A highly sensitive and specific luminescent MOF determines nitric oxide production and quantifies hydrogen sulfide-mediated inhibition of nitric oxide in living cells.

The synthesis of a novel carboxylate-type organic linker-based luminescent MOF (Zn(H2L) (L1)) (named PUC2) (H2L = 2-aminoterephtalic acid, L1 = 1-(3-aminopropyl) imidazole) is reported by the solvothermal method and comprehensively characterized using single-crystal XRD, PXRD, FTIR, TGA, XPS, FESEM, HRTEM, and BET. PUC2 selectively reacts with nitric oxide (▪NO) with a detection limit of 0.08 µM, and a quenching constant (0.5 × 104 M-1) indicating a strong interaction with ▪NO. PUC2 sensitivity remains unaffected by cellular proteins or biologically relevant metals (Cu2+/ Fe3+/Mg2+/ Na+/K+/Zn2+), RNS/ROS, or H2S to score ▪NO in living cells. Lastly, we used PUC2 to demonstrate that H2S inhibition increases ▪NO production by ~ 14-30% in various living cells while exogenous H2S suppresses ▪NO production, indicating that the modulation of cellular ▪NO production by H2S is rather generic and not restricted to a particular cell type. In conclusion, PUC2 can successfully detect ▪NO production in living cells and environmental samples with considerable potential for its application in improving the understanding of the role of ▪NO in biological samples and study the inter-relationship between ▪NO and H2S.

Selection of Ideal Reference Genes for Gene Expression Analysis in COVID-19 and Mucormycosis.

Selection of reference genes during real-time quantitative PCR (qRT-PCR) is critical to determine accurate and reliable mRNA expression. Nonetheless, not a single study has investigated the expression stability of candidate reference genes to determine their suitability as internal controls in SARS-CoV-2 infection or COVID-19-associated mucormycosis (CAM). Using qRT-PCR, we determined expression stability of the nine most commonly used housekeeping genes, namely, TATA-box binding protein (TBP), cyclophilin (CypA), ß-2-microglobulin (B2M), 18S rRNA (18S), peroxisome proliferator-activated receptor gamma (PPARG) coactivator 1 alpha (PGC-1α), glucuronidase beta (GUSB), hypoxanthine phosphoribosyltransferase 1 (HPRT-1), ß-ACTIN, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in patients with COVID-19 of various severities (asymptomatic, mild, moderate, and severe) and those with CAM. We used statistical algorithms (delta-CT [threshold cycle], NormFinder, BestKeeper, GeNorm, and RefFinder) to select the most appropriate reference gene and observed that clinical severity profoundly influences expression stability of reference genes. CypA demonstrated the most consistent expression irrespective of disease severity and emerged as the most suitable reference gene in COVID-19 and CAM. Incidentally, GAPDH, the most commonly used reference gene, showed the maximum variations in expression and emerged as the least suitable. Next, we determined expression of nuclear factor erythroid 2-related factor 2 (NRF2), interleukin-6 (IL-6), and IL-15 using CypA and GAPDH as internal controls and show that CypA-normalized expression matches well with the RNA sequencing-based expression of these genes. Further, IL-6 expression correlated well with the plasma levels of IL-6 and C-reactive protein, a marker of inflammation. In conclusion, GAPDH emerged as the least suitable and CypA as the most suitable reference gene in COVID-19 and CAM. The results highlight the expression variability of housekeeping genes due to disease severity and provide a strong rationale for identification of appropriate reference genes in other chronic conditions as well. IMPORTANCE Gene expression studies are critical to develop new diagnostics, therapeutics, and prognostic modalities. However, accurate determination of expression requires data normalization with a reference gene, whose expression does not vary across different disease stages. Misidentification of a reference gene can produce inaccurate results. Unfortunately, despite the global impact of COVID-19 and an urgent unmet need for better treatment, not a single study has investigated the expression stability of housekeeping genes across the disease spectrum to determine their suitability as internal controls. Our study identifies CypA and then TBP as the two most suitable reference genes for COVID-19 and CAM. Further, GAPDH, the most commonly used reference gene in COVID-19 studies, turned out to be the least suitable. This work fills an important gap in the field and promises to facilitate determination of an accurate expression of genes to catalyze development of novel molecular diagnostics and therapeutics for improved patient care.

Targeting endogenous gaseous signaling molecules as novel host-directed therapies against tuberculosis infection.

Tuberculosis (TB) is a chronic pulmonary disease caused by Mycobacterium tuberculosis which is a major cause of morbidity and mortality worldwide. Due to the complexity of disease and its continuous global spread, there is an urgent need to improvise the strategies for prevention, diagnosis, and treatment. The current anti-TB regimen lasts for months and warrants strict compliance to clear infection and to minimize the risk of development of multi drug-resistant tuberculosis. This underscores the need to have new and improved therapeutics for TB treatment. Several studies have highlighted the unique ability of Mycobacterium tuberculosis to exploit host factors to support its survival inside the intracellular environment. One of the key players to mycobacterial disease susceptibility and infection are endogenous gases such as oxygen, nitric oxide, carbon monoxide and hydrogen sulfide. Nitric oxide and carbon monoxide as the physiological gaseous messengers are considered important to the outcome of Mycobacterium tuberculosis infection. The role of hydrogen sulfide in human tuberculosis is yet not fully elucidated, but this gas has been shown to play a significant role in bacterial respiration, growth and pathogenesis. This review will focus on the host factors majorly endogenous gaseous signaling molecules which contributes to Mycobacterium tuberculosis survival inside the intracellular environment and highlight the potential therapeutic targets.

Synthesis, characterization and utility of a series of novel copper(II) complexes as excellent surface disinfectants against nosocomial infections.

Nosocomial infections are among the major public health concerns, especially during the ongoing Covid19 pandemic. There is a great demand for novel chemical agents that are capable of killing specific pathogens or augmenting the efficiency of existing disinfectants. Herein, we report the synthesis and comprehensive characterization (through FT-IR, HR-MS, SEM, TGA-DSC, CV, UV and SCXRD analyses) of six novel copper(II) complexes, [CuL(4X-An)] (5a-5d), [CuL(An)] (5e), and [CuL(benzhydrylamine)] (5f), and their evaluation as anti-microbial agents against WHO priority pathogens, confirming their possible use in hospital settings. The compounds were synthesized with a Schiff base (H2L) obtained by the condensation reaction of 3-acetyl-6-methyl-2H-pyran-2,4(3H)-dione (DHA) and benzohydrazide and further addition of different p-substituted aniline (An) molecules. Single crystal structure analyses revealed that the aniline derivatives are isostructural to the copper atom in a square planar coordination, while the benzhydrylamine complex forms a dimer (5f), with a square pyramidal coordination geometry for the metal. Time-kill kinetics and reduced microbial recovery studies revealed excellent bactericidal action against Staphylococcus aureus and Enterococcus faecalis. Particularly, the novel compound 5f significantly reduced microbial recovery compared to ethanol-based sanitisers. In fact, addition of 5f to 70% ethanol remarkably synergized the killing with >6-log reduction in microbial burden. Overall, our novel compounds would increase the disinfection efficacy in hospitals and industries, thereby improving the efficiency and minimizing the risk of infections.