NMR spectroscopy and theoretical calculations in the conformational analysis of 1-methylpyrrolidin-2-one 3-halo-derivatives.

This study reports the results of ab initio and density functional theory (DFT) electronic structure calculations as well as (3)J(HH) experimental and calculated coupling constant data obtained in the investigation of the conformational equilibrium of 3-halo-derivatives of 1-methylpyrrolidin-2-one. The five-membered ring assumes an envelope conformation owing to the plane of formation of the O═C-N-R bond, with C4 forming the "envelope lid". When the conformation changes, the "lid" alternates between positions above and below the amide plane. The α-carbonyl halogen assumes two positions: a pseudo-axial and a pseudo-equatorial. In the gaseous phase, the calculations indicate that the pseudo-axial conformer is more stable and preferable going down the halogen family. Natural bond orbital analysis showed that electronic delocalization is significant only for the iodo derivative. In the other derivatives, the electrostatic repulsion between oxygen and the halogen determines the conformational equilibrium. When the solvated molecule was taken into account, the pseudo-equatorial conformer population increased with the relative permittivity of the solvent. This variation was strong in the fluoro derivative, and the preference was inverted. In the chlorine derivative, the two populations became closer in methanol and acetonitrile. In the bromine and iodine derivatives, the percentage of pseudo-equatorial conformer increased only slightly owing to the dipole moment of the conformation: the pseudo-equatorial conformation has a greater dipole moment and thus is stable in media with high relative permittivity.

Utility of serum Galactomannan in diagnosing COVID-19 patients with suspected IPA: an observational study in resource limited settings.

OBJECTIVE: To study the utility of Galactomannan (GM) antigen as a screening marker for diagnosing invasive pulmonary aspergillosis (IPA) in coronavirus disease 2019 (COVID-19) patients. PATIENTS AND METHODS: The serum samples from patients with severe COVID-19 diseases admitted to the Critical Care Unit were collected on the 5th day of admission for GM screening. The samples were analysed by enzyme linked immune sorbent assay (ELISA) and GM index of more than 1 was considered as positive. All GM positive patients were serially followed until discharge or death. RESULTS: The GM was raised in serum of 12 out of 38 patients, indicating an incidence of possible COVID-19 associated IPA (CAPA) in 31.57% of patients. The median age of these CAPA patients was 56.5 years, males were significantly more affected than females. The inflammatory marker serum ferritin was raised in all 12 patients (median value of 713.74 ng/ml), while IL-6 was raised in 9 patients (median value of 54.13 ng/ml). None of these patients received antifungals. Their median length of hospital stay was 20 days (IQR: 12, 34 days). All these patients succumbed to the illness. CONCLUSIONS: The serum GM appears to be sensitive diagnostic tool to identify early IPA in COVID-19 patients and pre-emptive antifungal therapy could play a role in salvaging these patients.

Comparative genetic analysis of trichome-less and normal pod genotypes of Mucuna pruriens (Fabaceae).

Velvet bean (Mucuna pruriens) seeds contain the catecholic amino acid L-DoPA (L-3,4-dihydroxyphenylalanine), which is a neurotransmitter precursor and used for the treatment of Parkinson's disease and mental disorders. The great demand for L-DoPA is largely met by the pharmaceutical industry through extraction of the compound from wild populations of this plant; commercial exploitation of this compound is hampered because of its limited availability. The trichomes present on the pods can cause severe itching, blisters and dermatitis, discouraging cultivation. We screened genetic stocks of velvet bean for the trichome-less trait, along with high seed yield and L-DoPA content. The highest yielding trichome-less elite strain was selected and indentified on the basis of a PCR-based DNA fingerprinting method (RAPD), using deca-nucleotide primers. A genetic similarity index matrix was obtained through multivariant analysis using Nei and Li's coefficient. The similarity coefficients were used to generate a tree for cluster analysis using the UPGMA method. Analysis of amplification spectra of 408 bands obtained with 56 primers allowed us to distinguish a trichome-less elite strain of M. pruriens.

Terbinafine resistance in dermatophytes: Time to revisit alternate antifungal therapy.

Resistant superficial dermatophytic infections of the skin and its appendages have emerged as a major health problem in India. Mutations in Squalene epoxidase gene have led to increasing incidence of resistance to terbinafine in dermatophytic isolates. We examined six patients with recalcitrant dermatophytosis attending Dermatology OPD at a tertiary care hospital and demonstrated terbinafine resistance by molecular method. Immediate hyperitivity (IH) reaction to Trichophytin antigen was highlighted in these patients. The patients were treated with alternate antifungals after demonstration of resistance to terbinafine based on the antifungal susceptibility testing (AFST). On follow up the patients responded well to the substitute but the duration of therapy had to be prolonged beyond six weeks.

Evaluation of PCR, DNA hybridization and immunomagnetic separation - PCR for detection of Burkholderia mallei in artificially inoculated environmental samples.

Glanders is highly contagious disease of equines, caused by Burkholderia mallei. The disease though rare, can be transmitted to humans. Here, we report a strategy for rapid detection of B. mallei from environmental samples. Different bacteriological media were evaluated and brain heart infusion broth medium with selective supplements (BHIB-SS) of penicillin (200 U/ml) and crystal violet (1:10,00000) was found to support the maximum growth of B. mallei even in the presence of other bacteria like Escherichia coli and Staphylococcus aureus. A polymerase chain reaction (PCR) and a DNA hybridization method was standardized for 823 bp specific dNA sequence of B. mallei. To enable the quicker and direct enrichment of B. mallei bacteria from environmental samples, an immunomagnetic separation (IMS) method was also standardized. Water, husk, grass and gram samples were artificially contaminated by B. mallei bacteria and after enrichment of B. mallei in BHIB-SS, detection was carried out by PCR and DNA hybridization. PCR was found to be a better method of the two with a detection limit of 10(4)-10(6) CFU/ml (6 h enrichment in BHIB-SS) in water and other particulate matrices. Detection by PCR in the above samples without enrichment in BHIBSS was carried out following IMS where the detection limit was about 1-2 log higher than PCR following enrichment in BHIB-SS. We recommend PCR for 823 bp for detection of B. mallei from environmental samples either following enrichment in BHIB-SS or IMS. IMS-PCR method may be preferred in situations where numbers of B. mallei bacteria are expected to be high and results are required in short time.

Th1-type immune response to infection by pYV-cured phoP-phoQ null mutant of Yersinia pseudotuberculosis is defective in mouse model.

The PhoP-PhoQ two-component system of Yersinia pseudotuberculosis, a Gram-negative enteric pathogen which causes a variety of gastrointestinal and extraintestinal infections in humans, has been shown to be necessary for virulence. A phoP-phoQ null mutant of a strain of Y. pseudotuberculosis cured of its native plasmid pYV was obtained and studied for generation of immune response in mouse model following intravenous inoculation. The phoP-phoQ null mutant elicited much weaker IgG antibody response to whole cell sonicated (WCS) antigen, in particular that of IgG2a isotype. Interferon-gamma levels were also significantly reduced in cultured splenocytes of mice immunized with phoP-phoQ null mutant. The null mutant was found to be about 72-fold less virulent than the parent isogenic strain of Y. pseudotuberculosis. Average counts in spleen of mice inoculated with the null mutant were observed to reduce by at least four logs when compared with the counts in the spleen of mice inoculated with parent isogenic strain. We can thus suggest that the Th1-type immune response of the phoP-phoQ null mutant of Y. pseudotuberculosis is diminished in mice.

Development of a real-time loop-mediated isothermal amplification assay for detection of Burkholderia mallei.

Burkholderia mallei is the aetiological agent of glanders, a highly contagious and re-emerging zoonotic disease. Early diagnosis of glanders is critically important to ensure timely treatment with appropriate antibiotics in humans, and to prevent spread of infection in animals. Molecular detection of B. mallei has always been troublesome because of its genetic similarity with Burkholderia pseudomallei, the causative agent of melioidosis. In present investigation, a set of six B. mallei-specific primers were designed and a simple, rapid, specific and sensitive real-time loop-mediated isothermal amplification (LAMP) assay was developed for detection of B. mallei. The LAMP assay could detect as low as 1 pg of B. mallei genomic DNA and 5.5 × 103  CFU/ml of B. mallei in spiked human blood. The assay was highly specific for B. mallei as it did not cross-react with other bacterial strains used in the study. The established LAMP assay is field adaptable and can be a better and viable alternative to PCR-based techniques for detection of B. mallei in glanders endemic areas with resource-limited settings.

Candida auris in critically ill patients: Emerging threat in intensive care unit of hospitals.

Candida auris has emerged as the multi-drug resistant non-albican candida species in critically ill patients admitted to intensive care units (ICU) causing bloodstream and other infections. It has often been misidentified as Candida famata, Candida sake, Rhodotorula glutinis, or Saccharomyces cerevisiae and Candida haemulonii by automated identification systems. We, in this study, discuss three patients who acquired Candida auris infection after 15 to 20days of their stay in medical ICU. Medical equipment, use of multiple antibiotics, and poor hand hygiene are the most probable predisposing factors attributing to its colonization at multiple anatomical sites leading to bloodstream infection. Candida auris might substantially contaminate the environment of colonized or infected patients making its eradication difficult. Patient screening for Candida auris, especially during prolonged ICU stays, along with strict infection prevention and control strategies needs to be adopted to break its persistence.

Development of methodologies for the regioselective synthesis of four series of regioisomer isoxazoles from ß-enamino diketones.

Four methodologies are reported for the regioselective synthesis of four series of regioisomer isoxazoles from cyclocondensation of ß-enamino diketones and hydroxylamine hydrochloride. Regiochemical control was achieved by varying reaction conditions and substrate structure. The mild reaction conditions used to access 4,5-disubstituted, 3,4-disubtituted, and 3,4,5-trisubstituted regioisomer isoxazoles, as well as the pharmacological and synthetic potential of the products, make these novel methodologies very powerful.

Challenges in culture-negative cases of Madurella mycetomatis: A case report re-accentuating PCR as an essential diagnostic tool.

Identification of dematiaceous fungi responsible for black-grain mycetoma has remained cumbersome and time consuming for years leading to delayed diagnosis and thereby increased agony to patients. Moreover, difficult morphology of some of these fungi demanding enough expertise for species identification in addition to culture-negativity has often led to misdiagnosis and hence inapt treatment to the patients. We report the identification of Madurella mycetomatis from culture-negative black granules discharged from foot nodular lesions of a 27 years old male using PCR followed by sequencing of the internal transcribed spacer region. The patient's lesions were successfully treated using a combination of itraconazole (200mg) and terbinafine (250mg), confirming our diagnosis. Our case study proves the clinical value of PCR as the best, rapid and accurate diagnostic method for the identification of Madurella mycetomatis and related fungi, particularly in culture-negative cases.

Evaluation of Parmotrema reticulatum Taylor for Antibacterial and Antiinflammatory Activities.

Lichens produce variety of secondary metabolites including depsides, depsidones and dibenzofurans having multifunctional activity in response to external environmental condition. Present study provides evidence for in vitro antibacterial and in vivo antiinflammatory activity of acetone and ethanol extracts of Parmotrema reticulatum. In vitro antibacterial activity was investigated against gram positive and gram negative bacteria. Cotton pellet-induced granuloma, xylene-induced ear swelling, carragennan-induced edema, histamine-induced and carboxymethylcellulose sodium-induced leukocyte emigration in mice models were used to quantify the antiinflammatory activity. Acetone and ethanol extracts were showed antibacterial activity against Bacillus subtilis (minimal inhibitory concentration: 100 and 150 µg/ml) and Staphylococcus aureus (minimal inhibitory concentration: 100 and 200 µg/ml), Escherichia coli (minimal inhibitory concentration: 200 and 250 µg/ml), and Pseudomonasa eruginosa (minimal inhibitory concentration: 200 and 300 µg/ml). Acetone extract was inhibited edema significantly at 200 mg/kg with xylene, cotton pellet, carragennan and histamine induced edema in vivo models. Ethanol extract was found effective at dose of 300 mg/kg with all in vivo antiinflammatory models. The results showed significant (P<0.01) antiinflammatory effects at 200 and 300 mg/kg dose of acetone and ethanol extracts, respectively, which can be concluded that significant activity may be due to presence of flavanoids in ethanol extract and usnic acid in acetone extract.

Synthesis and evaluation against Leishmania amazonensis of novel pyrazolo[3,4-d]pyridazinone-N-acylhydrazone-(bi)thiophene hybrids.

A new series of pyrazolo[3,4-d]pyridazin-7-one derivatives were synthesised and evaluated for their in vitro antileishmanial activity against Leishmania amazonensis promastigote and axenic amastigote forms. The results showed that the pyrazolo[3,4-d]-pyridazin-7-one-N-acylhydrazone-(bi)thiophene hybrids 5b, 6b and 6d exhibit better antileishmanial activity with IC50 84.96, 3.63 and 10.79 µM, against the promastigote form and IC50 32.71, 2.32 and >100 µM against the axenic amastigote form, respectively. The active compounds had their cytotoxicity tested against macrophages and fibroblast cells with a higher selectivity index than 10 for compounds 6b and 6d. Molecular docking studies were performed for all active compounds using the enzyme trypanothione reductase (TR) to investigate a possible action mechanism. The results suggested that active compounds had interactions with the residues of amino acids Gly 13, Thr 51, Thr 160, Gly 161, Tyr 198, Arg 287, Asp 327, Thr 335, which may inhibit the enzyme TR.

Chitosan hydrochloride based microspheres of albendazole for colonic drug delivery.

Microspheres of chitosan hydrochloride (CH) were prepared in order to deliver albendazole specifically into the colon. Microspheres were prepared by an emulsion method using different ratios of drug and CH (1:1 to 1:5), agitation speeds (500 to 1500 rpm) and concentrations of glutaraldehyde in toluene as the cross-linking agent (0.25 to 1.0% w/v). The effect of polymer concentration, stirring rate and concentration of cross-linking agent on the particle size and drug loading was studied. With an increase in CH concentration, the average particle size was increased. Increased agitation speed reduced the size of the microspheres but higher agitation speed resulted in irregularly shaped microspheres. Increasing the concentration of cross-linking agent produced more regularly shaped microspheres of smaller size. The drug loading was highest at a drug: CH ratio of 1:3, stirring speed 1000 rpm and 0.75% w/v concentration of cross-linking agent. The effect of CH concentration on in vitro drug release from the microspheres was evaluated in simulated g.i.t fluids. A comparative in vitro drug release study of the optimized formulation was carried out in simulated colonic fluid, with and without 2% rat caecal content. The drug release in 24 h was 48.9% in colonic fluid without rat caecal content, and 76.5% in colonic fluid with rat caecal contents.

Cytokines in posterior uveitis: an update.

T helper (Th) lymphocytes differentiate into two distinct subsets--Th1 and Th2--as defined by functional abilities and cytokine profiles. The functional differences between Th subsets are explained primarily through the activities of the cytokines they secrete. Interferon-gamma (IFN-gamma) is the signature cytokine of Th1 cells, which also produce interleukin-2 (IL-2) and tumor necrosis factor-beta (TNF-beta). IL-4 is the corresponding signature cytokine of Th2 cells, which also secrete IL-5, IL-6, IL-9, and IL-13. Recently, a few transcription factors have been identified that not only control the expression of cytokines of a particular type but also repress cytokines of other types. Human Th1 and Th2 cells not only produce a different set of cytokines but also exhibit distinct functional properties and the preferential expression of some activation markers. Pathophysiologically, the two subsets have been found to be mutually antagonistic. Various Th1 and Th2 cytokines appear to play an important role in the etiopathogenesis of posterior uveitis and its animal model, experimental autoimmune uveitis (EAU). The exact contribution of these mediators to uveitis remains to be defined. Recent studies suggest that a shift from Th1- to Th2-dominated response could be of therapeutic benefit. This review evaluates various studies in which uveitopathogenic and therapeutic potentials of various Th1 and Th2 cytokines have been investigated.

Semi-inclusive lambda and K(S) production in p-Au collisions at 17.5 GeV/c

The first detailed measurements of the centrality dependence of strangeness production in p-A collisions are presented. Lambda and K(S) dn/dy distributions from 17.5 GeV/ c p-Au collisions are shown as a function of "grey" track multiplicity and the estimated number of collisions, nu, made by the proton. The nu dependence of the Lambda yield deviates from a scaling of p-p data by the number of participants, increasing faster than this scaling for nu

Erythrocyte sedimentation rate and disease in the elderly.

The erythrocyte sedimentation rate (ESR) is not always elevated in relation to disease. Of 188 elderly autopsy patients studied, 125 had nonmalignant disease and 63 had malignant disease. Of the patients with malignant disease, 13 percent had a clinical ESR of less than 10 mm/hr and 26 percent a rate of less than 20 mm/hr, the upper level of normal for the elderly. Of the patients with nonmalignant disease, 9.6 percent had an ESR of less than 10 mm/hr and 25.6 percent a rate of less than 20 mm/hr. Thus, in about a quarter of the patients, the ESR was below the top normal level for the elderly. Moreover, the ESR may be as high as 35--40 mm/hr in healthy aged persons. Therefore, the ESR is unreliable as a test for the presence of disease (malignant or nonmalignant) in the elderly.

The corneal reflex in elderly patients.

The corneal reflex was absent in 23.5 percent of 115 elderly patients, particularly in those with cerebrovascular disease. However, it was also absent in many of the patients without neurologic disease. Therefore, physicians should not attach too much importance to lack of the corneal reflex in elderly persons.

The "elephant man" of Cambridge. a case report of neurofibromatosis.

The case is presented of a 65-year-old man with neurofibromatosis manifesting facial and skeletal features resembling those of the "elephant man" described by Sir Frederick Treves. Autopsy revealed not only a pheochromocytoma (a common accompaniment of neurofibromatosis), but an enlarged infarcted spleen and a subphrenic abscess. These findings have not been described previously in a patient with neurofibromatosis.

Nephrotic syndrome in the elderly.

Study of hospital activity analysis data over a ten-year period in a district general hospital show that nephrotic syndrome is uncommon in the elderly. The spectrum of histology, however, is no different from the younger adults. The other interesting findings were: diabetic elderly with nephrotic syndrome may have minimal change, therefore should have renal biopsy; and occurrence of sero-negative lupus nephritis in a 90-year-old male and a focal glomerulo-nephritis in a 75-year-old female.

Copper and zinc levels in "healthy" and "sick" elderly.

Healthy elderly as a group have a wider range for serum copper, ceruloplasmin, and serum zinc than the adjusted ranges for the laboratory. In disease serum copper and/or ceruloplasmin act as acute-phase reactants in vascular disease, inflammation, and malignancy. Serum zinc values likewise are affected by the presence of inflammation and malignancy. The reported low level of copper in patients with osteoporosis was not confirmed by the present study. Neither was there any evidence found for zinc deficiency in patients with dementia.