Eco-friendly methodology for efficient synthesis and scale-up of 2-ethylhexyl-p-methoxycinnamate using Rhizopus oryzae lipase and its biological evaluation

Lipase-mediated synthesis of phenolic acid esters is a green and economical alternative to current chemical methods. Octyl methoxycinnamate, an important UVB-absorbing compound, was synthesized by the esterification of p-methoxycinnamic acid with 2-ethyl hexanol using Rhizopus oryzae lipase. A molar ratio of 1:2 of p-methoxycinnamic acid and 2-ethyl hexanol was found to give an optimum yield using cyclo-octane (50 ml) as reaction solvent, at a temperature of 45 °C, and 750 U of lipase, resulting in a yield of 91.3 % in 96 h. This reaction was successfully scaled up to 400-ml reaction size where 88.6 %bioconversion was achieved. The synthesized compound was found to have superior antioxidant activity as compared to ascorbic acid. The synthesized compound also exhibited good antimicrobial activity against Escherichia coli, Klebsiella pneumonia, Salmonella typhi, Staphylococcus aures, Candida albicans (yeast), Aspergillus niger, Alternaria solani, and Fussarium oxysporum by well diffusion method in terms of zone of inhibitions (in mm).     

Five-Year Field Results and Long-Term Effectiveness of 20 mg/kg Liposomal Amphotericin B (Ambisome) for Visceral Leishmaniasis in Bihar,India

Background

Visceral Leishmaniasis (VL; also known as Kala-azar) is an ultimately fatal disease endemic in Bihar. A 2007 observational cohort study in Bihar of 251 patients with VL treated with 20 mg/Kg intravenous liposomal amphotericin B (Ambisome) demonstrated a 98% cure rate at 6-months. Between July 2007 and August 2012, Médecins Sans Frontières (MSF) and the Rajendra Memorial Research Institute (RMRI) implemented a VL treatment project in Bihar, India—an area highly endemic for Leishmania donovani—using this regimen as first-line treatment.

Methods and Principal Findings

Intravenous Ambisome 20 mg/kg was administered in four doses of 5 mg/kg over 4–10 days, depending on the severity of disease. Initial clinical cure at discharge was defined as improved symptoms, cessation of fever, and recession of spleen enlargement. This observational retrospective cohort study describes 8749 patients with laboratory-confirmed primary VL treated over a 5-year period: 1396 at primary healthcare centers, 7189 at hospital, and 164 at treatment camps. Initial clinical cure was achieved in 99.3% of patients (8692/8749); 0.3% of patients (26/8749) defaulted from treatment and 0.4% (31/8749) died. Overall, 1.8% of patients (161/8749) were co-infected with HIV and 0.6% (51/8749) with tuberculosis. Treatment was discontinued because of severe allergic reactions in 0.1% of patients (7/8749). Overall, 27 patients (0.3%) were readmitted with post Kala-azar dermal leishmaniasis (PKDL). Risk factors for late presentation included female sex, age >15 years and being from a scheduled caste.In 2012, a long-term efficacy survey in the same area of Bihar determined relapse rates of VL after 5 years'' intervention with Ambisome. Of 984 immunocompetent patients discharged between September 2010 and December 2011, 827 (84.0%) were traced in order to determine their long-term outcomes. Of these, 20 patients (2.4%) had relapsed or received further treatment for VL. Of those completing 6, 12, and 15 month follow-up, 0.3% (2/767), 3.7% (14/383), and 2.4% (4/164), respectively, had relapsed. The mean ±SD time-to-relapse was 9.6±3.0 months.

Significance

This is the largest cohort of VL patients treated with 20 mg/kg Ambisome worldwide. The drug has high initial and long-term efficacy, and a low rate of adverse reactions when administered under field conditions in Bihar, India. Although challenging, its use as first line treatment in rural settings in Bihar is safe and feasible.     

Stationary-state mutagenesis inEscherichia coli: A model

Stationary-phase mutagenesis in nondividingE. coli cells exposed to a nonlethal stress was, a few years ago, claimed to be a likely case of a Lamarckian mechanism capable of producing exclusively useful mutations in a directed manner. After a heated debate over the last decade it now appears to involve a Darwinian mechanism that generates a transient state of hypermutagenesis, operating on a large number of sites spread over the entire genome, at least in a proportion of the resting cells. Most of the studies that clarified this position were on the reversion of a frameshift mutation present in alacI-lacZ fusion inE. coli strain FC40. Several groups have extensively examined both the sequence changes associated with these reversions and the underlying genetic requirements. On the basis of our studies on the genomic sequence analysis, we recently proposed a model to explain the specific changes associated with the reversion hotspots. Here we propose a more detailed version of this model that also takes into account the observed genetic requirements of stationary-state mutagenesis. Briefly, G:T/U mismatches produced at methylatable cytosines are preferentially repaired in nondividing cells by the very short patch mismatch repair (VSPMR) mechanism which is itself mutagenic and can produce mutations in very short stretches located in the immediate vicinity of these cytosine methylation sites. This mechanism requires a homologous or homeologous strand invasion step and an error-prone DNA synthesis step and is dependent on RecA, RecBCD and a DNA polymerase. The process is initiated near sequences recognized by Dcm and Vsr enzymes and further stimulated if these sequences are a part of CHI or CHI-like sequences, but a double-strand-break-dependent recombination mediated by the RecBCD pathways proposed by others seems to be nonessential. The strand transfer step is proposed to depend on RecA, RuvA, RuvB and RuvC and is opposed by RecG and MutS. The model also gives interesting insights into the evolution of theE. coli genome.     

Ventriculomegaly associated with ependymal gliosis and declines in barrier integrity in the aging human and mouse brain

Age‐associated ventriculomegaly is typically attributed to neurodegeneration; however, additional factors might initiate or contribute to progressive ventricular expansion. By directly linking postmortem human MRI sequences with histological features of periventricular tissue, we show that substantial lateral ventricle surface gliosis is associated with ventriculomegaly. To examine whether loss of ependymal cell coverage resulting in ventricle surface glial scarring can lead directly to ventricle enlargement independent of any other injury or degenerative loss, we modeled in mice the glial scarring found along the lateral ventricle surface in aged humans. Neuraminidase, which cleaves glycosidic linkages of apical adherens junction proteins, was administered intracerebroventricularly to denude areas of ependymal cells. Substantial ependymal cell loss resulted in reactive gliosis rather than stem cell‐mediated regenerative repair of the ventricle lining, and the gliotic regions showed morphologic and phenotypic characteristics similar to those found in aged humans. Increased levels of aquaporin‐4, indicative of edema, observed in regions of periventricular gliosis in human tissue were also replicated in our mouse model. 3D modeling together with volume measurements revealed that mice with ventricle surface scarring developed expanded ventricles, independent of neurodegeneration. Through a comprehensive, comparative analysis of the lateral ventricles and associated periventricular tissue in aged humans and mouse, followed by modeling of surface gliosis in mice, we have demonstrated a direct link between lateral ventricle surface gliosis and ventricle enlargement. These studies highlight the importance of maintaining an intact ependymal cell lining throughout aging.     

Cyclosporin A and rabbit mammary prolactin receptors

The binding of cyclosporin A and ovine prolactin to rabbit mammary gland membranes was determined. CsA bound with a Kd of 2.2 X 10(-6)M whereas prolactin bound with a Kd of 2 X 10(-10)M. The binding of each ligand was an independent event and neither ligand influenced the binding of the other ligand showing that CsA does not inhibit the binding of prolactin to its specific receptor in this system.     

Anionic Surfactant,Linear Alkyl Benzene Sulphonate Induced Oxidative Stress and Hepatic Impairments in Fish <Emphasis Type="Italic">Channa punctatus</Emphasis>

Linear alkyl benzene sulphonate (LAS), one of the main ingredients used in synthetic detergents to enhance their cleansing properties. Indiscriminate and untreated discharge of detergents and their residues in both lantic and lotic habitats pose a variety of ecological threats and also adversely affect aquatic fauna. In vivo, LAS metabolism and biotransformation occurs via monooxygenases in liver, leading to Reactive Oxygen Species, ROS, production and consequently oxidative stress by disturbing cellular antioxidant enzymatic equilibrium. Present study aims to evaluate the activities of two widely distributed antioxidant enzymes viz., catalase (CAT) and superoxide dismutase (SOD) and ROS induced histological impairments in liver of freshwater fish, Channa punctatus. For the estimation of oxidative stress and hepatic impairments, well acclimatized fishes were divided in three groups. Fish of group G1 serves as control whereas fish of the other two groups, G2 and G3 were exposed to two fractions, 1/20th and 1/10th of 96 h LC₅₀ of LAS for 24, 48, 72 and 96 h of exposure periods. Our results showed a significant induction in CAT and SOD activities in liver tissue of C. punctatus in a dose and time dependent manner. ROS induced histopathological impairments in hepatic tissues are characterized by loosely arranged, irregularly distributed and degenerated hepatocytes with increased vacuolization and pyknotic nuclei. The results are quite suggestive that LAS intoxication generates oxidative stress by ROS production which brings about histopathological impairments in exposed fish.     

Roles of the sequence encoding tobacco etch virus capsid protein in genome amplification: requirements for the translation process and a cis-active element.

The roles of the capsid protein (CP) and the CP coding sequence of tobacco etch potyvirus (TEV) in genome amplification were analyzed. A series of frameshift-stop codon mutations that interrupted translation of the CP coding sequence at various positions were introduced into the TEV genome. A series of 3' deletion mutants that lacked the CP coding sequence beyond each of the frameshift-stop codon mutations were also produced. In addition, a series of 5' CP deletion mutants were generated. Amplification of genomes containing either frameshift-stop codon insertions after codons 1, 59, 103, and 138 or genomes containing the corresponding 3' deletions of the CP coding sequence was reduced by 100- to 1,000-fold relative to that of the parental genome in inoculated protoplasts. In contrast, a mutant containing a frameshift-stop codon after CP position 189 was amplified to 27% of the level of the parental virus, but the corresponding 3' deletion mutant lacking codons 190 to 261 was nonviable. Deletion mutants lacking CP codons 2 to 100, 2 to 150, 2 to 189, and 2 to 210 were amplified relatively efficiently in protoplasts, but a deletion mutant lacking codons 2 to 230 was nonviable. None of the amplification-defective frameshift-stop codon or deletion mutants was rescued in transgenic cells expressing TEV CP, although the transgenic CP was able to rescue intercellular movement defects of replication-competent CP mutants. Coupled with previous results, these data led to the conclusions that (i) TEV genome amplification requires translation to a position between CP codons 138 and 189 but does not require the CP product and (ii) the TEV CP coding sequence contains a cis-active RNA element between codons 211 and 246. The implications of these findings on mechanisms of RNA replication and genome evolution are discussed.     

Laser power dependence of mechanoluminescence in metals

Mechanoluminescence (ML) glow is produced on the back side when the front of a metal sample is irradiated with infrared Nd:YAG laser pulses. An incident laser beam with a power density below the plasma‐flare onset threshold causes a rise in temperature in the studied metal. As the incident laser power density increases, the intensity of the ML glow signal also increases. On the basis of the laser power density‐induced temperature, an expression is derived for the temperature‐induced thermal stress. An expression is derived for the correlation between thermal stress and laser power density, which indicates that the temperature‐induced thermal stress is directly related to the incident laser power density. In the region of plastic deformation, temperature‐induced thermal stress is related to the strain and, consequently, to the emitted ML intensity. Finally, an expression is derived for the laser power dependence of the ML intensity, and good agreement is found between the theoretical and experimental results. Copyright © 2016 John Wiley & Sons, Ltd.