid bacteria against H9N2. Bacteria that produce lactic acid are commonly used in food processing. In addition, these bacteria are considered more affordable, effective, and safe "nutraceuticals" than other alternative medicines. We tested Lactiplantibacillus plantarum KAU007 against the low-pathogenic avian influenza virus (H9N2). As confirmed by the hemagglutination assay, KAU007 showed potent antiviral activity against H9N2 and vigorous antioxidant activity. The CFCS showed a dose-dependent reduction in the levels of IL-6 and IFN-γ. Thus, KAU007 might be considered a potential H9N2 target-based probiotic.

Characterization of the Secretome of Pathogenic Candida glabrata and Their Effectiveness against Systemic Candidiasis in BALB/c Mice for Vaccine Development.

Infections by non-albicans Candida species have increased drastically in the past few decades. Candida glabrata is one of the most common opportunistic fungal pathogens in immunocompromised individuals, owing to its capability to attach to various human cell types and medical devices and being intrinsically weakly susceptible to azoles. Immunotherapy, including the development of antifungal vaccines, has been recognized as an alternative approach for preventing and treating fungal infections. Secretory proteins play a crucial role in establishing host-pathogen interactions and are also responsible for eliciting an immune response in the host during candidiasis. Therefore, fungal secretomes can provide promising protein candidates for antifungal vaccine development. This study attempts to uncover the presence of immunodominant antigenic proteins in the C. glabrata secretome and delineate their role in various biological processes and their potency in the development of antifungal vaccines. LC-MS/MS results uncovered that C. glabrata secretome consisted of 583 proteins, among which 33 were identified as antigenic proteins. The protection ability of secretory proteins against hematogenously disseminated infection caused by C. glabrata was evaluated in BALB/c mice. After immunization and booster doses, all the animals were challenged with a lethal dose of C. glabrata. All the mice showing signs of distress were sacrificed post-infection, and target organs were collected, followed by histopathology and C. glabrata (CFU/mg) estimation. Our results showed a lower fungal burden in target organs and increased survival in immunized mice compared to the infection control group, thus revealing the immunogenic property of secreted proteins. Thus, identified secretome proteins of C. glabrata have the potential to act as antigenic proteins, which can serve as potential candidates for the development of antifungal vaccines. This study also emphasizes the importance of a mass-spectrometry approach to identifying the antigenic proteins in C. glabrata secretome.

Human ß defensins-1, an antimicrobial peptide, kills Candida glabrata by generating oxidative stress and arresting the cell cycle in G0/G1 phase.

Candida glabrata is the most frequently isolated non-albicans Candida species in clinical samples and is known to develop resistance to commonly used antifungal drugs. Human ß defensins (hBDs) are antimicrobial peptides of immune systems and are active against a broad range of pathogens including Candida species. Herein, the antifungal effect of hBD-1 and its mechanism of action in C. glabrata was studied. The antifungal susceptibility of hBD-1 against C. glabrata was calculated by broth microdilution assay. To study the mechanism of antifungal action, the impact of hBD-1 on cell cycle, expression of oxidative stress enzymes, and membrane disintegration were assessed. The susceptibility results confirmed that hBD-1 possessed the minimum inhibitory concentration of 3.12 µg/mL and prevented the growth and caused yeast cell death to various extents. The peptide at subinhibitory and inhibitory concentrations blocked the cell cycle in C. glabrata in G0/G1 phase and disturbed the activity of primary and secondary antioxidant enzymes. Furthermore, at higher concentrations disruption of membrane integrity was observed. Altogether, hBD-1 showed candidicidal activity against C. glabrata and was able to induce oxidative stress and arrested cell cycle in C. auris and therefore has a potential to be developed as an antifungal drug against C. glabrata.

Triazole Derivatives Target 14α-Demethylase (LDM) Enzyme in Candida albicans Causing Ergosterol Biosynthesis Inhibition.

Candida albicans is the most dominant and prevalent cause of fungal infections in humans. Azoles are considered as first-line drugs for the treatment of these infections. However, their prolonged and insistent use has led to multidrug resistance and treatment failures. To overcome this, modification or derivatization of the azole ring has led to the development of new and effective antifungal molecules. In a previous study, we reported on the development of new triazole-based molecules as potential antifungal agents against Candida auris. In this study, the most potent molecules from the previous study were docked and simulated with lanosterol 14-alpha demethylase enzyme. These compounds were further evaluated for in vitro susceptibility testing against C. albicans. In silico results revealed favorable structural dynamics of the compounds, implying that the compounds would be able to effectively bind to the target enzyme, which was further manifested by the strong interaction of the test compounds with the amino acid residues of the target enzyme. In vitro studies targeting quantification of ergosterol content revealed that pta1 was the most active compound and inhibited ergosterol production by >90% in both drug-susceptible and resistant C. albicans isolates. Furthermore, RT-qPCR results revealed downregulation of ERG11 gene when C. albicans cells were treated with the test compound, which aligns with the decreased ergosterol content. In addition, the active triazole derivatives were also found to be potent inhibitors of biofilm formation. Both in silico and in vitro results indicate that these triazole derivatives have the potential to be taken to the next level of antifungal drug development.

Combination Effect of Novel Bimetallic Ag-Ni Nanoparticles with Fluconazole against Candida albicans.

The increasing frequency of antifungal drug resistance among pathogenic yeast "Candida" has posed an immense global threat to the public healthcare sector. The most notable species of Candida causing most fungal infections is Candida albicans. Furthermore, recent research has revealed that transition and noble metal combinations can have synergistic antimicrobial effects. Therefore, a one-pot seedless biogenic synthesis of Ag-Ni bimetallic nanoparticles (Ag-Ni NPs) using Salvia officinalis aqueous leaf extract is described. Various techniques, such as UV-vis, FTIR, XRD, SEM, EDX, and TGA, were used to validate the production of Ag-Ni NPs. The antifungal susceptibility of Ag-Ni NPs alone and in combination with fluconazole (FLZ) was tested against FLZ-resistant C. albicans isolate. Furthermore, the impacts of these NPs on membrane integrity, drug efflux pumps, and biofilms formation were evaluated. The MIC (1.56 µg/mL) and MFC (3.12 µg/mL) results indicated potent antifungal activity of Ag-Ni NPs against FLZ-resistant C. albicans. Upon combination, synergistic interaction was observed between Ag-Ni NPs and FLZ against C. albicans 5112 with a fractional inhibitory concentration index (FICI) value of 0.31. In-depth studies revealed that Ag-Ni NPs at higher concentrations (3.12 µg/mL) have anti-biofilm properties and disrupt membrane integrity, as demonstrated by scanning electron microscopy results. In comparison, morphological transition was halted at lower concentrations (0.78 µg/mL). From the results of efflux pump assay using rhodamine 6G (R6G), it was evident that Ag-Ni NPs blocks the efflux pumps in the FLZ-resistant C. albicans 5112. Targeting biofilms and efflux pumps using novel drugs will be an alternate approach for combatting the threat of multi-drug resistant (MDR) stains of C. albicans. Therefore, this study supports the usage of Ag-Ni NPs to avert infections caused by drug resistant strains of C. albicans.

CRISPR-based systems for sensitive and rapid on-site COVID-19 diagnostics.

The COVID-19 pandemic has strained healthcare systems. Sensitive, specific, and timely COVID-19 diagnosis is crucial for effective medical intervention and transmission control. RT-PCR is the most sensitive/specific, but requires costly equipment and trained personnel in centralized laboratories, which are inaccessible to resource-limited areas. Antigen rapid tests enable point-of-care (POC) detection but are significantly less sensitive/specific. CRISPR-Cas systems are compatible with isothermal amplification and dipstick readout, enabling sensitive/specific on-site testing. However, improvements in sensitivity and workflow complexity are needed to spur clinical adoption. We outline the mechanisms/strategies of major CRISPR-Cas systems, evaluate their on-site diagnostic capabilities, and discuss future research directions.