A low-background, fluorescent assay to evaluate inhibitors of diverse viral proteases.

Multiple coronaviruses (CoVs) can cause respiratory diseases in humans. While prophylactic vaccines designed to prevent infection are available for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), incomplete vaccine efficacy, vaccine hesitancy, and the threat of other pathogenic CoVs for which vaccines do not exist have highlighted the need for effective antiviral therapies. While antiviral compounds targeting the viral polymerase and protease are already in clinical use, their sensitivity to potential resistance mutations as well as their breadth against the full range of human and preemergent CoVs remain incompletely defined. To begin to fill that gap in knowledge, we report here the development of an improved, noninfectious, cell-based fluorescent assay with high sensitivity and low background that reports on the activity of viral proteases, which are key drug targets. We demonstrate that the assay is compatible with not only the SARS-CoV-2 Mpro protein but also orthologues from a range of human and nonhuman CoVs as well as clinically reported SARS-CoV-2 drug-resistant Mpro variants. We then use this assay to define the breadth of activity of two clinically used protease inhibitors, nirmatrelvir and ensitrelvir. Continued use of this assay will help define the strengths and limitations of current therapies and may also facilitate the development of next-generation protease inhibitors that are broadly active against both currently circulating and preemergent CoVs. IMPORTANCE Coronaviruses (CoVs) are important human pathogens with the ability to cause global pandemics. Working in concert with vaccines, antivirals specifically limit viral disease in people who are actively infected. Antiviral compounds that target CoV proteases are already in clinical use; their efficacy against variant proteases and preemergent zoonotic CoVs, however, remains incompletely defined. Here, we report an improved, noninfectious, and highly sensitive fluorescent method of defining the sensitivity of CoV proteases to small molecule inhibitors. We use this approach to assay the activity of current antiviral therapies against clinically reported SARS-CoV-2 protease mutants and a panel of highly diverse CoV proteases. Additionally, we show this system is adaptable to other structurally nonrelated viral proteases. In the future, this assay can be used to not only better define the strengths and limitations of current therapies but also help develop new, broadly acting inhibitors that more broadly target viral families.

Order-Disorder Transitions in (Ca_{x}Sr_{1-x})_{3}Rh_{4}Sn_{13}.

The classification of structural phase transitions as displacive or order-disorder in character is usually based on spectroscopic data above the transition. We use single crystal x-ray diffraction to investigate structural correlations in the quasiskutterudites, (Ca_{x}Sr_{1-x})_{3}Rh_{4}Sn_{13}, which have a quantum phase transition at x∼0.9. Three-dimensional pair distribution functions show that the amplitudes of local atomic displacements are temperature independent below the transition and persist to well above the transition, a signature of order-disorder behavior. The implications for the associated electronic transitions are discussed.

A Screen of Natural Product Extracts Identifies Moenomycin as a Potent Antigonococcal Agent.

Increasing multidrug resistance in Neisseria gonorrheae is a growing public health crisis. Resistance to the last line therapies, cephalosporins and azithromycin, are of particular concern, fueling the need to discover new treatments. Here, we identified the phosphoglycolipid moenomycin from a screen of microbial natural products against drug-resistant N. gonorrheae as a potent antigonococcal agent. Moenomycin demonstrates excellent activity (MIC = 0.004-0.03 µg/mL) against a variety of multidrug-resistant N. gonorrheae. Importantly, moenomycin, thought to be a Gram-positive specific antibiotic, penetrates the Gram-negative gonococcal outer membrane. Moenomycin causes intracellular accumulation of peptidoglycan precursors, cell blebbing, and rupture of the cell envelope, all consistent with cell wall biosynthesis inhibition. Serial bacterial exposure to moenomycin for 14 days revealed slow development of resistance (MICDay14 = 0.03-0.06 µg/mL), unlike the clinically used drug azithromycin. Our results offer the potential utility of moenomycin as a lead for antigonococcal therapeutic candidates and warrant further investigation.

Mimicking the human environment in mice reveals that inhibiting biotin biosynthesis is effective against antibiotic-resistant pathogens.

To revitalize the antibiotic pipeline, it is critical to identify and validate new antimicrobial targets1. In Mycobacteria tuberculosis and Francisella tularensis, biotin biosynthesis is a key fitness determinant during infection2-5, making it a high-priority target. However, biotin biosynthesis has been overlooked for priority pathogens such as Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa. This can be attributed to the lack of attenuation observed for biotin biosynthesis genes during transposon mutagenesis studies in mouse infection models6-9. Previous studies did not consider the 40-fold higher concentration of biotin in mouse plasma compared to human plasma. Here, we leveraged the unique affinity of streptavidin to develop a mouse infection model with human levels of biotin. Our model suggests that biotin biosynthesis is essential during infection with A. baumannii, K. pneumoniae and P. aeruginosa. Encouragingly, we establish the capacity of our model to uncover in vivo activity for the biotin biosynthesis inhibitor MAC13772. Our model addresses the disconnect in biotin levels between humans and mice, and explains the failure of potent biotin biosynthesis inhibitors in standard mouse infection models.

Dependence of DNA looping on Escherichia coli culture density.

Repression of a promoter by entrapment within a tightly bent DNA loop is a common mechanism of gene regulation in bacteria. Besides the mechanical properties of the looped DNA and affinity of the protein that anchors the loop, cellular energetics and DNA negative supercoiling are likely factors determining the stability of the repression loop. E. coli cells undergo numerous highly regulated and dynamic transitions as resources are depleted during bacterial growth. We hypothesized that the probability of DNA looping depends on the growth status of the E. coli culture. We utilized a well-characterized repression loop model assembled from elements of the lac operon to measure loop length-dependent repression at three different culture densities. Remarkably, even with changes in supercoiling, there exists a dynamic compensation in which the contribution of DNA looping to gene repression remains essentially constant.

Clinical profile of human immunodeficiency virus patients with opportunistic infections: A descriptive case series study.

BACKGROUND: Human immunodeficiency virus (HIV) virus, causative agent in acquired immunodeficiency syndrome, is fast becoming a major threat in the Indian subcontinent, with an estimated 3.7 million persons being infected with HIV. HIV infection is complicated by various opportunistic infections (OIs) such as tuberculosis (TB), candidiasis, herpes zoster, Pneumocystis jirvoceii, cytomegalovirus (CMV) etc., This study carried out to know the clinical profile of HIV patients with OIs. METHODS: A case series study was carried out at a tertiary care hospital in Bellary, Karnataka, India. A hospital based case series study was conducted among 164 HIV patients with OIs admitted to various wards as well as attending outpatient department at Vijayanagara Institute of Medical Sciences Hospital, Bellary during Jan 2013 to Nov 2013. Both primary and secondary data was collected to gather information on clinical profile. The statistical tests used were descriptive statistics and independent t test. RESULTS: Among 164 patients, 29.3% were females and 68.3% males. High proportions of patients were observed in 28-37 years of age group and heterosexual route was the most common mode of transmission. TB (50%) is the most frequent OI followed by candidiasis (49%), pneumocystis (16%) and others. The mean CD4 cell count in TB was 237.02/mL and in candidiasis 189.07/mL. Low values were observed in promyelocytic leukemia (18.10/mL), CMV (18.5/mL) and in toxoplasmosis (73.1/mL). CONCLUSIONS: Respiratory system was the most common system involved by OIs and most of patients with OIs had CD4 T cell count below 200/mL, whereas there were no patients in the study with counts above 500/mL.

Socio-demographic and other risk factors of pre eclampsia at a tertiary care hospital, karnataka: case control study.

BACKGROUND: Pre-eclampsia is one of the leading causes of maternal and infant morbidity and mortality worldwide. The aetiopathogenesis of this condition involves combination of genetic predisposition and environmental factors. The aim of the study was to determine the socio demographic and other risk factors of pre-eclampsia. MATERIALS AND METHODS: A case control study was conducted at a tertiary care hospital, Karnataka among 100 cases of pre-eclampsia and 200 controls without pre eclampsia. Non probability purposive sampling technique was adopted to select the study subjects. Data was collected by using a pre tested semi structured questionnaire which included information related to socio-demographic and other known risk factors of pre eclampsia. Primary data was collected by interviewing study subjects and secondary data of cases was obtained from case records. Data was analysed using SPSS. RESULTS: Study subjects included 100 cases and 200 controls. Age of less than 20 y (OR=3.8), monthly income of less than Rs4000 (OR=6.8), age of menarche of less than 12 y (OR=13.1), family h/o pre eclampsia (OR=36.0), family h/o Diabetes (OR=44.9), family h/o hypertension (OR=16.7) and previous h/o PIH (OR=58.5) are found to be significant risk factors of pre eclampsia. CONCLUSION: The significant risk factors may be used for screening pre-eclampsia during registration of pregnancy.