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.

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.

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.

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.

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.

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.

Detection of fetal cocaine exposure by analysis of amniotic fluid.

OBJECTIVE: To determine whether analysis of amniotic fluid (AF) using solid-phase extraction and high-performance liquid chromatography is useful in detecting fetal exposure to cocaine and its metabolites. METHODS: Amniotic fluid and urine samples were obtained from 23 subjects with documented cocaine abuse during pregnancy and five subjects without a history of cocaine abuse. Urine samples were also collected from all newborns. Urine samples were screened using conventional immunoassay techniques and confirmed with thin-layer chromatography. Using a modified solid-phase extraction procedure, we removed cocaine and its metabolites from all AF samples and from the newborn urine samples in which the initial screening was positive. Subsequently, we analyzed the extracts with high-performance liquid chromatography. RESULTS: Cocaine or benzoylecgonine was detected in 74% of AF samples taken from the 23 known cocaine abusers. In these subjects, conventional maternal and neonatal urine toxicology screens were positive in 61 and 35%, respectively. Concentrations in AF ranged from 400 to greater than 5000 ng/mL for benzoylecgonine and from trace to 250 ng/mL for cocaine. Mean benzoylecgonine recovery from AF was significantly greater than from newborn urine (1800 versus 280 ng/mL, respectively; P < .0001). CONCLUSION: Analysis of AF appears to be useful in detecting gestational cocaine exposure.

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.

Conidia as a Substrate for Internal Transcribed Spacer-Based PCR Identification of Members of the Leptosphaeria maculans Species Complex.

ABSTRACT The blackleg disease of oilseed rape is caused by an ascomycete species complex termed Leptosphaeria maculans (anamorph Phoma lingam). L. maculans isolates collected worldwide were gathered in the International Blackleg of Crucifers Network (IBCN) collection. Representative IBCN isolates, along with one P. nigrificans isolate, were further analyzed using polymerase chain reaction (PCR) amplification of the internal transcribed spacer (ITS) region. ITS size polymorphism discriminated three groups: (i) P. nigrificans, (ii) Tox(+) and 'Lepidium' isolates, and (iii) NA1, NA2, NA3, 'Thlaspi', and 'Erysimum' isolates. Digestion of the ITS region with 19 selected endonucleases showed restriction site polymorphism between the different subgroups: digestion with RsaI could discriminate Tox(+) from 'Lepidium' isolates, whereas digestion with four enzymes, i.e., HaeIII, EcoRII, RsaI, and AluI, was needed to discriminate between NA1, NA2, NA3, 'Thlaspi', and 'Erysimum' isolates. No restriction site polymorphism was observed between isolates within the 'Thlaspi', Tox(+), NA1, and NA2 subgroups. Direct amplification of the ITS region could be achieved using intact conidia, collected either in axenic cultures or on leaf lesions, with only a 4-min 95 degrees C denaturation step prior to PCR reaction. A routine identification protocol requiring no DNA extraction and a sequential use of a few restriction enzymes following PCR has been used successfully for large-scale identification of French field isolates.

Cardiopulmonary resuscitation of newborns. Its application to transport medicine.

The outcome of sick infants requiring transport to tertiary care centers is critically dependent on the stabilization of these infants at the hospital of delivery and the quality of care during transport. The American Heart Association/American Academy of Pediatrics guidelines for neonatal resuscitation emphasize a structured, step-wise approach to resuscitation. This article highlights the application of these guidelines to resuscitation of newborns during transport and reviews the experience of a large perinatal network in this area.

Effect of pregnancy-induced and chronic hypertension on pregnancy outcome.

OBJECTIVES: To compare the impact of pregnancy-induced hypertension (PIH) and chronic hypertension (CHH) on pregnancy outcome. STUDY DESIGN: Retrospective study of data from a large perinatal data base with 109,428 consecutive deliveries from 1982 to 1987. RESULTS: Among 109,428 women who delivered during the study period, 8019 (7.3%) were hypertensive. The hypertensive group included 5971 (74.5%) with PIH and 2048 (25.5%) with CHH. Hypertension was associated with a significantly higher risk for adverse pregnancy outcome. Compared with PIH, CHH was more likely to result in an adverse outcome as shown by increased odds ratio (OR) for fetal death (OR, 1.9 for PIH, 2.9 for CHH), prematurity (OR 1.6 for PIH, 1.8 for CHH), and intrauterine growth retardation (OR 2.8 for PIH, 3.7 for CHH). Chronic hypertension was associated with a 184 gm reduction in birth weight compared with 168 gm reduction with PIH. Mothers with CHH were more likely to be black (CHH, 50.1%; PIH, 40.2%), receive public assistance (CHH, 41.6%; PIH, 34.5%), and have no prenatal care (CHH, 3.2%; PIH 2.1%). CONCLUSIONS: In this study population, hypertension during pregnancy was associated with significantly increased morbidity and mortality rates. Women with chronic hypertension are at greater risk for adverse outcome than those with pregnancy-induced hypertension.

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.

Iatrogenic disorders in modern neonatology.

Rather than give a detailed listing of all the iatrogenic problems in modern neonatology, this article seeks to explain the process of iatrogenicity and separate those problems that are preventable from those that currently are unpreventable.

Determination of cocaine and benzoylecgonine in human amniotic fluid using high flow solid-phase extraction columns and HPLC.

A new solid-phase extraction procedure for the determination of cocaine and benzoylecgonine in amniotic fluid, using high flow co-polymeric sorbents is reported. The recoveries of cocaine and benzoylecgonine within the range 0.1-1 mg/l were 95.7% and 50.3%, respectively. The use of high-flow sorbents allowed the easy extraction of amniotic fluid regardless of sample viscosity or physical nature. The use of these solid-phase columns provided many advantages over the more commonly used solvent extraction, including an increase in extraction speed and efficiency, reduced operator time, reduced solvent use and disposal volumes and exceptional extract quality. Further, the determination of amniotic fluid obtained from pregnant cocaine users may provide important information about handling of cocaine by the fetus at various gestational ages. The procedure was successfully applied to amniotic fluid from suspected cocaine abusers.

Cross-validation with active pattern selection for neural-network classifiers.

We propose a new approach for leave-one-out cross-validation of neural-network classifiers called "cross-validation with active pattern selection" (CV/APS). In CV/APS, the contribution of the training patterns to network learning is estimated and this information is used for active selection of CV patterns. On the tested examples, the computational cost of CV can be drastically reduced with only small or no errors.

Degree of familiarity ART2 in knowledge-based landmine detection.

The self-organizing network ART2 is extended to provide a fuzzy output value, which indicates the degree of familiarity of a new analog input pattern to previously stored patterns in the long-term memory of the network. The outputs of the multilayer perceptron and this modified ART2 provide an analog value to a fuzzy rule-based fusion technique which also uses a processed polarization resolved image as its third input. In real-time situations these two classifier outputs indicate the likelihood of a surface landmine target when presented with a number of multispectral and textural bands. Due to the modifications in ART2 this updated alternative architecture (to that of a previous network in [3]) has improved real-time landmine detection capabilities although the registration of all bands is more critical to the accuracy of results in this case. The real-time fuzzy rule-based system in preliminary tests has detected two of the three landmines and the landmine surrogate with two false alarms. Advanced tests on 30 images using the fuzzy rule-based system further confirmed the distinct advantages of fusion and improved detection rates.

Thorny foreign bodies of upper airway.

Twenty cases of unusual thorny foreign bodies (seeds of Xanthium strumarium) in laryngo-pharynx area are reported. The aetiopathogenesis, clinical features and management are briefly discussed.