HTS driven by fluorescence lifetime detection of FRET identifies activators and inhibitors of cardiac myosin.

Small molecules that bind to allosteric sites on target proteins to alter protein function are highly sought in drug discovery. High-throughput screening (HTS) assays are needed to facilitate the direct discovery of allosterically active compounds. We have developed technology for high-throughput time-resolved fluorescence lifetime detection of fluorescence resonance energy transfer (FRET), which enables the detection of allosteric modulators by monitoring changes in protein structure. We tested this approach at the industrial scale by adapting an allosteric FRET sensor of cardiac myosin to high-throughput screening (HTS), based on technology provided by Photonic Pharma and the University of Minnesota, and then used the sensor to screen 1.6 million compounds in the HTS facility at Bristol Myers Squibb. The results identified allosteric activators and inhibitors of cardiac myosin that do not compete with ATP binding, demonstrating high potential for FLT-based drug discovery.

Clinical profile and risk factors associated with severe scrub typhus infection among non-ICU patients in semi-urban south India.

BACKGROUND & OBJECTIVES: Scrub typhus is as an emerging infectious disease that generally causes acute febrile illness, with disease spectrum ranging from mild illness to multiorgan dysfunction. This study was aimed to report the clinical profile, complications and risk factors associated with severe illness in patients with scrub typhus, outside the intensive care setting. METHODS: It was a prospective study, which involved recruitment of patients with acute febrile illness and diagnosed to have scrub typhus, who were admitted to the general medical wards of a tertiary care centre in Kanchipuram district, in semi-urban south India, over a 12 month period between June 2015 and May 2016. The diagnosis was established both clinically (with or without pathognomonic eschar) and by a positive test of IgM antibodies against scrub typhus by ELISA. The severity of scrub typhus was determined by the presence of organ dysfunction, and the factors associated with it were analyzed. RESULTS: A total of 50 patients with mean age of 39.6±20.5 yr (mean ± SD) were admitted. The mean duration of illness before presentation was 9.10 ± 8.6 days. The mean duration of hospital stay was 7.7±3.6 days. The symptoms included fever, abdominal symptoms, headache, dysuria, breathlessness and altered sensorium. Most common findings on physical examination were eschar (58%), crepitations in the chest (36%), hepatomegaly (34%) and lymphadenopathy (30%). Thirty two percent had respiratory complications, 4% required mechanical ventilation, 24% had shock, 16% had acute kidney injury, and 6% had dysfunction of ≥2 organs. Age of >50 yr, longer duration of illness (>7 days), residence in a rural area and the absence of eschar were found to be independent risk factors for development of severe illness. INTERPRETATION & CONCLUSION: Severe scrub typhus infection among non-ICU patients is more likely to occur in elderly patients and in those with longer duration of illness prior to presentation. The subset of patients without eschar might be more prone to develop complications.

Enicostemma littorale prevents tumor formation in 7,12-dimethylbenz(a)anthracene-induced hamster buccal pouch carcinogenesis.

Oral cancer is one of the most common malignancies worldwide, and India has recorded the highest annual incidence of oral cancer in comparison with other countries. Altered lipid peroxidation and antioxidant status along with defect in detoxification cascade have been implicated in the pathogenesis of several cancers including oral cancer. The aim of this study was to investigate the chemopreventive potential of ethanolic extract of Enicostemma littorale leaves (ElELet) in 7,12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch carcinogenesis. Oral tumor was developed in the buccal pouches of male golden Syrian hamsters by painting with 0.5% DMBA three times a week for 14 weeks. We observed 100% tumor formation with increase in tumor volume and tumor burden in the hamsters treated with DMBA alone. Imbalance in phase I (cytochrome P450 and cytochrome b5) and phase II (glutathione reductase, glutathione-S-transferase, glutathione, and Deoxythymidine-diaphorase (DT)-diaphorase) detoxification agents and lipid peroxidation by-products (thiobarbituric acid reactive substances) and antioxidant (superoxide dismutase, catalase, glutathione peroxidase, and vitamins E and C) status was noticed in hamsters treated with DMBA alone. Oral administration of ElELet at a dose of 250 mg/kg body weight to hamsters treated with DMBA significantly prevented both precancerous and cancerous lesions in the oral cavity. ElELet modulated the status of phase I and II detoxification agents and antioxidants in favor of the suppression of oral carcinogenesis. This study thus suggests that E. littorale might have inhibited the oral carcinogenesis in DMBA-treated hamsters through its antioxidant potential. The present findings are also substantiated by histological studies during DMBA-induced oral carcinogenesis.

Lupeol, a bioactive triterpene, prevents tumor formation during 7,12-dimethylbenz(a)anthracene induced oral carcinogenesis.

The oral cancer chemopreventive efficacy of lupeol, a bioactive triterpene, was assessed by monitoring the tumor incidence and using the status of phase I and II xenobiotic metabolizing enzymes, lipid peroxidation and antioxidants as biochemical end points during 7,12-dimethylbenz(a)anthracene (DMBA) induced hamster buccal pouch carcinogenesis. Oral tumors were developed in the buccal pouch of golden Syrian hamsters by painting with 0.5 % DMBA three times a week for 14 weeks. Well differentiated oral squamous cell carcinoma with marked abnormalities in the status of biochemical markers were noticed in hamsters treated with DMBA alone. Oral administration of lupeol at a dose of 50 mg/kg bw completely inhibited the formation of oral tumors and restored the status of biochemical markers during DMBA induced oral carcinogenesis. The present study thus demonstrates the chemopreventive potential of lupeol in DMBA induced oral carcinogenesis. The chemopreventive potential of lupeol is probably due to its antioxidant or free radical scavenging property and modulating effect on phase I and II xenobiotic metabolizing enzymes in favour of the excretion of carcinogenic metabolites during DMBA induced hamster buccal pouch carcinogenesis.

An interesting case of cardiotoxicity due to bufotoxin (toad toxin).

Consumption of toads for their aphrodisiac effect is a common practice in Laos, China and in some parts of India. Toad secretions from parotid and skin contains toxin similar to cardiac glycosides. It results in bradycardia and cardiac dysfunction leading on to death in some cases. We report a case of toad poisoning in a young previously healthy male.

Central and peripheral demyelination with immune polymyositis in a patient with Bardet Biedl syndrome.

We report extremely rare combination of central and peripheral demyelination with immune polymyositis in a patient with Bardet-Biedl syndrome. The inciting factor postulated was mycoplasma pneumonia infection.

Guillain-Barre syndrome due to organophosphate compound poison.

Acute manifestations of Organophosphate Compound (OPC) poison are due to effect cholinergic excess. Others are intermediate syndrome [IMS], organophosphate induced delayed neuropathy [OPIND] and chronic organophosphate induced neuropsychiatric disorder [COPIND]. All these manifestation have specific period of occurrence and duration. There are very sparse reports of toxic demylination due to OPC poisoning. We report a case of Guillain-Barre Syndrome (GBS) due to toxic demyelination following OPC poison.

Acute pancreatitis presenting as seizures and blindness.

Acute pancreatitis presenting as seizures and blindness, is very rare. Purtscher's retinopathy as the cause of blindness in acute pancreatitis; is not well known and rarely diagnosed condition by physicians. Here we report a case of acute pancreatitis presenting as seizures and Purtscher's retinopathy.

Drug related crisis in myasthenia gravis.

Myasthenia gravis is an autoimmune disorder where antibodies against the nicotinic Ach receptor resulting in impaired transmission at the NM junction. A number of drugs have been reported to cause neuromuscular blockade and/or to increase weakness in myasthenia gravis. We report a case of myasthenia gravis in which the calcium channel blocker-nifedipine caused the worsening of the symptoms.

Oncogenicity studies of the cognition-enhancing agent nefiracetam in mice and rats.

Oncogenicity studies of nefiracetam (N-(2,6-dimethylphenyl)-2-(2-oxo-1-pyrrolidinyl)acetamide, DM-9384, CAS 77191-36-7), a new cognition-enhancing agent, were carried out in male and female mice and rats. The compound was administered in diet for 104 weeks at dosage levels of 30, 90 and 270 mg/kg/d for mice and of 200, 600 and 1800 ppm for rats. The administration of nefiracetam produced no effects on survival, appearance or behavior. Body weights of the high dose male mice and rats were occasionally significantly decreased when compared to the controls. When calculated on a g/animal/d basis, food consumption was sometimes decreased for these male groups. At necropsy, there was no evidence of treatment related changes, nor were these seen on histopathological examination. All microscopic changes seen in mice and rats were of the usual type commonly occurring in untreated aged B6C3F1 mice and F344 rats. In conclusion, the administration of nefiracetam for 24 months to B6C3F1 mice and F344/DuCrj rats produced only slight effects on body weight in the high dose males with a no-effect level of 90 mg/kg/d for mice or 600 ppm for rats. There was no evidence of an oncogenic effect of nefiracetam.

Subchronic oral toxicology of 4-fluoro-3-nitroaniline in the rat.

4-Fluoro-3-nitroaniline (4-F-3-NA), an intermediate used in the production of commercial hair dyes, was administered daily for 90 days via po intubation to Charles River CD rats at doses of 40, 120, or 360 mg/kg to determine its subchronic toxicology. All animals receiving 360 mg/kg died prior to termination of the study. The principal effect to rats of 4-F-3-NA exposure was alteration of the hematopoietic system characteristic of hemolytic anemia. Hematocrit, hemoglobin concentration, and erythrocyte count were decreased and reticulocytes increased in a dose-related pattern in both male and female animals. Histopathological change consistent with hemolytic anemia was observed in the bone marrow (erythroid hyperplasia), kidneys (deposition of biliverdin and pigment-laden macrophages in the tubules), liver (pigmented Kupffer cells), and spleen (increased hematopoiesis) in males and females. Heinz bodies and an increase in methemoglobinemia were not observed in this study. In the testes, aspermia and testicular degeneration were seen in isolated tubules, but these changes were significant only at the high dose. Additional pathologic changes noted at the high dose included colloid depletion and follicular cell hypertrophy of the thyroid gland, hypertrophy of the zone fasciculata in the adrenal glands, and lymphoid cell depletion in mesenteric lymph nodes. The calcification of the myocardium and coronary arteries was considered metastatic in nature, secondary to kidney damage. Serum ALT and BUN were elevated in males and AP was elevated in females from the high dose group. Glycosuria, bilirubinuria, and urobilinogenuria were also increased in both sexes at this dose.