IL-1ß induces changes in expression of core circadian clock components PER2 and BMAL1 in primary human chondrocytes through the NMDA receptor/CREB and NF-κB signalling pathways.

The circadian clock is a specialised cell signalling circuit present in almost all cells. It controls the timing of key cell activities such as proliferation and differentiation. In osteoarthritis, expression of two components of the circadian clock, BMAL1 and PER2 is altered in chondrocytes and this change has been causally linked with the increase in proliferation and altered chondrocyte differentiation in disease. IL-1ß, an inflammatory cytokine abundant in OA joints, has previously been shown to induce changes in BMAL1 and PER2 expression in chondrocytes. The purpose of this study is to identify the mechanism involved. We found IL-1ß treatment of primary human chondrocytes led to activation of NMDA receptors as evidenced by an increase in phosphorylation of GluN1 and an increase in intracellular calcium which was blocked by the NMDAR antagonist MK801. Levels of phosphorylated CREB were also elevated in IL-1ß treated cells and this effect was blocked by co-treatment of cells with IL-1ß and the NMDAR antagonist MK-801. Knockdown of CREB or inhibition of CREB activity prevented the IL-1ß induced increase in PER2 expression in chondrocytes but had no effect on BMAL1. Phosphorylated p65 levels were elevated in IL-1ß treated chondrocytes indicating increased NF-κB activation. Inhibition of NF-κB activity prevented the IL-1ß induced reduction in BMAL1 expression and partially mitigated the IL-1ß induced increase in PER2 expression in chondrocytes. These data indicate that the NMDAR/CREB and NF-κB signalling pathways regulate the core circadian clock components PER2 and BMAL1 in chondrocytes. Given that changes in expression of these clock components have been observed in a wide range of diseases, these findings may be broadly relevant for understanding the mechanism leading to circadian clock changes in pathology.

DNA methylation epi-signature is associated with two molecularly and phenotypically distinct clinical subtypes of Phelan-McDermid syndrome.

BACKGROUND: Phelan-McDermid syndrome is characterized by a range of neurodevelopmental phenotypes with incomplete penetrance and variable expressivity. It is caused by a variable size and breakpoint microdeletions in the distal long arm of chromosome 22, referred to as 22q13.3 deletion syndrome, including the SHANK3 gene. Genetic defects in a growing number of neurodevelopmental genes have been shown to cause genome-wide disruptions in epigenomic profiles referred to as epi-signatures in affected individuals. RESULTS: In this study we assessed genome-wide DNA methylation profiles in a cohort of 22 individuals with Phelan-McDermid syndrome, including 11 individuals with large (2 to 5.8 Mb) 22q13.3 deletions, 10 with small deletions (< 1 Mb) or intragenic variants in SHANK3 and one mosaic case. We describe a novel genome-wide DNA methylation epi-signature in a subset of individuals with Phelan-McDermid syndrome. CONCLUSION: We identified the critical region including the BRD1 gene as responsible for the Phelan-McDermid syndrome epi-signature. Metabolomic profiles of individuals with the DNA methylation epi-signature showed significantly different metabolomic profiles indicating evidence of two molecularly and phenotypically distinct clinical subtypes of Phelan-McDermid syndrome.

Monitoring steady production of 1,3-propanediol during bioprospecting of glycerol-assimilating soil microbiome using dye-based pH-stat method.

AIM: In this investigation, a dye-based pH-stat method was devised for monitoring steady production of 1,3-propanediol (1,3-PDO) during bioprospecting of glycerol-assimilating soil microbiome. METHODS AND RESULTS: Soil samples were collected from two potential sites of CSIR-IIP, India. Selective enrichment of microbial consortia was done using the glycerol-based medium at initial stage, followed by purification to isolated colonies, after positive high-performance liquid chromatography detection of 1,3-PDO in the fermentation broth. When the purified isolated were re-tested for 1,3-PDO production, only two isolates namely Isolate 1 and Isolate 3 were capable of producing the targeted product preferably under anaerobic conditions. Based on better 1,3-PDO fermentation efficiency (Isolate 3, 22% vs Isolate 1, 4·48%) and acetic acid as the only major by-product, Isolate 3 was shortlisted for further studies. A dye-based technique was devised in which bromothymol blue was incorporated into the medium to monitor the pH drop due to acetic acid formation and hence change in colour. Visual change in colour helped in intermittent pH restoration. During fermentation, with pH stat being 8-8·5, Isolate 3 at 32°C yielded 0·67 mol mol-1 1,3-PDO within a short span of 12 h only with an initial concentration of glycerol being 20 g l-1 . Phylogenetic analysis revealed that Isolate 3 shared 95·8% homology with Citrobacter freundii CFNIH1 and hence designated as C. freundii IIP DR3. CONCLUSION: This study demonstrated that during bioprospecting glycerol-assimilating microbiome, dye-based technique can be successfully employed. This technique can further be exploited to monitor consistent production of all microbial secondary metabolites that accompanies acid production. SIGNIFICANCE AND IMPACT OF THE STUDY: Incorporation of 'Bromothymol blue' can visually help in the identification of pH drop in the medium, so that pH stat can be easily maintained during 1,3-PDO production from glycerol especially under shake flask conditions.

18F-Fluorodeoxyglucose positron emission tomography-computed tomography imaging in HIV-infected patients with lymphadenopathy, with or without fever and/or splenomegaly.

We audited whether 18F-Fluorodeoxyglucose positron emission tomography-computed tomography (18FDG PET-CT) imaging could discriminate between different diagnoses in HIV-infected patients presenting with lymphadenopathy, with or without fever and/or splenomegaly. Maximum standardised uptake (SUVmax) values were similar in lymphoma and mycobacterial and fungal infections and were lower but similar in those with human herpesvirus (HHV) 8-associated disease and HIV-associated reactive lymphadenopathy. Nodal 18FDG avidity, with SUVmax ≥10, excluded diagnoses of HHV 8-associated disease and miscellaneous conditions, and HIV-associated reactive lymphadenopathy was additionally excluded in those who had undetectable plasma HIV viral loads. This audit suggests 18FDG PET-CT imaging did not permit discrimination between specific diagnoses but has utility in identifying lymph nodes with increased avidity that could be targeted for biopsy and in ruling out significant pathology.

Outcome of universal newborn eye screening with wide-field digital retinal image acquisition system: a pilot study.

PurposeTo evaluate the outcome of universal newborn eye screening with wide-field digital retinal imaging (WFDRI) system.MethodsIn this pilot study, we examined 1152 apparently healthy newborn infants in the obstetrics and gynecology ward of a civil hospital in Eastern India over 1.5 years. The examination included external eye examination, red reflex test and fundus imaging by WFDRI (RetCam II, Clarity medical system, Pleasanton, CA, USA) by a trained optometrist. The pathologies detected, net monetary gain and skilled manpower saved were documented. The results were compared with three similar studies thus far published in the literature.ResultsOcular abnormality of any kind was seen in 172 (14.93%) babies. Retinal hemorrhage in 153 babies (88.9% of all abnormal findings) was the most common abnormality; it was bilateral in 118 (77.12%) babies and 4 babies had foveal hemorrhage. Other abnormalities included vitreous hemorrhage (n=1), congenital glaucoma (n=2), uveal coloboma (n=2), retinopathy mimicking retinopathy of prematurity (n=2), and cystic fovea (n=3). The retinal hemorrhages resolved spontaneously in all eyes. One baby with congenital glaucoma received surgery and the other was treated medically. The benefits included savings in skilled manpower, a net monetary gain of INR 4.195 million (US$ 62,612) and skilled manpower saving by 319.4 h.ConclusionsThe universal neonatal eye screening using WFDRI detected pathologies that needed immediate care or regular follow up; saved skilled manpower with a net monetary gain. But compared to a red reflex test the benefits were marginal in terms of detecting treatment warranting ocular pathologies.

Impact of completion of primary biliary procedure on outcome of endoscopic retrograde cholangiopancreatographic related perforation.

BACKGROUND AND AIMS: Perforation is one of the worst complications of therapeutic endoscopic retrograde cholangiopancreatography (ERCP). We aimed to study the epidemiology of ERCP related perforation and the impact of completion of intended procedure on the outcome of this complication. METHODS: ERCP records from January 2007 to April 2012 were independently evaluated by two investigators for the occurrence of procedure related perforations. A total of 11 500 patients underwent therapeutic ERCP during the study period. The case records of 171 (1.5 %) patients with ERCP related perforations were reviewed to analyze the epidemiology and risk factors associated with poor outcome. RESULTS: Of the 171 patients included in this study, the majority of perforations (n = 129, 75.4 %) were related to use of the needle-knife precut technique. Female gender (1.9 % vs 0.7 %, P  < 0.001), age > 40 years (1.7 % vs 1.1 %, P  < 0.01), and benign disease (1.7 % vs. 1.1 %, P  < 0.01) were risk factors for ERCP related perforation. Most of the perforations (n = 135, 79 %) were detected during the procedure. The majority of patients were managed conservatively (n = 164, 96 %). Although 159 patients recovered, 12 patients (7 %) did not survive. Completion of intended biliary procedure for primary disease was associated with low risk of mortality (2 % vs 15.4 %, P  < 0.001). CONCLUSIONS: ERCP related perforation is uncommon. The majority of patients can be managed conservatively. The risk of mortality is low and completion of the intended biliary procedure decreases the risk of mortality.

Emergence of dengue in tribal villages of Mandla district, Madhya Pradesh, India.

BACKGROUND & OBJECTIVES: Dengue (DEN) is a rapidly spreading arboviral disease transmitted by Aedes mosquitoes. Although it is endemic in India, dengue virus (DENV) infection has not been reported from tribal areas of Madhya Pradesh. Investigations were conducted to establish the aetiology of sudden upsurge of cases with febrile illness in June 2013 from tribal villages of Mandla district of Madhya Pradesh, India. METHODS: The rapid response team of the National Institute for Research in Tribal Health, Jabalpur, conducted clinical investigations and field surveys to collect the samples from suspected cases. Samples were tested using molecular and serological tools. Collected mosquitoes were identified and tested for the presence of virus using semi nested reverse transcriptase-polymerase chain reaction (nRT-PCR). The sequences were analysed to identify serotype and genotype of the virus. RESULTS: Of the 648 samples collected from 18 villages of Mandla, 321 (49.53%) were found to be positive for dengue. The nRT-PCR and sequencing confirmed the aetiology as dengue virus type 2. Eighteen per cent of patients needed hospitalization and five deaths were attributed to dengue. The virus was also detected from Aedes aegypti mosquito, which was incriminated as a vector. Phylogenetic analysis revealed that the dengue virus 2 detected belonged to cosmopolitan genotype of the virus. INTERPRETATION & CONCLUSIONS: Dengue virus serotype 2 was detected as the aetiological agent in the outbreak in tribal villages of Mandla district of Madhya Pradesh. Conducive man-made environment favouring mosquitogenic conditions and seeding of virus could be the probable reasons for this outbreak. Urgent attention is needed to control this new threat to tribal population, which is already overburdened with other vector borne diseases.

Microscopic visualization of intravasal spermatozoa is positively associated with patency after bilateral microsurgical vasovasostomy.

We evaluated pre-operative and intraoperative factors associated with successful patency following bilateral microsurgical vasovasostomy (VV). We retrospectively reviewed the charts of 1331 men who underwent bilateral VV by two surgeons between 2006 and 2013. Vasal fluid was examined intraoperatively for gross quality (i.e., clear or opaque and creamy/thick) and for the presence of spermatozoa on microscopy (i.e., whole spermatozoa, sperm fragments, or azoospermia). Post-operative patency was assessed by semen analysis or patient report of conception. Perioperative factors were explored using descriptive statistics and examined in logistic regression models for associations with post-operative patency. The median age at VV was 39 years [interquartile range (IQR): 35-44] and the median obstructive interval (OI) was 7 years (IQR: 4-11). Overall, 1307 patients achieved post-operative patency (98%) while 24 remained obstructed (2%). Among those who became patent, 410 reported conception. After adjustment for potential confounders, only microscopic examination of the intravasal fluid for the presence of spermatozoa (bilateral or unilateral whole spermatozoa vs. sperm parts/azoospermia) at the time of VV was significantly associated with post-operative patency with an odds ratio (OR) of 14.2 (95% CI: 5.8-34.9; p = <1 × 10(-8) ). Identification of bilateral or unilateral sperm fragments vs. azoospermia was also associated with increased odds of post-operative patency with an OR of 3.5 (95% CI: 0.9-13.6; p = 0.08). There was no statistically significant association between age at VV, OI, presence of granuloma, gross fluid quality, or surgeon and post-operative patency after controlling for potential confounders. Identification of whole spermatozoa in the vasal fluid at the time of VV was positively associated with post-operative patency. Our findings stress the need for intraoperative microscopy to aid in post-operative patient counseling.

Pilomatrixoma masquerading as metastatic adenocarcinoma. A diagnostic pitfall on cytology.

Pilomatrixoma (Px) is a relatively uncommon slowly growing, benign skin appendageal tumor with a wide morphological spectrum, and can pose diagnostic difficulties especially in cases with unusual clinical presentation. We report a case of young male who presented with a nodule over anterior abdominal wall, fine needle aspiration cytology (FNAC) revealed predominantly small cells in focal acinar pattern suggestive of metastatic carcinoma; however, histopathological diagnosis was Px. The case highlights the diagnostic pitfalls on cytology due to limited morphology.

Office of clinical pharmacology science day: a forum to stimulate innovation in clinical pharmacology.

The Office of Clinical Pharmacology (OCP) of the US Food and Drug Administration (FDA) has hosted an office-wide event called Science Day (SD) since 1999. SD 2012 included presentations on physiologically based pharmacokinetic models, exposure-response analyses, and other research projects. SD provides a forum for showcasing clinical pharmacology (CP) research within the OCP and provides an opportunity for professional development. This article discusses the evolution of Science Day and then focuses on SD 2012 as an example platform for promotion of regulatory research.

Bilateral symmetrical facial swelling owing to tuberculous gummas.

Metastatic tuberculous abscesses and gummas are unusual forms of cutaneous tuberculosis. They result from haematogeneous spread of the mycobacterium from a primary focus during a period of impaired immunity. A 5-year-old boy is reported who presented with spinal tuberculosis and bilateral subcutaneous swelling of the cheeks owing to tuberculous gummas.

Implications of obesity for drug therapy: limitations and challenges.

Obesity has become a worldwide challenge with significant health and socioeconomic implications. One of the major implications is its impact on drug therapy. In order to gain a better understanding of this impact, we surveyed the regulatory guidances, the newly approved molecular entity drug products, and drug product labels in the Physician's Desk Reference. This review summarizes the findings of the survey along with the existing knowledge on pharmacokinetic and pharmacodynamic changes associated with obesity.

Antifungal activity of the alkaloids from Eschscholzia californica.

The isoquinoline alkaloids hunnemanine and norsanguinarine have been isolated from methanolic extract of the whole plant of Eschscholzia californica. These two alkaloids were checked for their antifungal activity against phytopathogenic fungi Alternaria melongenae, A. brassicola, A. brassicae, Curvularia lunata, C. maculans, Helminthosporium pennisetti, H. oryzae, H. turcicum, Fusarium undum and F. lini. Hunnemanine exhibited 100 % inhibition of spore germination of A. brassicae, H. pennisetti and F. lini at 1000 ppm whereas norsanguinarine exhibited 100 % inhibition of A. brassicicola and C. maculans at this concentration.

Expression of highly selective sodium channels in alveolar type II cells is determined by culture conditions.

Alveolar fluid clearance in the developing and mature lungs is believed to be mediated by some form of epithelial Na channels (ENaC). However, single-channel studies using isolated alveolar type II (ATII) cells have failed to demonstrate consistently the presence of highly selective Na+ channels that would be expected from ENaC expression. We postulated that in vitro culture conditions might be responsible for alterations in the biophysical properties of Na+ conductances observed in cultured ATII cells. When ATII cells were grown on glass plates submerged in media that lacked steroids, the predominant channel was a 21-pS nonselective cation channel (NSC) with a Na+-to-K+ selectivity of 1; however, when grown on permeable supports in the presence of steroids and air interface, the predominant channel was a low-conductance (6.6 +/- 3.4 pS, n = 94), highly Na+-selective channel (HSC) with a P(Na)/P(K) >80 that is inhibited by submicromolar concentrations of amiloride (K(0.5) = 37 nM) and is similar in biophysical properties to ENaC channels described in other epithelia. To establish the relationship of this HSC channel to the cloned ENaC, we employed antisense oligonucleotide methods to inhibit the individual subunit proteins of ENaC (alpha, beta, and gamma) and used patch-clamp techniques to determine the density of this channel in apical membrane patches of ATII cells. Overnight treatment of cells with antisense oligonucleotides to any of the three subunits of ENaC resulted in a significant decrease in the density of HSC channels in the apical membrane cell-attached patches. Taken together, these results show that when grown on permeable supports in the presence of steroids and air interface, the predominant channels expressed in ATII cells have single-channel characteristics resembling channels that are associated with the coexpression of the three cloned ENaC subunits alpha-, beta-, and gamma-ENaC.

Ethanol ingestion via glutathione depletion impairs alveolar epithelial barrier function in rats.

We determined that rats fed a liquid diet containing ethanol (36% of calories) for 6 wk had decreased (P < 0.05) net vectorial fluid transport and increased (P < 0.05) bidirectional protein permeability across the alveolar epithelium in vivo compared with rats fed a control diet. However, both groups increased (P < 0.05) fluid transport in response to epinephrine (10(-5) M) stimulation, indicating that transcellular sodium transport was intact. In parallel, type II cells isolated from ethanol-fed rats and cultured for 8 days formed a more permeable monolayer as reflected by increased (P < 0.05) leak of [(14)C]inulin. However, type II cells from ethanol-fed rats had more sodium-permeant channels in their apical membranes than type II cells isolated from control-fed rats, consistent with the preserved response to epinephrine in vivo. Finally, the alveolar epithelium of ethanol-fed rats supplemented with L-2-oxothiaxolidine-4-carboxylate (Procysteine), a glutathione precursor, had the same (P < 0.05) net vectorial fluid transport and bidirectional protein permeability in vivo and permeability to [(14)C]inulin in vitro as control-fed rats. We conclude that chronic ethanol ingestion via glutathione deficiency increases alveolar epithelial intercellular permeability and, despite preserved or even enhanced transcellular sodium transport, renders the alveolar epithelium susceptible to acute edematous injury.

Physiology of infants with very low birth weight.

Advances in neonatal management have resulted in dramatic increases in survival in infants with birth weights less than 1,500 g. Extensive basic science and clinical research has led to a more comprehensive understanding of the physiological differences between the VLBW infant and larger neonates. Meticulous attention must be paid to appropriate fluid, electrolyte, nutrition, and temperature maintenance to achieve homeostasis and growth. Additionally, the clinician must be aware of the diagnostic and treatment modalities for the common complications seen in the premature infant to minimize mortality and long-term morbidity.

A cysteine-free firefly luciferase retains luminescence activity.

A mutant of Photinus pyralis luciferase in which all four native cysteine residues are converted to serines retains about 10% of wild-type activity. This mutant should prove useful as a starting point for the introduction of biophysical probes of conformational changes associated with enzyme function. The activities of the cysteine-free mutant and others in which two or three cysteines are converted to serines suggest, however, that small chemical changes can have substantial and interdependent effects on bioluminescence. The introduction of probes should therefore be approached cautiously.

Alveolar fluid clearance in developing lungs and its role in neonatal transition.

Successful resuscitation of a fetus and a smooth transition to air breathing is a complex process that requires the orchestration of numerous events. The ability of the newborn to establish adequate ventilation and oxygenation is critically dependent on alveolar expansion and clearing of lung fluid. This transition is aided by changes, including a decrease in lung fluid production, that occur in the immediate period prior to birth. In neonates who face difficulty in establishing air exchange after birth, failure of adequate clearance of lung fluid is an important factor. Such infants are likely to benefit from efforts directed at increasing lung epithelial sodium reabsorption, which in turn will drive water out of the alveolar spaces. We are beginning to understand how these physiologic events occur and some of the factors that can speed up the process. Therapeutic modalities aimed at rescuing infants whose lungs continue to stay flooded are likely to reduce pulmonary morbidity and improve outcome.