RAD51-WSS1-dependent genetic pathways are essential for DNA-protein crosslink repair and pathogenesis in Candida albicans.

Genetic analyses in Saccharomyces cerevisiae suggest that nucleotide excision repair (NER), homologous recombination (HR), and protease-dependent repair pathways coordinately function to remove DNA-protein crosslinks (DPCs) from the genome. DPCs are genomic cytotoxic lesions generated because of the covalent linkage of proteins with DNA. Although NER and HR processes have been studied in pathogenic Candida albicans, their roles in DPC repair (DPCR) are yet to be explored. Proteases like Wss1 and Tdp1 (tyrosyl-DNA phosphodiesterase-1) are known to be involved in DPCR; however, Tdp1 that selectively removes topoisomerase-DNA complexes is intrinsically absent in C. albicans. Therefore, the mechanism of DPCR might have evolved differently in C. albicans. Herein, we investigated the interplay of three genetic pathways and found that RAD51-WSS1-dependent HR and protease-dependent repair pathways are essential for DPC removal, and their absence caused an increased rate of loss of heterozygosity in C. albicans. RAD1 but not RAD2 of NER is critical for DPCR. In addition, we observed truncation of chromosome #6 in the cells defective in both RAD51 and WSS1 genes. While the protease and DNA-binding activities are essential, a direct interaction of Wss1 with the eukaryotic DNA clamp proliferating cell nuclear antigen is not a requisite for the function of Wss1. DPCR-defective C. albicans cells exhibited filamentous morphology, reduced immune cell evasion, and attenuation in virulence. Thus, we concluded that RAD51-WSS1-dependent DPCR pathways are essential for genome stability and candidiasis development. Since no vaccine against candidiasis is available for human use yet, we propose to explore DPCR-defective attenuated strains (rad51ΔΔwss1ΔΔ and rad2ΔΔrad51ΔΔwss1ΔΔ) for whole-cell vaccine development.

A chemically induced attenuated strain of Candida albicans generates robust protective immune responses and prevents systemic candidiasis development.

Despite current antifungal therapy, invasive candidiasis causes >40% mortality in immunocompromised individuals. Therefore, developing an antifungal vaccine is a priority. Here, we could for the first time successfully attenuate the virulence of Candida albicans by treating it with a fungistatic dosage of EDTA and demonstrate it to be a potential live whole cell vaccine by using murine models of systemic candidiasis. EDTA inhibited the growth and biofilm formation of C. albicans. RNA-seq analyses of EDTA-treated cells (CAET) revealed that genes mostly involved in metal homeostasis and ribosome biogenesis were up- and down-regulated, respectively. Consequently, a bulky cell wall with elevated levels of mannan and ß-glucan, and reduced levels of total monosomes and polysomes were observed. CAET was eliminated faster than the untreated strain (Ca) as found by differential fungal burden in the vital organs of the mice. Higher monocytes, granulocytes, and platelet counts were detected in Ca- vs CAET-challenged mice. While hyper-inflammation and immunosuppression caused the killing of Ca-challenged mice, a critical balance of pro- and anti-inflammatory cytokines-mediated immune responses are the likely reasons for the protective immunity in CAET-infected mice.

Immunogenicity and efficacy of CNA25 as a potential whole-cell vaccine against systemic candidiasis.

Disseminated fungal infections account for ~1.5 million deaths per year worldwide, and mortality may increase further due to a rise in the number of immunocompromised individuals and drug-resistance fungal species. Since an approved antifungal vaccine is yet to be available, this study explored the immunogenicity and vaccine efficacy of a DNA polymerase mutant strain of Candida albicans. CNA25 is a pol32ΔΔ strain that exhibits growth defects and does not cause systemic candidiasis in mice. Immunized mice with live CNA25 were fully protected against C. albicans and C. parapsilosis but partially against C. tropicalis and C. glabrata infections. CNA25 induced steady expression of TLR2 and Dectin-1 receptors leading to a faster recognition and clearance by the immune system associated with the activation of protective immune responses mostly mediated by neutrophils, macrophages, NK cells, B cells, and CD4+ and CD8+ T cells. Molecular blockade of Dectin-1, IL-17, IFNγ, and TNFα abolished resistance to reinfection. Altogether, this study suggested that CNA25 collectively activates innate, adaptive, and trained immunity to be a promising live whole-cell vaccine against systemic candidiasis.

Facial nerve electrical motor evoked potential in cerebellopontine angle tumors for its anatomical and functional preservation.

Background: Among the technical measures to preserve facial nerve (FN) function, intraoperative neuromonitoring has become mandatory and is constantly being scrutinized. Hence, to determine the efficacy of FN motor evoked potentials (FNMEPs) in predicting long-term motor FN function following cerebellopontine angle (CPA) tumor surgery, an analysis of cases was done. Methods: In 37 patients who underwent CPA surgery, FNMEPs through corkscrew electrodes positioned at C5-C6 and C6-C5 (C is the central line of the brain as per 10-20 EEG electrode placement) were used to deliver short train stimuli and recorded from the orbicularis oculi, oris, and mentalis muscles. Results: In 58 patients, triggered electromyography (EMG) was able to identify the FN during resection of tumor, but 8 out of these (4.64%) patients developed new facial weakness, whereas 3 out of 38 (1.11%) patients who had intact FN function MEP (decrement of FN target muscles - CMAPs amplitude peak to peak >50-60%), developed new facial weakness (House and Brackmann grade II to III). Conclusion: The FNMEP has significant superiority over triggered EMG when tumor is giant and envelops the FN.

Critical roles of Dpb3-Dpb4 sub-complex of DNA polymerase epsilon in DNA replication, genome stability, and pathogenesis of Candida albicans.

DNA polymerase ε (Polε) is an essential replicative polymerase consisting of Pol2, Dpb2, Dpb3, and Dpb4 subunits and has not been explored in the pathogenic yeast Candida albicans. C. albicans is accountable for >40% of deaths due to systemic candidiasis per year worldwide. Genome plasticity is one of the adaptive mechanisms associated with virulence, and as it is associated with DNA polymerase function, this study explored the role of Polε in genome stability and pathogenesis of C. albicans. POL2 and DPB2 are haploinsufficient, but DPB3 and DPB4 are dispensable for cell survival in diploid C. albicans. However, unlike in Saccharomyces cerevisiae, loss of any or both of the nonessential subunits or defective interaction between the two resulted in slow growth and temperature-sensitive phenotypes. Knockout strains of C. albicans (dpb3ΔΔ and dpb4ΔΔ and dpb3ΔΔdpb4ΔΔ) also exhibited sensitivity to genotoxic agents and delayed cell cycle progression. Reduced processive DNA synthesis and increased rate of mutagenesis were observed in dpb3 and dpb4 null strains. Whole-genome sequencing further confirmed the accumulation of indels and SNPs majorly in the intergenic repeat regions of the chromosomes of dpb3ΔΔdpb4ΔΔ. Polε-defective strains were constitutively filamentous and non-pathogenic in mice models of systemic candidiasis. Altogether, this study showed that the function of the Dpb3-Dpb4 subcomplex is critical for fungal morphogenesis and virulence besides its role as a structural component of Polε in DNA replication and genome stability; thus, their interacting interface may be targeted to develop antifungal drugs. IMPORTANCE: This study explored the role of DNA polymerase epsilon, especially its non-essential structural subunits in Candida albicans biology. Apart from their role in DNA replication and genome stability, the Dpb3-Dpb4 subcomplex regulates morphological switching and virulence. Since the defective strain is locked in filamentous form and is avirulent, the complex may be targeted for anti-fungal drug development.

4-nitroquinoline 1-oxide induces immune cells death to onset early immunosuppression during oral squamous cell carcinoma development.

4-Nitroquinoline N-oxide (4-NQO) and its derivatives react with genomic DNA to form stable quinolone monoadducts, which are highly mutagenic and genotoxic. While the chronic high-dose exposure of epithelial cells to a carcinogen such as 4-NQO leads to tumor development, its effect on other cells has not been explored yet. Since the immunosuppression due to aberrant immunological profile is recognized as a significant cause in tumors, here we determine the interaction between 4-NQO and immune cells both in vivo and in vitro, and its effect on oral squamous cell carcinoma (OSCC) progression in a murine model. Immune cell profiling of the spleen and peripheral blood revealed a significant decrease in the B-cell population in 4-NQO-exposed mice than the untreated group. Additionally, γδ T and CD5+ B lymphocyte populations decreased at both pre- and post-cancerous stages of OSCC. These results suggested that 4-NQO induced tumor transition from pre-malignant lesions to OSCC by altering certain immune cells systemically. Next, to establish the effect of 4-NQO on immune cells, human B- and T-cell lines were subjected to 4-NQO; the reduction in cell viability, increase in DNA damage response marker, and induction of apoptosis were more pronounced in B than T cells. Altogether, our results indicated that in addition to the genotoxicity of oral epithelial cells, 4-NQO potentiates long-range effects on specific immune cells to induce cell death to cause very-early immunosuppressive response during oral carcinogenesis, and thus immunosuppression and tumor development are coevolved.

Vaccines against candidiasis: Status, challenges and emerging opportunity.

Candidiasis is a mycosis caused by opportunistic Candida species. The occurrence of fungal infections has considerably increased in the last few years primarily due to an increase in the number of immune-suppressed individuals. Alarming bloodstream infections due to Candida sp. are associated with a higher rate of morbidity and mortality, and are emerged as major healthcare concerns worldwide. Currently, chemotherapy is the sole available option for combating fungal diseases. Moreover, the emergence of resistance to these limited available anti-fungal drugs has further accentuated the concern and highlighted the need for early detection of fungal infections, identification of novel antifungal drug targets, and development of effective therapeutics and prophylactics. Thus, there is an increasing interest in developing safe and potent immune-based therapeutics to tackle fungal diseases. In this context, vaccine design and its development have a priority. Nonetheless, despite significant advances in immune and vaccine biology over time, a viable commercialized vaccine remains awaited against fungal infections. In this minireview, we enumerate various concerted efforts made till date towards the development of anti-Candida vaccines, an option with pan-fugal vaccine, vaccines in the clinical trial, challenges, and future opportunities.

First report on transferrin in the silkworm, Antheraea mylitta, with a putative role in antioxidant defense: Insights from proteomic analysis and immunodetection.

Transferrin is a highly conserved multifunctional protein involved in iron metabolism, oxidative stress and immune response. However, very little is known about its context-dependent functional role in insects. The present study adopts a proteomic approach to identify an abundant hemolymph protein of silkworm (Antheraea mylitta) as transferrin. SDS-PAGE coupled to LC-MS/MS was followed to identify this protein (75.7 kDa) in the advanced larvae of A. mylitta as transferrin (AmTsf). Antibody developed against this protein enabled us to determine its tissue-specific expression and functional relevance during development. Its immunodetection was optimized by western blotting, immunohisto- and cytochemistry in the larval and pupal tissues of this insect. AmTsf exhibited a considerable homology with that of other related insects. It was found to be expressed constitutively in hemolymph, fat body, midgut and silk gland of this insect during development. Abundance of transferrin in the hemolymph of this species and its upregulation in response to oxidant challenges indicated its tissue-specific role in the antioxidant protection. The present work adds to the existing knowledge of multiple roles of transferrin in insects and provides an insight into a relatively unexplored aspect of iron-associated H2O2 metabolism and redox homeostasis in the apparent absence of catalase and glutathione peroxidase. Therefore, A. mylitta can be an attractive acatalasemic model for the study of in vivo redox regulation. In addition, this study provides a novel hypothesis to explore the role of transferrin in limiting oxidative stress during larval rearing of silkworm.

Curcumin restores hepatic epigenetic changes in propylthiouracil(PTU)Induced hypothyroid male rats: A study on DNMTs, MBDs, GADD45a, C/EBP-ß and PCNA.

6-n-propyl-2-thiouracil (PTU), a thioamide drug, is used as an effective anti-thyroid agent to treat hyperthyroidism and Graves' disease. However, acute liver oxidative damage is an important side effect of the drug. In the present study, we report that PTU administration to rat induces hepatic epigenetic changes by upregulating expression of DNMT1, DNMT3a, DNMT3b, MBD4, MeCP2, p53 and Gadd45a and down-regulation of PCNA and C/EBP-ß. This is accompanied by decrease in the cell population and augmentation of cellular lipid peroxidation, an index of oxidative stress, in liver. On the other hand, co-administration of curcumin, a polyphenol extract from the rhizome of Curcuma longa L, along with PTU ameliorates PTU- induced oxidative stress and epigenetic parameters except for the expression of MBD4. Also, co-administration of curcumin with PTU resulted in restoration of hepatic cell population and histoarchitecture. The protective effect of curcumin to PTU-induced hepatotoxicity is attributed to its antioxidative properties.

Host plant-derived allelochemicals and metal components are associated with oxidative predominance and antioxidant plasticity in the larval tissues of silkworm, Antheraea mylitta: Further evidence of joint effects hypothesis.

Tasar silkworm, Antheraea mylitta is a polyphagous insect that primarily feeds on Terminalia arjuna, Terminalia tomentosa and Shorea robusta. However, larval rearing on S. robusta results in poor performance for the reasons unexplored. Oxidative burden imposed by host plants is presumed to be a determining factor for larval fitness. With this hypothesis we have analyzed the foliar constituents of the respective host plants, the levels of oxidative stress and antioxidant protection in the larval tissues in response to their altered feeding on different host plants for different durations (2 and 10 days). Results indicate that S. robusta leaves contain the highest amount of tannin and redox active metals compared to those of other host plants. Consequently, hemolymph and midgut tissues of the larvae shifted to S. robusta exhibited oxidative predominance. Increased activities of superoxide dismutase, catalase and glutathione S-transferase in the larval tissues indicated an adaptive response to host plant driven oxidative assault. Our in vitro study also strongly supplements the in vivo findings indicating S. robusta foliages as a strong inducer of lipid peroxidation (LPx). Copper and Iron were found to be more potent in inducing LPx in the midgut tissues of the larvae compared to Zinc and Manganese. This study for the first time demonstrates the combined implications of host plant derived allelochemicals and elements on oxidative stress and antioxidant plasticity in this insect. The overall findings also brace up the newly emerging concept on joint effects hypothesis (organic and elemental defence) for enhanced plant defence.

Traumatic Cerrebral Fungus: Experience From an Institution in North East India.

BACKGROUND: Traumatic brain fungus is manifestation of neglected head injury. Although rare it is not uncommon. The patients are usually intact with good Glasgow coma (GCS) score inspite of complex injuries and exposed brain parenchyma but morbidity and mortality is very high with time if no proper and timely management is offered. There is very less study on traumatic brain fungus with no defined management protocols. So an attempt was made to explain in details the surgical strategies and other management techniques in patients with traumatic brain fungus. AIMS: To study and evaluate the pattern of causation, clinical presentations, modalities of management of traumatic brain fungus and outcome after treatment. METHODS: All patients with fungus cerebri, admitted to our centre from January 2012 to December 2015 were studied prospectively. All the patients were examined clinically and triaged urgently for surgery. CT head was done in all patients to look for any brain parenchymal injury. All patients were managed surgically. Outcome was assessed as per the Glassgow Outcome Score. RESULTS: Total 10 patients were included in the study. 8 were men and 2 women. The patients' ages ranged from 3-48 years (mean 31.6 years). The interval between initial injury and protrusion ranged from 3 days to 6 days (mean 4.1 days). Mean GCS at the time of presentation was 13.2.60% of the patients (n = 6) sustained moderate head injury. (GCS-9-13). Size of the fungus ranged from 5cm×3cm to 8cm×10cm. CONCLUSION: Early and proper local wound treatment prevents fungus formation. Pre-emptive antibiotics, AEDs and cerebral decongestants are recommended. Loose water-tight duroplasty prevents CSF leak. But mortality and morbidity can be reduced significantly if brain fungus is managed properly by applying basic surgical principles and antibiotic protocols combined with newer surgical modalities.