CRISPR-Cas9 mediated editing in a dermatophyte via NHEJ repair
Dermatophytes are the fungi, which invade the outer layer of skin and are responsible for athlete’s foot, ringworm, and jock itch – a few examples of fungal skin infections. One of the dermatophytes is Trichophyton rubrum which causes 80-90% of superficial fungal infections. The survivability of T. rubrum during the pH shift it encounters (i.e. from slightly acidic pH of human skin/ nail to alkaline pH) while causing an infection depends on PacC, a major pH regulatory transcription factor. Downregulation of PacC has been shown to reduce the growth of T. rubrum on nails.
Absence of gene manipulation tools due to low homologous recombination in non-model fungal species like T. rubrum makes it difficult to study pathogenesis and molecular mechanisms of infection or design and develop newer treatment strategies. In this study, Bhupesh Taneja’s lab reports a CRISPR-Cas9-based gene editing method for fungi like T. rubrum mediated via NHEJ repair. A dual-plasmid-based CRISPR-Cas9 system harboring GFP in one of the plasmids for screening was developed to target PacC of T. rubrum, thus, opening up future avenues of research for understanding basic mechanisms in such infectious yet poorly understood fungal strains and aid in the development of newer therapeutics for them.
(With inputs from Sanchita Dey)
A dual-plasmid-based CRISPR/Cas9-mediated strategy enables targeted editing of pH regulatory gene pacC in a clinical isolate of Trichophyton rubrum. Dey SS, Ramalingam S, Taneja B. J. Fungi 2022, 8(12), 124
https://www.mdpi.com/2309-608X/8/12/1241
Alu, LINE1 and lncRNAs in COVID-19 breakthrough infections
As SARS CoV2 evolved with time, the ability to escape the immune system even in vaccinated individuals was seen, called vaccination breakthrough infections. In this study, Rajesh Pandey’s lab using a transcriptomic sequencing approach identifies the possible reason for such breakthrough infections. By comparing vaccination breakthrough and unvaccinated COVID-19 patients, lncRNAs and mRNAs that were differentially expressed and co-expressed were identified. The study found that the interactions between lncRNAs and mRNAs of topologically associated domain genes are being mediated by homologous regions of Alu and LINE1 repeats. The suppression of innate immune response/inflammation and activation of adaptive immune response via such lncRNA and mRNA interactions in vaccination breakthrough infections explains the milder symptoms observed in comparison to unvaccinated COVID-19 individuals.
Transcriptomic study reveals lncRNA-mediated downregulation of innate immune and inflammatory response in the SARS-CoV-2 vaccination breakthrough infections. Chattopadhyay P, Mishra P, Mehta P, Soni J, Gupta R, Tarai B, Budhiraja S, Pandey R. Front. Immunol., 18 November 2022
https://www.frontiersin.org/articles/10.3389/fimmu.2022.1035111/full
Overcoming efflux of anti-epileptic drugs to counter drug unresponsiveness
There is a high percentage (40-50%) of failure in epilepsy treatment using the first line anti-epileptic drugs. Overexpression of multidrug transporters (ABC efflux transporters) – which cause efflux of the drugs – at the blood brain barrier have been known to be one of the reasons for this unresponsiveness to anti-epileptic drugs. In this study, Ritushree Kukreti’s lab looked at the effect of various anti-epileptic drugs on human cerebral microvascular endothelial cells to understand their interactions with ABC efflux transporters. They found that first and second line anti-epileptic drugs – phenytoin, valproic acid, and lamotrigine- acted as substrates for ABC efflux transporter BCRP. Valproic acid was also found to increase the expression of BCRP in these cells at mRNA and protein levels via inducing expression and transport of PPAR alpha from the cytoplasm to the nucleus. Intervention at the level of PPAR alpha thus could be of importance for overcoming anti epileptic drug unresponsiveness.
Valproic acid-induced upregulation of multidrug efflux transporter ABCG2/BCRP via PPARα-dependent mechanism in human brain endothelial cells. Kukal S, Bora S, Kanojia N, Singh P, Paul PR, Rawat C, Sagar S, Bhatraju NK, Grewal GK, Singh A, Kukreti S, Satyamoorthy K, Kukreti R. Mol Pharmacol. 2022 Nov 22:MOLPHARM-AR-2022-000568.
https://pubmed.ncbi.nlm.nih.gov/36414374
Efficacy of diagnostics for monkey pox genetic variants
COVID-19 pandemic followed by monkey pox (mpox) virus outbreak are reminders to the high evolution rate of pathogens in our environment. The current diagnostic tools have made it easier for identifying the infected and containing the spread. While the need to keep monitoring the tools used by diagnostic tests is shown in this paper from Vindod Scaria’s lab. An in-silico analysis of the known oligos that are used for detection of mpox virus was done to find whether any of these coincide the emerging variants of mpox. Using thermodynamic parameters, Gibbs free energy and melting temperature the effectiveness of the oligos in detection was assessed. They further report that five oligos out of the 114 included in the analysis were shown to have a decrease melting temperature because of the presence of genetic variants in the mpox genome, which may be hampering the diagnostic efficacy of these oligos. They thus, emphasize the need for continuous monitoring of the current diagnostic tools.
Systematic in-silico evaluation of the diagnostic impact of mpox genome variants in the current outbreak. Aastha Vatsyayan, V. R. Arvinden & Vinod Scaria. Molecular Diagnosis & Therapy (2022)
https://link.springer.com/article/10.1007/s40291-022-00629-8