Phf5a regulates DNA repair in class switch recombination via p400 and histone H2A variant deposition.

Antibody class switch recombination (CSR) is a locus-specific genomic rearrangement mediated by switch (S) region transcription, activation-induced cytidine deaminase (AID)-induced DNA breaks, and their resolution by non-homologous end joining (NHEJ)-mediated DNA repair. Due to the complex nature of the recombination process, numerous cofactors are intimately involved, making it important to identify rate-limiting factors that impact on DNA breaking and/or repair. Using an siRNA-based loss-of-function screen of genes predicted to encode PHD zinc-finger-motif proteins, we identify the splicing factor Phf5a/Sf3b14b as a novel modulator of the DNA repair step of CSR. Loss of Phf5a severely impairs AID-induced recombination, but does not perturb DNA breaks and somatic hypermutation. Phf5a regulates NHEJ-dependent DNA repair by preserving chromatin integrity to elicit optimal DNA damage response and subsequent recruitment of NHEJ factors at the S region. Phf5a stabilizes the p400 histone chaperone complex at the locus, which in turn promotes deposition of H2A variant such as H2AX and H2A.Z that are critical for the early DNA damage response and NHEJ, respectively. Depletion of Phf5a or p400 blocks the repair of both AID- and I-SceI-induced DNA double-strand breaks, supporting an important contribution of this axis to programmed as well as aberrant recombination.

Anti-biofilm activity of a sophorolipid-amphotericin B niosomal formulation against Candida albicans.

Sophorolipids (SLs) have gained interest in the pharmaceutical industries due to their anti-microbial, anti-adhesive and anti-biofilm properties. In the present study, the production of SL was increased by using low-cost media components. The potential of a SL-based niosomal formulation of amphotericin B (AmB) was determined against biofilm of the opportunistic fungal pathogen Candida albicans. In-house prepared SL-AmB niosomes were characterized by different microscopic techniques. The mean entrapment efficiency of AmB within SL-AmB niosome was 63.20% ± 3.86. The cytotoxicity of SL-AmB on mature C. albicans biofilm was compared with an expensive, marketed drug, viz. phosome (a liposomal formulation of AmB). Fewer hyphae were observed in C. albicans biofilm treated with SL-AmB niosome whereas more budding cells were found in phosome treated biofilm. The present study has established the affordable production of SL and the suitability of this approach for delivery of poorly soluble drugs such as AmB against candidiasis infections.

Rhamnolipids from Pseudomonas aeruginosa strain W10; as antibiofilm/antibiofouling products for metal protection.

Industrial biofouling-problems associated with the accumulation of microorganisms from flowing water and fluids on processing surfaces can cause severe problems. A Pseudomonas aeruginosa strain W10 was isolated from industrial setting and found to produce predominantly di-rhamnolipids (Rha-Rha-C10-C10) with a yield of around 10 g L-1 and a critical micelle concentration (CMC) of 80 mg L-1 . P. aeruginosa W10 rhamnolipids were able to disrupt up to 99% of 48 h pre-formed biofilms of the Gram-positive organisms Bacillus licheniformis CAN55, Staphylococcus capitis SH6, and a mixed culture (strains CAN55, SH6, and W10), under static conditions, at concentrations of 0.1, 0.5, and 1 mg ml-1 on a stainless steel surface commonly used in industrial process pipelines. CFU measurements and LIVE/DEAD BacLight staining confirmed these observations. Furthermore, a purified di-rhamnolipid fraction was found to be responsible for the microbial inhibition of B. licheniformis strain CAN55. This study provides evidence that rhamnolipids may have valuable applications in preventing biofilms and biofouling in industrial plants and, in a wider context, may also apply to metal medical devices.

XLID syndrome gene Med12 promotes Ig isotype switching through chromatin modification and enhancer RNA regulation.

The transcriptional coactivator Med12 regulates gene expression through its kinase module. Here, we show a kinase module-independent function of Med12 in CSR. Med12 is essential for super-enhancer activation by collaborating with p300-Jmjd6/Carm1 coactivator complexes. Med12 loss decreases H3K27 acetylation and eRNA transcription with concomitant impairment of AID-induced DNA breaks, S-S synapse formation, and 3'RR-Eµ interaction. CRISPR-dCas9-mediated enhancer activation reestablishes the epigenomic and transcriptional hallmarks of the super-enhancer and fully restores the Med12 depletion defects. Moreover, 3'RR-derived eRNAs are critical for promoting S region epigenetic regulation, synapse formation, and recruitment of Med12 and AID to the IgH locus. We find that XLID syndrome-associated Med12 mutations are defective in both 3'RR eRNA transcription and CSR, suggesting that B and neuronal cells may have cell-specific super-enhancer dysfunctions. We conclude that Med12 is essential for IgH 3'RR activation/eRNA transcription and plays a central role in AID-induced antibody gene diversification and genomic instability in B cells.

ROS-Mediated Necrosis by Glycolipid Biosurfactants on Lung, Breast, and Skin Melanoma Cells.

Cancer is one of the major leading causes of death worldwide. Designing the new anticancer drugs is remained a challenging task due to ensure complexicity of cancer etiology and continuosly emerging drug resistance. Glycolipid biosurfactants are known to possess various biological activities including antimicrobial, anticancer and antiviral properties. In the present study, we sought to decipher the mechanism of action of the glycolipids (lactonic-sophorolipd, acidic-sophorolipid, glucolipid, and bolalipid) against cancer cells using lung cancer cell line (A549), breast cancer cell line (MDA-MB 231), and mouse skin melanoma cell line (B16F10). Scratch assay and fluorescence microscopy revealed that glycolipids inhibit tumorous cell migration possibly by inhibiting actin filaments. Fluorescence activated cell sorter (FACS) analysis exhibited that lactonic sophorolipid and glucolipid both induced the reactive oxygen species, altered the mitochondrial membrane potential (ΔΨ) and finally led to the cell death by necrosis. Furthermore, combinatorial effect of lactonic-sophorolipd and glucolipid demonstrated synergistic interaction on A549 cell line whereas additive effect on MDA-MB 231 and B16F10 cell lines. Our study has highlighted that lactonic-sophorolipd and glucolipid could be useful for developing new anticancer drugs either alone or in combination.

Sophorolipid exhibits antifungal activity by ROS mediated endoplasmic reticulum stress and mitochondrial dysfunction pathways in Candida albicans.

In the present study, we investigated the mechanism of cell death in C. albicans due to treatment with sophorolipid (SL). SL is an extracellular glycolipid biosurfactant produced by various species of non-pathogenic yeasts and is known to inhibit the growth and biofilm formation of C. albicans. This study revealed that treatment of C. albicans cells with SL increases the ROS production and expression of oxidative stress-related genes significantly (SOD1, CAT1). Increased ROS level within the cells causes ER stress and release of Ca2+ in the cytoplasm and alteration of the mitochondrial membrane potential (MMP). Quantitative real time-polymerase chain reaction (qRT-PCR) data showed that SL also upregulates the Endoplasmic Reticulum (ER) stress marker HAC1. Flow cytometric analysis (AnnexinV/PI) indicated that the cell death may have occurred due to necrosis which was further confirmed by LDH release assay and transmission electron microscopy (TEM). Further experiments with the null mutant Δ hog1 strain of C. albicans SC5314 indicated the activation of the osmotic stress response pathway (HOG-MAPK) and SAP9. This study gave an insight into the mechanism of cell death initiation by glycolipids and indicated that further modification of these molecules can lead to the development of new therapeutic agent against C. albicans.

Inhibitory Effect of Sophorolipid on Candida albicans Biofilm Formation and Hyphal Growth.

Candida albicans causes superficial and life-threatening systemic infections. These are difficult to treat often due to drug resistance, particularly because C. albicans biofilms are inherently resistant to most antifungals. Sophorolipid (SL), a glycolipid biosurfactant, has been shown to have antimicrobial and anticancer properties. In this study, we investigated the effect of SL on C. albicans biofilm formation and preformed biofilms. SL was found to inhibit C. albicans biofilm formation as well as reduce the viability of preformed biofilms. Moreover, SL, when used along with amphotericin B (AmB) or fluconazole (FLZ), was found to act synergistically against biofilm formation and preformed biofilms. Effect of SL on C. albicans biofilm formation was further visualized by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM), which revealed absence of hyphae, typical biofilm architecture and alteration in the morphology of biofilm cells. We also found that SL downregulates the expression of hypha specific genes HWP1, ALS1, ALS3, ECE1 and SAP4, which possibly explains the inhibitory effect of SL on hyphae and biofilm formation.