Development and validation of a stability-indicating RP-HPLC method for the identification, determination of related substances and assay of dimethyl fumarate in drug substance.

OBJECTIVE: The objective of current study was to develop and validate a short, economical, accurate, precise stability-indicating RP-HPLC method for identification, quantitation of related substances (fumaric acid and mono methyl fumarate) and assay of dimethyl fumarate (DMF) drug substance. MATERIAL AND METHODS: The RP-HPLC method was developed by using liquid chromatography (waters 2695 with PDA detector & Agilent 1200 with DAD) with Symmetry C18 column. Pharmaceutical grade of high pure materials of DMF, MMF, FA and HPLC grade water, acetonitrile and orthophosphoric acid were used for this study. The mobile phase consists of 0.1% of ortho-phosphoric acid in water: acetonitrile (55:45% v/v). RESULTS: The developed method was validated according to ICH guidelines. To prove the stability indicating potential, stress studies performed using acid, base, peroxide and thermal. After sufficient exposure, these solutions were injected in to HPLC and found that all degradants formed during stress study were well separated from the main peak and resolution between all impurities was more than ICH requirements. CONCLUSION: A simple, short and stability indicating RP-HPLC method was developed and validated for simultaneous estimation of DMF and its related substances in drug substance. Based on literature survey it was evident that many methods were published for determination of DMF individually or its related substances, however short run time methods were not available for simultaneous estimation of DMF and its related substances. The intended method would support to industries for quick quantitation of DMF and its related substances without compromising quality parameters like precision and accuracy.

Demodex canis targets TLRs to evade host immunity and induce canine demodicosis.

Widespread incidence of Demodex mites throughout the mammalian class and occasional serious and fatal outcomes in dogs warrant an insight into the host-parasite interface especially. Therefore, this study was aimed to unravel the interplay between innate immune response and canine demodicosis. The dogs diagnosed to have natural clinical demodicosis were allocated into two groups; dogs with localized demodicosis (LD) and with generalized demodicosis (GD). The expression of toll-like receptors (TLRs) 2, 4 and 6 genes in peripheral blood mononuclear cells of these dogs was quantified by real-time PCR. Significantly increased TLR2 gene expression, while significantly diminished TLR4 and TLR6 gene expressions were observed in demodicosed dogs (LD and GD) as compared with the healthy ones. Even the expression of TLR2 gene was found to differ significantly between the dogs with LD and GD. Therefore, it can be inferred that clinical demodicosis in dogs is coupled with an up-regulation of TLR2 and down-regulation of TLR4 and TLR6 gene expressions. Overexpression of TLR2 gene might be responsible for Demodex-induced clinical manifestations, while TLR4 and TLR6 gene down-regulations could be the paramount strategy of Demodex mites to elude the host-immune interface.

Demodex canis regulates cholinergic system mediated immunosuppressive pathways in canine demodicosis.

Demodex canis infestation in dogs remains one of the main challenges in veterinary dermatology. The exact pathogenesis of canine demodicosis is unknown but an aberration in immune status is considered very significant. No studies have underpinned the nexus between induction of demodicosis and neural immunosuppressive pathways so far. We have evaluated the involvement of cholinergic pathways in association with cytokines regulation as an insight into the immuno-pathogenesis of canine demodicosis in the present study. Remarkable elevations in circulatory immunosuppressive cytokine interleukin-10 and cholinesterase activity were observed in dogs with demodicosis. Simultaneously, remarkable reduction in circulatory pro-inflammatory cytokine tumour necrosis factor-alpha level was observed in dogs with demodicosis. Findings of the present study evidently suggest that Demodex mites might be affecting the cholinergic pathways to induce immunosuppression in their host and then proliferate incessantly in skin microenvironment to cause demodicosis.

Seasonal variations in seminal plasma proteins of buffalo.

The study was designed to evaluate the influence of season on semen characteristics and seminal plasma protein profile of buffalo bull semen. Thirty-six ejaculates were collected in three seasons (winter, summer and rainy) from six adult Bhadawari bulls, and semen characteristics were evaluated immediately after collection. The seminal plasma was harvested by centrifugation and protein profiling, and percentage protein fractions were analysed by SDS-PAGE. The significant effect of season was observed on ejaculate volume, sperm concentration, progressive motility, percentage live spermatozoa, hypo-osmotic swelling test (HOST) and acrosomal integrity. The electrophoretogram of seminal plasma proteins revealed 20 protein bands in winter, 23 bands in rainy and 25 bands in summer seasons, illustrating the significant effect of seasons on seminal plasma proteins. Among these protein bands, 18 bands were observed common in semen samples of all three seasons while protein bands of 46, 55, 58, 144 and 160 kDa were found in rainy and summer seasons. The protein bands of 48 and 60 kDa were observed only in winter season, whereas 184 and 200 kDa were reported in summer season only. The protein fractions (protein%) of common protein bands observed in three seasons revealed a significant effect of season on protein bands of 24.5, 66, 70, 72, 84 and 86 kDa. From the study, it was pertinent that bull seminal plasma contains specific proteins in particular season, which may be associated with some of the semen characteristics, and these proteins could be used as markers of the semen quality of buffalo bulls.

Automated detection, delineation and quantification of whole-body bone metastasis using FDG-PET/CT images.

Non-small cell lung cancer (NSCLC) patients with the metastatic spread of disease to the bone have high morbidity and mortality. Stereotactic ablative body radiotherapy increases the progression free survival and overall survival of these patients with oligometastases. FDG-PET/CT, a functional imaging technique combining positron emission tomography (PET) with 18 F-fluorodeoxyglucose (FDG) and computer tomography (CT) provides improved staging and identification of treatment response. It is also associated with reduction in size of the radiotherapy tumour volume delineation compared with CT based contouring in radiotherapy, thus allowing for dose escalation to the target volume with lower doses to the surrounding organs at risk. FDG-PET/CT is increasingly being used for the clinical management of NSCLC patients undergoing radiotherapy and has shown high sensitivity and specificity for the detection of bone metastases in these patients. Here, we present a software tool for detection, delineation and quantification of bone metastases using FDG-PET/CT images. The tool extracts standardised uptake values (SUV) from FDG-PET images for auto-segmentation of bone lesions and calculates volume of each lesion and associated mean and maximum SUV. The tool also allows automatic statistical validation of the auto-segmented bone lesions against the manual contours of a radiation oncologist. A retrospective review of FDG-PET/CT scans of more than 30 candidate NSCLC patients was performed and nine patients with one or more metastatic bone lesions were selected for the present study. The SUV threshold prediction model was designed by splitting the cohort of patients into a subset of 'development' and 'validation' cohorts. The development cohort yielded an optimum SUV threshold of 3.0 for automatic detection of bone metastases using FDG-PET/CT images. The validity of the derived optimum SUV threshold on the validation cohort demonstrated that auto-segmented and manually contoured bone lesions showed strong concordance for volume of bone lesion (r = 0.993) and number of detected lesions (r = 0.996). The tool has various applications in radiotherapy, including but not limited to studies determining optimum SUV threshold for accurate and standardised delineation of bone lesions and in scientific studies utilising large patient populations for instance for investigation of the number of metastatic lesions that can be treated safety with an ablative dose of radiotherapy without exceeding the normal tissue toxicity.

Untangling the effect of organic matter and dissolved oxygen on living benthic foraminifera in the southeastern Arabian Sea.

We studied the relationship between organic carbon in sediments (%Corg), bottom water dissolved oxygen and living benthic foraminifera in a marginal marine upwelling setting from the southeastern Arabian Sea to develop proxy indicator for marine productivity as well as oxygen deficient zones. The surface sediments from 43 stations covering a depth range of 25 to 2980 m were used. The relationship between living benthic foraminifera and ambient environmental parameters (seawater temperature, salinity, dissolved oxygen, organic carbon and Corg/TN and depth) was assessed by multivariate analysis. A majority of the living benthic foraminifera were significantly affected by several ambient parameters. We report that the living benthic foraminiferal assemblage comprising of Epistominella umbonifera, Uvigerina auberiana, Reophax longicollis and Osangularia bengalensis is significantly affected only by %Corg in the sediment. Additionally, we also found that the assemblage including Bolivina obscuranta, Bulimina arabiensis, Bulimina pseudoaffinis and Cancris penangensis is significantly affected only by the bottom water dissolved oxygen. The living benthic foraminifera assemblages can be used to reconstruct marine productivity and dissolved oxygen concentration in the past.

Lack of denitrification causes a difference in benthic foraminifera living in the oxygen deficient zones of the Bay of Bengal and the Arabian Sea.

Despite being located at the same latitudes, the Bay of Bengal oxygen deficient zone (ODZ) is markedly different than the Arabian Sea ODZ. The uptake of oxygen in the Bay of Bengal does not lead to denitrification as in the Arabian Sea. This difference in ODZ of the Bay of Bengal and the Arabian Sea is expected to support different benthic fauna. We report that the living benthic foraminifera in the Bay of Bengal ODZ are markedly different than that in the Arabian Sea ODZ. Only four species (Brizalina spathulata, Eubuliminella exilis, Uvigerina peregrina and Rotaliatinopsis semiinvoluta) dominant in the Bay of Bengal ODZ have also been reported from the Arabian Sea oxygen deficient waters. The difference in living benthic foraminifera dominant in the ODZ of the Bay of Bengal and the Arabian Sea, is attributed to the lack of denitrification and associated processes in the Bay of Bengal.

Focal therapy for localized prostate cancer in the era of routine multi-parametric MRI.

BACKGROUND: Prostate cancer focal therapy aims to minimize the side-effects of whole gland treatments, such as radical prostatectomy and radiotherapy without compromising oncological efficacy. However, concerns exist regarding the multifocal nature of prostate cancer and the lack of long-term oncological data for this form of treatment. In recent years, the routine adoption of multi-parametric magnetic resonance imaging (mpMRI) of the prostate has improved our ability to select candidates for focal therapy and to accurately deliver this form of prostate cancer treatment. METHODS: We performed a review of the literature to provide a summary of the oncological and functional outcomes of men receiving primary prostate focal therapy. Furthermore, we discuss the impact of the routine implementation of mpMRI as part of the initial prostate cancer diagnostic pathway on the selection of candidates and delivery of focal therapy. Finally, we summarize knowledge gaps in the field and highlight active clinical trials in this arena. RESULTS: Primary focal therapy involves the application of one of a number of energies that ablate tissue, such as cryotherapy and high intensity focused ultrasound (HIFU). Success is principally dependent on highly accurate patient selection and disease localization underpinned in large part by the routine integration of pre-biopsy mpMRI. Prospective medium-term follow-up data for primary HIFU and cryotherapy for men with intermediate-risk disease have shown acceptable cancer control with low risk of side effects and complications. Additional research is needed to clearly define an appropriate follow-up approach and to guide the management of in- and out-of-field recurrences. Multiple comparative trials with randomization against standard care are currently underway in men with intermediate- and high-risk prostate cancer. CONCLUSION: The widespread adoption of prostate mpMRI has led to improved disease localization, enabling the performance of focal therapy as a viable treatment strategy for men with low volume intermediate-risk prostate cancer.

The Demographic Study of Otorhinolaryngological Trauma Among Patients with Head and Neck Trauma and Their Management in a Tertiary Care Centre.

The trauma has been increasing in frequency, especially in recent decades because of the higher number of automobile accidents and violence. The ear, nose and face region is the area in the body that is commonly involved in head and neck injury. Trauma to this region is often associated with mortality and varying degree of physical, functional and psychological damage. To study the demography, aetiology, clinical presentation, outcome of ENT trauma in our tertiary care centre and to compare our study with previous studies to see the changing trends with passage of time. A prospective study was conducted on 200 head and neck patients with ENT injuries during a period of 1 year from July 2014 to July 2015, who were admitted in ENT Department and Trauma Centre of Gajra Raja Medical College and J.A. Group of Hospital, Gwalior. Most of the patients were young adult males of age group 18-40 years (45, 22.5%) with a male:female ratio 1.78:1. Among them 53% patients were from rural area while 47% were from urban area. RTA is the most prevalent cause of ENT trauma (37%), followed by interpersonal violence (25%). 27% of the patients had facial injury and multiple region injury each. Among 200 patients 33% were managed conservatively on the other hand 66% patients required surgical interventions. Mortality rate was 8%. Among all 184 survivors 25.5% suffered from hearing loss, 14.5% had tympanic membrane perforation with hearing loss, 12% had pinna disfigurement, 10% had change in voice, 6.5% had permanent nasal deformity, 2% had facial nerve palsy. Young male adults were the most prevalent victims of ENT trauma, and Road traffic accidents were responsible for majority of the ENT injuries. Screening of all the patients with head and neck injuries for the presence of trauma in the ENT region should be introduced to enable early detection and therefore prevention of complications.

Algorithm for perturbing thermodynamically infeasible metabolic networks.

Networks of reactions inside the cell are constrained by the laws of mass and energy balance. The second law of thermodynamics further constrains the topology of the metabolic network, by disallowing interactions that can sustain themselves when they are isolated from the rest of the network. We present an algorithm that identifies such subnetworks and makes them feasible by deleting reactions. This perturbs the network topology, but preserves the optimality of the mass balance solutions. This method has been applied to a full network of Escherichia coli.

[Urethral centralization and pseudo-glans formation after partial penectomy]. / Zentralisierung der Harnröhre und Pseudoglansbildung nach partieller Penektomie.

The management of penile cancer has altered dramatically over the last decade. Confidence to excise lesions safely with smaller margins has led to the adoption of penile-preserving techniques and in turn improved the functional and cosmetic results. Patients undergoing partial penectomy (PP) find that the urethral meatus is located in an abnormal ventral position. In addition, there is a high risk of meatal stenosis. We describe our novel technique that allows the urethral meatus to be centralised after PP and creation of a pseudo-glans and wide meatus and therefore maintain the cosmetic appearance of the penis after split thickness skin grafting. The UCAPP technique allows the restoration of the normal meatal location and creation of a pseudo-glans in case of partial penectomy and therefore can improve the overall cosmetic appearance and reduce the psychological morbidity.

Application of foraminifers in ecotoxicology: retrospect, perspect and prospect.

In view of the extensive use of foraminifers for the pollution monitoring especially their unmatched utility in deciphering the temporal variation in the type and concentration of pollutants at a given location, the literature dealing with the application of foraminifers for pollution studies has been reviewed. The pollution monitoring through foraminifers started with attribution of peculiar foraminiferal features at any location to the circumstantial presence of pollutants at that site. These peculiar features of the foraminifers in polluted areas included, variation in the abundance of total, calcareous and agglutinated foraminifers, species diversity, abnormal tests, etc. The abnormalities of tests included stunted growth, abraded margins, dissolved ornamentations, etc. Initially, much attention was paid to the effects of sewage pollution on the foraminifers but later on all kinds of human induced as well as natural pollutants came under the preview of foraminiferologists working on pollution aspects. The advantage of application of foraminifers, over other chemical and biological techniques, for pollution monitoring lies in their potentiality to decipher temporal variation in type and concentration of pollutants at any site even in the absence of pre-pollution studies, based on the recovery of foraminifers from the sediment core samples. Realizing the potential application of characteristic foraminiferal features from the polluted areas to decipher the variation in introduction and concentration of pollutants at any given location with time, efforts were made to characterize the specific types of foraminiferal features to the specific pollutants. It was at this point that the need of culture studies was widely felt which resulted in numerous lab and field culture studies where foraminifers were subjected to specific pollutants in order to document their response to these pollutants and to develop effective foraminiferal proxies for pollution monitoring through time. Such studies are still going on and it is felt that culture studies need to be supplemented with advanced crystallographic and molecular studies in order to find the mechanism through which foraminifers respond to the pollutants.

Androgen receptor mutations in prostate cancer.

We analyzed the frequency and relevance of mutations in the coding region of the androgen receptor (AR) in genomic DNA extracted from 137 specimens of prostate cancer. The specimens were obtained from the primary tumors of patients affected by stage B disease [15 nonmicrodissected (group 1A) and 84 microdissected (group 1B)] and from the metastatic deposits of individuals with stage D1 disease [8 nonmicrodissected (group 2A) and 30 microdissected (group 2B)] who had not undergone androgen ablation therapy. The study was conducted by PCR-single strand conformational polymorphism (SSCP) analysis of exons 2-8 in the four groups and direct sequence analysis of exon 1 in group 1B. As positive and negative controls, we used genomic DNA extracted from genital skin fibroblasts of patients affected by various forms of androgen resistance with known mutations in the AR. To control for genetic instability, PCR-SSCP analysis of exon 2 of the human progesterone receptor was carried out on each specimen. The overall number of mutations detected was 11 (8%). No mutations were detected in any of the 99 patients with stage B disease. Eleven mutations were detected in exons 2-8 in 8 of the 38 patients with stage D1 disease (all in group 2B). Simultaneous analysis of exon 2 of the progesterone receptor was carried out, and no SSCP changes were identified. These data suggest that AR mutations are rare and presumably do not play a role in the initial phase of prostatic carcinogenesis. The presence of a significant number of AR mutations in metastatic disease indicates that mutations of this molecule may play a role in the most advanced phases of the natural history of this disease, either by facilitating growth or acquisition of the metastatic phenotype.

Association of oxidative status and semen characteristics with seminal plasma proteins of buffalo semen.

To study the influence of season on oxidative status of buffalo semen and their association with semen characteristics and seminal plasma proteins, ejaculates were collected twice a week in winter, summer and rainy seasons from six adult Bhadawari buffalo bulls. The neat semen was analyzed for semen characteristics immediately after collection and oxidative status viz. lipid peroxidation (LPO), catalase (CAT), super oxide dismutase (SOD) activity, and total protein (TP) were estimated in seminal plasma. The protein profiling was carried out by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The significant effect of season was observed on TP, SOD activity and % protein fractions of seminal plasma proteins of buffalo bulls. The TP values showed positive correlation with ejaculate volume (EV), sperm concentration (SC), and % live-dead (LD) and negative correlation with progressive motility (PM), and hypo-osmotic swelling test (HOST). The SOD activity showed positive correlation with PM, LD, HOST and % acrosoamal integrity (AI). Besides that, results showed correlation of TP with 6.5, 38 and 66 kDa proteins, LPO with 70, 72, 84 and 86 kDa proteins, CAT with 70 kDa and 86 kDa proteins, and SOD with 6.5, 24.5, 44.5, 70 and 72 kDa proteins. In conclusion, this study indicated that TP and SOD activity of seminal plasma of buffalo bulls were influenced by season and were found to be associated with some of the semen characteristics and expression of seminal plasma proteins.

Cholesterol: free radical peroxidation and transfer into phospholipid membranes.

Cholesterol, when sequestered in saturated liposomes of dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC), undergoes peroxidation thermally initiated either by a lipid-soluble or a water-soluble azo initiator and in both cases the reaction is inhibited effectively by the water-soluble antioxidant, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylate (Trolox). Quantitative kinetic methods of autoxidation show that the oxidizability, kp/(2kt)1/2 (where kp and 2kt are the rate constants of radical chain propagation and termination, respectively) of cholesterol in DMPC or DPPC multilamellar liposomes, where kp/(2kt)1/2 is 3.0.10(-3) to 4.3.10(-3) M-1/2 s-1/2 at 37-45 degrees C, is similar to that measured in homogeneous solution in chlorobenzene, where kp/(2kt)1/2 is 3.32.10(-3). However, its oxidizability in smaller unilamellar vesicles of DMPC or DPPC increases by at least 3-times that measured in multilamellar systems. Autoxidation/antioxidant methods show that cholesterol partitions directly from the solid state into DMPC or DPPC liposomes by shaking and this is confirmed by 31P and 2H quadrupole NMR spectra of deuterated cholesterol when membrane bound. Analytical studies indicate that up to 21 mol% cholesterol will partition into the membranes by shaking.

Biotransformation of organic nitrates and vascular smooth muscle cell function.

The organic nitrates are interesting examples of drugs that undergo biotransformation at their site of action to generate the active form of the drug. Furthermore, tolerance to the vasodilator effects of organic nitrates is associated with impairment of this metabolic activation process. Despite considerable research effort, the intracellular processes and the chemical reaction pathways by which organic nitrates are converted to their active form are still unresolved. This review by Brian Bennett and colleagues summarizes the characteristics of organic-nitrate biotransformation in vascular smooth muscle, the difficulties encountered when assessing this biotransformation, and the evidence for the role of two identified vascular biotransformation systems (glutathione-S-transferases and the cytochrome P450 system) in the metabolic activation of organic nitrates.

Bupropion sustained release as a smoking cessation treatment in remitted depressed patients maintained on treatment with selective serotonin reuptake inhibitor antidepressants.

BACKGROUND: Patients with depressive disorders smoke tobacco more often than the population at large and find quitting more difficult. Furthermore, when they quit smoking, they are more likely to suffer a relapse of depression. We evaluated the addition of bupropion sustained release (SR) for smoking cessation among patients with a history of depressive disorders being maintained in a euthymic state with selective serotonin reuptake inhibitor (SSRI) antidepressants. METHOD: Twenty-five adults with DSM-IV major depressive disorder or depressive disorder NOS currently receiving SSRI maintenance treatment and smoking > or = 15 cigarettes per day participated in the 9-week study. Bupropion SR, 150 mg/day, was added to SSRI treatment and increased to 300 mg/day. Subjects were counseled on smoking cessation measures and chose a target quit date 2 or 4 weeks after the initiation of bupropion SR. Self-reported smoking status, expired carbon monoxide (CO) measurements, Hamilton Rating Scales for Depression and Anxiety scores, and weight were measured at each visit. Subjects were abstinent if they reported not smoking during the prior 7 days, confirmed with an expired-air CO value of < or = 10 ppm. RESULTS: Eight (32%) of 25 subjects were abstinent after 9 weeks. At 3-month follow-up, 3 subjects remained abstinent, 3 relapsed, and 2 were lost to follow-up. Eleven subjects (44%) were nonresponders, and 6 (24%) dropped out prior to 3 weeks of treatment due to side effects (N = 3) or were lost to follow-up (N = 3). Mean weight gain was approximately 0.5 lb (0.2 kg) for those completing 9 weeks of bupropion SR treatment. During the 9-week study and the 3-month follow-up, there was no evidence of emergent depression in any subject. Four subjects (16%) spontaneously reported an improvement in SSRI-associated sexual dysfunction. CONCLUSION: These open data suggest modest effectiveness for and the safety of bupropion SR as a smoking cessation agent in individuals with depression maintained on treatment with SSRIs. Minimal weight gain, lack of emergent depressive episodes, and improvement of SSRI-associated sexual dysfunction are added advantages.

Treatment of dual diagnosis patients: a relapse prevention group approach.

The authors describe the successful use of an adjunctive group psychotherapy for substance-abusing patients with major psychiatric disorders (bipolar, schizophrenia, schizoaffective, psychotic depression, and atypical psychosis). The group utilizes a psychoeducational approach that focuses on substance abuse causes and consequences, principles of recovery, and relapse prevention strategies. Eight patients with prolonged histories of abuse of cocaine, alcohol, marijuana, or other drugs were enrolled in this weekly group treatment at a community mental health center drug treatment program, while continuing in treatment with their current case manager or primary therapist. Six of the eight patients achieved periods of stable abstinence, documented by self-report, urine toxicology screens, continued group attendance, and improved social functioning. Case examples are utilized to illustrate the group process.

Episodic release of luteinizing hormone and testosterone in Surti and Marwari bucks younger than one year of age.

Studies on the episodic release of luteinizing hormone (LH) and testosterone in Surti (n=2) and Marwari (n=2) bucks younger than one year of age (6 to 8 months) were carried out by collecting blood plasma during the breeding season. The studies revealed that definite pulsatile releases of LH and testosterone occur in both breeds of bucks. The overall number of LH and testosterone pluses over a 24 hour period was 9.1+/-1.00 and 7.5+/-0.28, respectively. The peak, basal and mean LH and testosterone concentrations did not show significant differences between the two breeds. The duration and interval of LH and testosterone pulses differed during light and dark hours. The time interval between LH peak followed by the testosterone peak was significantly (P<0.05) longer during the night than the day hours for both the breeds. The physiological basis of the findings are discussed.