Identification and validation of potential genotoxic impurities, 1,3-dichloro-2-propanol, and 2,3-dichloro-1-propanol, at subtle levels in a bile acid sequestrant, colesevelam hydrochloride, using hyphenated GC-MS technique.

Potential genotoxic impurities (PGI) and N-nitrosamine impurities in active pharmaceutical ingredients (APIs) and their determination at low levels are substantial challenges for cholesterol-lowering agents in recent years. Herein we developed a robust, reliable, rapid, accurate and validated technique of gas chromatography equipped with a mass spectrometer (GC-MS) for quantifying subtle levels of 1,3-dichloro-2-propanol (PGI-I) and 2,3-dichloro-1-propanol (PGI-II) in colesevelam hydrochloride drug substance (bile acid sequestrant). The separation of colesevelam hydrochloride, PGI-I and PGI-II was executed with chromatographic technique using a capillary column, DB-624 measuring with 30 m × 0.32 mm × 1.8 µm specification of 6% cyanopropylphenyl-94% dimethylpolysiloxane copolymer and helium carrier gas. This developed technique gave a good intensity peak without any interference and extra masses at the retention times of 11.17 min for PGI-I and 11.59 min for PGI-II, which was adequate, with mass spectra (m/z) of 79 and 62, respectively. The method's sensitivity and linearity are demonstrated by its detection and quantification limits at subtle levels with correlation coefficients of 0.9965 for PGI-I and 0.9910 for PGI-II. The determination is mainly focused on improving sensitivity with the limits of detection and quantitation far below the specifications, which can support tighter limits. This results in a cost-effective and easily adoptable methodology having precise and accurate results in colesevelam hydrochloride API at subtle levels.

A randomized, double-blind, parallel-group, single-dose comparative pharmacokinetic study of DRL_TZ, a candidate biosimilar of trastuzumab, with Herceptin® (EU) in healthy adult males.

BACKGROUND & OBJECTIVES: Trastuzumab (TZ) is a recombinant DNA-derived humanized monoclonal antibody approved for human epidermal growth factor receptor 2 positive early breast cancer, metastatic breast and gastric cancers. For the development of TZ biosimilars, establishing pharmacokinetic equivalence is required. The primary objective of this study was to compare the pharmacokinetics (PK) of Dr Reddy's Laboratories TZ (DRL_TZ) with that of EU-approved Reference Medicinal Product (RMP), Herceptin® in healthy adult male subjects. METHODS: In this double-blind, parallel-group, phase I study (TZ-01-003), healthy male subjects aged 18-55 yr were randomized 1:1 to receive a single intravenous infusion of 6 mg/kg of TZ as DRL_TZ or RMP. Similarity for primary PK parameters was defined as the 90 per cent confidence intervals (CIs) for the geometric mean ratios (GMRs) falling within 75-133 per cent limits. Primary endpoints included area under the concentration-time curve - from time zero (pre-dose) to the last quantifiable concentration [AUC(0-t)] and from time zero (pre-dose) extrapolated to infinity [AUC(0-∞)], and maximum observed serum concentration (Cmax). Secondary objectives were to compare the safety and immunogenicity of DRL_TZ with that of the RMP. RESULTS: Thirty two subjects were dosed (DRL_TZ, 16; RMP, 16). Primary PK parameters were found to be comparable with their 90 per cent CIs for the GMR falling within the usual more stringent limits of 80-125 per cent. The number of subjects reporting at least one TEAE in both the arms was similar. No serious adverse events were reported. Fifteen subjects, eight in DRL_TZ arm and seven in Herceptin® arm, tested positive for anti-drug antibodies (ADAs), none of the ADAs were neutralizing in nature. INTERPRETATION & CONCLUSIONS: In this study, DRL_TZ demonstrated PK equivalence with the RMP and had comparable safety and immunogenicity profiles in healthy adult male subjects.

Molecular mechanisms in grass-Epichloë interactions: towards endophyte driven farming to improve plant fitness and immunity.

All plants harbor many microbial species including bacteria and fungi in their tissues. The interactions between the plant and these microbes could be symbiotic, mutualistic, parasitic or commensalistic. Mutualistic microorganisms are endophytic in nature and are known to play a role in plant growth, development and fitness. Endophytes display complex diversity depending upon the agro-climatic conditions and this diversity could be exploited for crop improvement and sustainable agriculture. Plant-endophyte partnerships are highly specific, several genetic and molecular cascades play a key role in colonization of endophytes in host plants leading to rapid changes in host and endophyte metabolism. This results in the accumulation of secondary metabolites, which play an important role in plant defense against biotic and abiotic stress conditions. Alkaloids are one of the important class of metabolites produced by Epichloë genus and other related classes of endophytes and confer protection against insect and mammalian herbivory. In this context, this review discusses the evolutionary aspects of the Epichloë genus along with key molecular mechanisms determining the lifestyle of Epichloë endophytes in host system. Novel hypothesis is proposed to outline the initial cellular signaling events during colonization of Epichloë in cool season grasses. Complex clustering of alkaloid biosynthetic genes and molecular mechanisms involved in the production of alkaloids have been elaborated in detail. The natural defense and advantages of the endophyte derived metabolites have also been extensively discussed. Finally, this review highlights the importance of endophyte-arbitrated plant immunity to develop novel approaches for eco-friendly agriculture.

In situ reversible redox switching of first hyperpolarizability of bimetallic ruthenium complexes.

In this article we report the reversible redox switching of first hyperpolarizability of bimetallic ruthenium complexes bridged by bipyridyl tetrazine (bptz) ligands by second harmonic light scattering experiments (SHLS). We have synthesised [RuII(acac)2(CH3CN)2] and [(acac)2Ru-bptz-Ru(acac)2] complexes and measured their first hyperpolarizabilities as a function of in situ electrochemical oxidation/reduction of the metal centres. As a result of the oxidation of ruthenium centre from Ru(ii) to Ru(iii), the molecular hyperpolarizability of the complexes went up. The mixed-valence intermediate bimetallic complex and not the fully oxidized complex exhibit the highest ß value of 780 × 10-30 esu. We also demonstrated that the hyperpolarizability is reversible with the change of the oxidation state of the metal and both the complexes are stable for several cycles of redox switching. The experimental results are also supported by calculations.

An immunoinformatics approach to define T cell epitopes from polyketide and non-ribosomal peptide synthesis proteins of Mycobacterium tuberculosis as potential vaccine candidates.

The role of polyketide and non-ribosomal proteins from the class of small molecule metabolism of Mycobacterium tuberculosis is well documented in envelope organization, virulence, and pathogenesis. Consequently, the identification of T cell epitopes from these proteins could serve to define potential antigens for the development of vaccines. Fourty-one proteins from polyketide and non-ribosomal peptide synthesis of small molecule metabolism proteins of M tuberculosis H37Rv were analyzed computationally for the presence of HLA class I binding nanomeric peptides. All possible overlapping nanomeric peptide sequences from 41 small molecule metabolic proteins were generated through in silico and analyzed for their ability to bind to 33 alleles belonging to A, B, and C loci of HLA class I molecule. Polyketide and non-ribosomal protein analyses revealed that 20% of generated peptides were predicted to bind HLA with halftime of dissociation T1/2  ≥ 100 minutes, and 77% of them were mono-allelic in their binding. The structural bases for recognition of nanomers by different HLA molecules were studied by structural modeling of HLA class I-peptide complexes. Pathogen peptides that could mimic as self-peptides or partially self-peptides in the host were excluded using a comparative study with the human proteome; thus, subunit or DNA vaccines will have more chance of success.

Biochemical markers assisted screening of Fusarium wilt resistant Musa paradisiaca (L.) cv. puttabale micropropagated clones.

An efficient protocol was standardized for screening of panama wilt resistant Musa paradisiaca cv. Puttabale clones, an endemic cultivar of Karnataka, India. The synergistic effect of 6-benzyleaminopurine (2 to 6 mg/L) and thidiazuron (0.1 to 0.5 mg/L) on MS medium provoked multiple shoot induction from the excised meristem. An average of 30.10 +/- 5.95 shoots was produced per propagule at 4 mg/L 6-benzyleaminopurine and 0.3 mg/L thidiazuron concentrations. Elongation of shoots observed on 5 mg/L BAP augmented medium with a mean length of 8.38 +/- 0.30 shoots per propagule. For screening of disease resistant clones, multiple shoot buds were mutated with 0.4% ethyl-methane-sulfonate and cultured on MS medium supplemented with Fusarium oxysporum f. sp. cubense (FOC) culture filtrate (5-15%). Two month old co-cultivated secondary hardened plants were used for screening of disease resistance against FOC by the determination of biochemical markers such as total phenol, phenylalanine ammonia lyase, oxidative enzymes like peroxidase, polyphenol oxidase, catalase and PR-proteins like chitinase, beta-1-3 glucanase activities. The mutated clones of M. paradisiaca cv. Puttabale cultured on FOC culture filtrate showed significant increase in the levels of biochemical markers as an indicative of acquiring disease resistant characteristics to FOC wilt.

WS2-Nanosheet-Modified Electrodes as an Efficient Electrochemical Sensing Platform for the Nonenzymatic Detection of the Insecticide Imidacloprid.

Imidacloprid (IMI) is a systemic insecticide, which is widely used for seed treatment and pest control in vegetables. The unwarranted presence of traces of IMI in vegetables and groundwater is a matter of grave concern which needs to be detected and quantified in order to effect remedial measures for the sake of food safety. In this work, we communicate the fabrication of tungsten sulfide (WS2) nanosheets and the construction of an amperometric sensor for the precise determination of IMI. The sensor performances were evaluated by using cyclic voltammetry (CV). The presence of surface-active sites and the fast electron transfer on WS2/GCE favored the electrochemical reduction of the aromatic nitro group in IMI. The developed IMI sensor displayed a linear range of IMI detection from 10 to 90 µM with a detection limit of 0.28 µM. The developed WS2/GCE sensor also displayed good sensitivity, with a value of 3.98 µA µM-1 cm-2. The electrochemical measurements demonstrated the superior selectivity of the constructed WS2/GCE sensor for IMI detection, which makes it suitable for practical applications.

Determination and quantification of related substances and degradation products in bictegravir by full factorial design evaluated HPLC and mass spectrometry.

Determining and quantifying novel impurities and degraded impurities of a drug product is always a continuous challenge to enhancing the drug quality for patients' safety. Herein, our work deals with (i) developing a rapid, accurate, and reliable high-performance liquid chromatographic validation method to quantify the bictegravir drug (integrase inhibitors of antiretroviral drugs) and its novel related impurities at low levels, and (ii) the liquid chromatography-mass spectrometry (LC-MS) method to identify degraded impurities. Separation of bictegravir acid (impurity-I) and methyl bictegravir (impurity-II) impurities which are identified by LC-MS in the bictegravir drug was executed by developing a method and the same method performance evaluated by using full factorial design. This developed analytical technique gave a well-separated peak of bictegravir and related analytes such as bictegravir acid (impurity-I) and methyl bictegravir (impurity-II), adequate with the peak properties as per USP guidelines. The method's sensitivity and linearity are demonstrated by its detection and quantification limits at low levels with a correlation coefficient of 0.998. The method's repeatability, specificity, and accuracy suggest that this developed technique is a reliable determination strategy for the bictegravir drug substance and its related impurities (impurity-I and impurity-II) in a simple, feasible, and affordable way.

Direct and indirect method of plant regeneration from root explants of Caesalpinia bonduc (L.) Roxb.--a threatened medicinal plant of Western Ghats.

An in vitro regeneration protocol has been standardized via direct and indirect methods from excised root explants of C. bonduc, a threatened woody legume used for the treatment of contagious diseases, inflammation, leprosy, antiperiodic, febrifuge, anthelmenthic, urinary disorders, leucorrhoea, piles and to heal wounds. MS medium supplemented with 17.75 micromol BAP and 2.46 micromol IBA, induced a mean of 3.40 +/- 1.07 shoots directly from the surface of excised root explant. Subsequently, the shoots rooted readily on MS half strength medium with out growth regulators. In indirect organogenesis, callogenic frequency was optimized (96.66%) at the concentration of 9.04 micromol 2, 4-D and 0.88 micromol BAP. An average, 15.30 +/- 5.25 shoots were differentiated from the root callus at the concentration of 17.57 micromol BAP and 2.85 micromol IAA. Shoots regenerated through callus were rooted well on MS half strength medium with growth regulators at 2.95 micromol IBA. Rooted plantlets were transferred to the pots containing sterilized soil and were successfully hardened at greenhouse condition for three weeks then exposed to the natural environment. Survival rate was more (95%) in plantlets derived through direct organogenesis than (60%) the plantlets regenerated through root calli.

Regeneration of plantlets from mature embryo calli of Western Ghats land race cultivar of rice, Oryza sativa L.

The Malnad region located in the Western Ghats of Karnataka is known for the cultivation of indigenous rain fed land race cultivar of rice. The present study was to investigate the callogenic and caulogenic potentialities of the two indigenous rice cultivar namely Karimundaga and Kanadatumba using dehusked mature embryo explants. For callus and shoot bud differentiation, the explants were cultured on Murashige and Skoog (MS) medium supplemented with 2,4-D (1-3 mg/L), IAA (1-2 mg/L), Kn (1-4 mg/L) and BAP (1-4 mg/L). The morphogenic potentialities of the two rice cultivar differed in texture of callus. In both the cultivar callogenic frequency was optimized at 1 mg/L 2,4-D concentration, it was 94% in Karimundaga and 58% in Kanadatumba. Supplementation of IAA either alone (1-2 mg/L) or in combination with Kn or BAP at 1 to 4 mg/L concentration of each induces shoot bud differentiation from the calli. In the cultivar Karimundaga caulogenic frequency was highest (10.60 +/- 2.55) at 1.0 mg/L IAA and 4.0 mg/L BAP concentration. While in the cultivar Kanadatumba highest number of shoot buds (7.90 +/- 2.69) was differentiated at 1.0 mg/L IAA and 4.0 mg/L Kn concentration. The calli derived regenerants were successfully acclimatized in the greenhouse and agro-morphological variations were evaluated. The growth characteristics and yield related parameters exhibited by in vitro plants were lower than the in vivo plants.

Stress and Strain Created Around Miniscrews during En Masse Retraction - A Finite Element Method.

Background: En masse retraction of the anterior teeth may create anchorage problems. The effective means of obtaining anchorage in en masse retraction is by the usage of temporary anchorage devices. Aims and Objectives: The purpose of this study is focused on stress and strain created around miniscrew during en masse retraction using the cone beam computed tomography (CBCT) data obtained at two intervals and by the finite element analysis done using the CBCT data obtained. This will show us the stress and strain created around miniscrews and the withstanding capacity of miniscrews. Conclusion: Stress and strain was higher in TADs as compared to cortical and alveolar bone.

Eco-Friendly Synthesis of MnO2 Nanorods Using Gmelina arborea Fruit Extract and Its Anticancer Potency Against MCF-7 Breast Cancer Cell Line.

INTRODUCTION: Cancer disease is known due to its unregulated proliferation of cells that have evolved from the body's regular cells. The disease develops as a result of epigenetic and genetic modifications, tumor suppressor gene inactivation, and oncogene activation. The present work describes an environmentally benign approach for the synthesis of manganese oxide nanoparticles (MnO2 NPs) using Gmelina arborea fruit extract (GAE) in an aqueous medium. METHODS: The study evaluated the formation of MnO2 NPs and their anticancer efficacy against MCF-7 breast cancer cell line. RESULTS: The formation of MnO2 NPs was confirmed through powder X-ray diffractometer (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HR-TEM). The crystalline nature of as-prepared MnO2 NPs was evident from XRD pattern. The morphology of the material was studied using SEM analysis, which suggested a rod-like nature with an average diameter of 50 nm. Further, the TEM and HR-TEM images confirmed the rod shape of the as-prepared MnO2 NPs with an interplanar distance of 0.271 nm. In addition, the concentric rings from selected area electron diffraction (SAED) analysis show the crystalline nature of the as-prepared material, which further supports the obtained XRD pattern. The anticancer efficacy of MnO2 NPs was evaluated against MCF-7 breast cancer cell line, which showed up to 96% inhibition of the cells at 400 µg/mL concentration. CONCLUSION: Bio-conjugation of MnO2 NPs can provide enough scope for the therapeutic use of Gmelina arborea, assuming appropriate mechanistic evaluations are conducted.

Functional Outcome of Open Latarjet Procedure in Non-Athletic Middle-Aged Patients.

INTRODUCTION: The movement and steadiness of the shoulder joint is due to both the dynamic and static stabilisers. Recurrent anterior shoulder instability is common due to the Bankart lesion or the Hill Sachs lesion. The bone loss and soft tissue failure due to these lesions causing instability is well compensated by Latarjet procedure which acts by triple blocking effect of the bone graft, the sling effect of the conjoint tendon of subscapularis and the ligament of the coracoacromial ligament stump. MATERIALS AND METHODS: Middle-aged patients with recurrent anterior shoulder dislocation and a mid-range instability on clinical assessment with an isolated glenoid bone loss of 20% or Bankart lesion with engaging Hill Sachs lesion were selected for the study. The surgical procedure included a subscapularis split to expose the glenoid. The coracoid graft harvested was prefixed with Kirschner wires and placed flush over the glenoid ensuring no medial or lateral overhang and fixed with 4.0mm cancellous screws with the washer. The functional outcome was measured with the ROWE score and ASES score and the movements were evaluated. RESULTS: A total of 24 patients fulfilled the inclusion criteria. Post-operatively at final follow-up, the mean ROWE score was 97.08 ±8.45 and the mean ASES score was 94.4±9.10. One patient had screw breakage as a complication and another had restriction of movement which was managed with physiotherapy. CONCLUSION: Open Latarjet is an effective procedure for recurrent anterior shoulder instability in non-athletic middle-aged patients as a excellent functional outcome was achieved with this technique. We therefore recommend open Latarjet as an alternative to arthroscopic treatment in developing countries where patient affordability and the availability of the resources are the issues.

Isolation and characterization of densonucleosis virus from Aedes aegypti mosquitoes and its distribution in India.

OBJECTIVES: Mosquito densonucleosis viruses (DNVs) are known to persistently infect the insect cell line and mosquito population in nature, causing mortality in mosquitoes. Here we report the isolation and characterization of a DNV from Aedes aegypti and its distribution among different Ae. aegypti populations from India. METHODS: We screened Ae. aegypti mosquito populations from different states of India by PCR. Virus isolation and purification was performed using a cesium chloride gradient from a positive mosquito colony. Characterization of this isolate was carried out by electron microscopy, Western blot and sequencing. RESULTS: Electron microscopy showed the presence of parvovirus-like particles, and Western blot showed the presence of 2 viral proteins of 40 and 41 kDa. A total of 3,776 bases of genome were sequenced, which included a 3'UTR of 128 bases, a coding region of 3,507 bases and a 5'UTR of 141 bases. Three open reading frames (ORFs) were identified and characterized. The NIVDNV genome showed 95% similarity with Culex pipiens pallens DNV and 93% similarity with Ae. aegypti DNV. CONCLUSION: Phylogenetic analysis of all 3 ORFs showed that this new isolate falls in the lineage of Brevidensovirus along with other mosquito DNVs.

Tuberculin skin test results in HIV-infected patients in India: implications for latent tuberculosis treatment.

OBJECTIVE: To evaluate the utility of the tuberculin skin test (TST) in detecting latent and active tuberculosis (TB) among human immunodeficiency virus (HIV) infected patients in South India. DESIGN: TSTs and CD4 counts were collected from 631 HIV-infected individuals without active TB and 209 antiretroviral and anti-tuberculosis treatment-naïve HIV-infected patients with TB. We calculated the proportion of TST-positive individuals, as well as the sensitivity, specificity, positive predictive value (PPV) and negative predictive value of TST in the diagnosis of TB. RESULTS: Among subjects without active TB, 28% with a CD4 count <100 cells/microl vs. 43% of the total cohort had a TST >5 mm (P = 0.14), while the proportions with induration >10 mm were 14% vs. 36%, respectively (P < 0.01). Among those with active TB, using a 5 mm cut-off, the sensitivity was 42% for those with CD4 counts <200 cells/mul compared to 70% for those with CD4 counts >or=200 cells/microl (P < 0.001). The PPV for detecting active TB was 29%. CONCLUSIONS: TST is a poor predictor of both latent and active TB in HIV-infected individuals in TB endemic countries. Programmes offering treatment for latent TB should consider including all HIV-positive patients regardless of TST status, or use other indicators, such as CD4 count.

A hot probe setup for the measurement of Seebeck coefficient of thin wires and thin films using integral method.

An experimental setup is developed for the measurement of the Seebeck coefficient of thin wires and thin films in the temperature range of 300-650 K. The setup makes use of the integral method for measuring the Seebeck voltage across the sample. Two pointed copper rods with in-built thermocouples serve as hot and cold probes as well as leads for measuring the Seebeck voltage. The setup employs localized heating and enables easy sample loading using a spring loaded mounting system and is fully automated. Test measurements are made on a constantan wire and indium tin oxide (ITO) thin film for illustration. The Seebeck voltage obtained for constantan wire is in agreement with the NIST data for copper constantan couple with an error of 1%. The calculated carrier concentration of ITO film from the Seebeck coefficient measurement is comparable with that obtained by electrical transport measurements. The error in the Seebeck coefficient is estimated to be within 3%.

Efficacy of Alpha Glucosidase Inhibitor from Marine Actinobacterium in the Control of Postprandial Hyperglycaemia in Streptozotocin (STZ) Induced Diabetic Male Albino Wister Rats.

The current study was carried out to evaluate the in-vitro and in-vivo efficiency of alpha glucosidase inhibitor of marine actinobacteria in the control of postprandial hyperglycaemia. Soil samples were collected from salterns, coastal area in Kothapatnam, Ongole, Andhra Pradesh, India. Among the actinobacterial isolates tested for yeastα-glucosidase inhibitory activity, only three isolates showed prominent inhibition. The patient isolate was selected and identified as Streptomyces coelicoflavus SRBVIT13 using 16S r-RNA gene sequencing. In in-vitro studies, the chloroform extract of Streptomyces coelicoflavus SRBVIT13 showed significant enzyme inhibitory activity against yeast and mammalian α-glucosidaseenzymes. In animal studies, the oral ingestion of chloroform extract (600 mg/kg) of S. coelicoflavus SRBVIT13 in maltose and sucrose loaded diabetic rats, showed significant regulation of postprandial blood glucose by 82.25% and a 77.25% reduction, respectively. The lead compound from S. coelicoflavusSRBVIT13 was isolated, purified, characterized, and identified by stranded analytical techniques as 2-t-butyl-5-chloromethyl-3-methyl-4-oxoimidazolidine-1-carboxylic acid, t-butyl ester. The results obtained in the present study are promising and the bioactive compound from S. coelicoflavusSRBVIT13 may be considered as a potential agent in regulating the postprandial hyperglycaemia.

EphB4 receptor tyrosine kinase is expressed in bladder cancer and provides signals for cell survival.

We sought to evaluate the biological function of the receptor tyrosine kinase EphB4 in bladder cancer. All of the nine bladder cancer cell lines examined express EphB4 and the receptor could be phosphorylated following stimulation with its cognate ligand, EphrinB2. Out of the 15 fresh bladder cancer specimens examined, 14 expressed EphB4 with a mean sevenfold higher level of expression compared to adjacent normal urothelium. EphB4 expression was regulated by several mechanisms: EPHB4 gene locus was amplified in 27% tumor specimens and 33% cell lines studied; inhibition of EGFR signaling downregulated EphB4 levels; and forced expression of wild-type p53 reduced EphB4 expression. EphB4 knockdown using specific siRNA and antisense oligodeoxynucleotides molecules led to a profound inhibition in cell viability associated with apoptosis via activation of caspase-8 pathway and downregulation of antiapoptotic factor, bcl-xl. Furthermore, EphB4 knockdown significantly inhibited tumor cell migration and invasion. EphB4 knockdown in an in vivo murine tumor xenograft model led to a nearly 80% reduction in tumor volume associated with reduced tumor proliferation, increased apoptosis and reduced tumor microvasculature. EphB4 is thus a potential candidate as a predictor of disease outcome in bladder cancer and as target for novel therapy.

Clearance of Alzheimer's amyloid-ss(1-40) peptide from brain by LDL receptor-related protein-1 at the blood-brain barrier.

Elimination of amyloid-ss peptide (Ass) from the brain is poorly understood. After intracerebral microinjections in young mice, (125)I-Ass(1-40) was rapidly removed from the brain (t(1/2)