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Purpose: Both Shigella and enteroinvasive Escherichia coli (EIEC) can cause enterocolitis, but they have a distinct epidemiology and public health relevance. Current culture-independent testing (CIT) methods to identify Shigella in faecal samples rely on the ipaH gene as the target, which is also found in EIEC genomes. The aim of this study was to design an assay that can identify EIEC in cultures from CIT ipaH-positive samples., Methodology: Shigella and EIEC genomes were screened to find unique regions present in EIEC genomes using a comparative genomics approach and differentiating genetic loci that are suitable PCR targets were identified. The primers for these loci were designed and tested in 6501 and 104 genomes of Shigella and EIEC, respectively., Results: An assay with two sets of multiplex PCR reactions that differentiates Shigella and EIEC based on the presence/absence of at least two out of six loci was developed and evaluated. The majority of Shigella genomes lacked all six loci, while at least two loci were present in most EIEC genomes. This assay successfully differentiated clinical EIEC from Shigella with a limit of detection of 10 5  cells ml -1 . The sensitivity and specificity were over 95 and 99%, respectively. The assay can further subtype EIEC genomes into their genetic lineages., Conclusion: This new highly specific assay can assist in the identification of EIEC in ipaH PCR-positive samples and augment the public health laboratory surveillance of EIEC and shigellosis.

Competing Interests: There are no conflicts of interest

Purpose of Review: Unlike immunocompetent hosts, solid organ transplant (SOT) recipients with posttransplant histoplasmosis (PTH) often present with disseminated disease and have an attributable mortality of approximately 10%. In this review, we discuss currently available diagnostic tests and treatment strategies in PTH., Recent Findings: None of the available tests have a 100% diagnostic accuracy. Histoplasma antigen assays are the most sensitive commercially available tests. However, crossreactivity of histoplasma antigen with aspergillus galactomannan and false positive histoplasma antigen tests because of rabbit antithymocyte globulin may cause difficulty in interpreting positive test results in transplant recipients. Molecular assays such as amplification and sequencing of 'panfungal' portions of the 28S ribosomal RNA from clinical specimens appear to be promising.Lipid formulations of amphotericin B and itraconazole are the drugs of choice in the treatment of PTH. Other extended spectrum azoles also appear to be effective, but, like itraconazole, problems with drug interactions and prolongation of the QTc interval (except for isavuconazole, which shortens the QTc interval) remain. Mycophenolate therapy is associated with severe disease and should be stopped during active disease and, if feasible, calcineurin inhibitors and steroids should be reduced., Summary: A combination of various tests (culture, antigen tests, nucleic amplification tests, etc.) should be used to optimize diagnostic yield. The role of unbiased next generation sequencing for early diagnosis and newer azoles in the treatment needs to be further explored.

Purpose of Review: Unlike in orthotopic heart transplant (OHT) setting where toxoplasma prophylaxis is a standard practice in pretransplant toxoplasma seronegative recipients who have received donor hearts from seropositive donors (D+/R-), there is no consensus regarding prophylaxis in non-OHT recipients., Recent Findings: The incidence of toxoplasma disease in non-OHT recipients is less than 1% but its true burden is underestimated. Among 31 cases of toxoplasma disease reported from 2004 through 2017, renal and liver transplant recipients comprised of 90% of cases. A total of 94% of 18 recipients with known pretransplant serology were seronegative recipients (mostly D+/R-). Out of 16 recipients with adequate information, 10 (63%) and five (31%) were deemed to be donor derived and nondonor-derived primary toxoplasmosis respectively. Tissue invasive reactivation was uncommon. Almost all cases were described in patients not on prophylaxis at the time of presentation. Universal screening of donor/recipient toxoplasma serology for risk stratification is beneficial as illustrated by reports of fatal cases of toxoplasmosis due to unavailability of positive donor serology results., Summary: Toxoplasma disease in non-OHT predominantly occurs in pretransplant seronegative recipients- mostly in D+/R- group and is rare in seropositive recipients. Posttransplant prophylaxis should be targeted against the high-risk D+/R- group and should be considered in seropositive recipients in whom unusually high immunosuppression is implemented. Toxoplasma serologies and PCR should be used in combination for the diagnosis of toxoplasmosis in non-OHT patients.

The current study aimed at genomic characterization and improved understanding of genetic diversity of two Indian mithun populations (both farm, 48 animals and field, 24 animals) using genome wide genotype data generated with Illumina BovineHD BeadChip. Eight additional populations of taurine cattle (Holstein and NDama), indicine cattle (Gir) and other evolutionarily closely related species (Bali cattle, Yak, Bison, Gaur and wild buffalo) were also included in this analysis (N = 137) for comparative purposes. Our results show that the genetic background of mithun populations was uniform with few possible signs of indicine admixture. In general, observed and expected heterozygosities were quite similar in these two populations. We also observed increased frequencies of small-sized runs of homozygosity (ROH) in the farm population compared to field mithuns. On the other hand, longer ROH were more frequent in field mithuns, which suggests recent founder effects and subsequent genetic drift due to close breeding in farmer herds. This represents the first study providing genetic evidence about the population structure and genomic diversity of Indian mithun. The information generated will be utilized for devising suitable breeding and conservation programme for mithun, an endangered bovine species in India.

Intramolecular [4 + 2]-cycloaddition of heteroallene salts gives polycyclic tetrahydrocinnoline structures that contain an N-aminoiminium motif. Deprotonation with mild base gives the corresponding azomethine imines, which readily participate in 1,3-dipolar cycloaddition reactions. The structurally complex tetracyclic 1,2,3,4-tetrahydrocinnoline products typically form as a mixture of two separable diastereomers. The major diastereomer is generally formed by reaction of the dipolarophile with the convex face of the dipole.

We present a microfabricated spiral-coupled passive resonator sensor realized through integrated passive device (IPD) technology for the sensitive detection and characterization of water-ethanol solutions. In order to validate the performance of the proposed device, we explicitly measured and analyzed the radio frequency (RF) characteristics of various water-ethanol solution compositions. The measured results showed a drift in the resonance frequency from 1.16 GHz for deionized (DI) water to 1.68 GHz for the solution containing 50% ethanol, whereas the rejection level given by the reflection coefficient decreased from -29.74 dB to -14.81 dB. The obtained limit of detection was 3.82% volume composition of ethanol in solution. The derived loaded capacitance was 21.76 pF for DI water, which gradually decreased to 8.70 pF for the 50% ethanol solution, and the corresponding relative permittivity of the solution decreased from 80.14 to 47.79. The dissipation factor increased with the concentration of ethanol in the solution. We demonstrated the reproducibility of the proposed sensor through iterative measures of the samples and the study of surface morphology. Successive measurement of different samples had no overlapping and had very minimum bias between RF characteristics for each measured sample. The surface profile for bare sensors was retained after the sample test, resulting a root mean square (RMS) value of 11.416 nm as compared to 10.902 nm for the bare test. The proposed sensor was shown to be a viable alternative to existing sensors for highly sensitive water-ethanol concentration detection., Competing Interests: The authors declare no conflicts of interest.

Few public sector rice breeders have the capacity to use NGS-derived markers in their breeding programmes despite rapidly expanding repositories of rice genome sequence data. They rely on > 18,000 mapped microsatellites (SSRs) for marker-assisted selection (MAS) using gel analysis. Lack of knowledge about target SNP and InDel variant loci has hampered the uptake by many breeders of Kompetitive allele-specific PCR (KASP), a proprietary technology of LGC genomics that can distinguish alleles at variant loci. KASP is a cost-effective single-step genotyping technology, cheaper than SSRs and more flexible than genotyping by sequencing (GBS) or array-based genotyping when used in selection programmes. Before this study, there were 2015 rice KASP marker loci in the public domain, mainly identified by array-based screening, leaving large proportions of the rice genome with no KASP coverage. Here we have addressed the urgent need for a wide choice of appropriate rice KASP assays and demonstrated that NGS can detect many more KASP to give full genome coverage. Through re-sequencing of nine indica rice breeding lines or released varieties, this study has identified 2.5 million variant sites. Stringent filtering of variants generated 1.3 million potential KASP assay designs, including 92,500 potential functional markers. This strategy delivers a 650-fold increase in potential selectable KASP markers at a density of 3.1 per 1 kb in the indica crosses analysed and 377,178 polymorphic KASP design sites on average per cross. This knowledge is available to breeders and has been utilised to improve the efficiency of public sector breeding in Nepal, enabling identification of polymorphic KASP at any region or quantitative trait loci in relevant crosses. Validation of 39 new KASP was carried out by genotyping progeny from a range of crosses to show that they detected segregating alleles. The new KASP have replaced SSRs to aid trait selection during marker-assisted backcrossing in these crosses, where target traits include rice blast and BLB resistance loci. Furthermore, we provide the software for plant breeders to generate KASP designs from their own datasets.

Candida glabrata can rapidly acquire mutations that result in drug resistance, especially to azoles and echinocandins. Identification of genetic mutations is essential, as resistance detected in vitro can often be correlated with clinical failure. We examined the feasibility of using whole genome sequencing (WGS) for genome-wide analysis of antifungal drug resistance in C. glabrata. The aim was torecognize enablers and barriers in the implementation WGS and measure its effectiveness. This paper outlines the key quality control checkpoints and essential components of WGS methodology to investigate genetic markers associated with reduced susceptibility to antifungal agents. It also estimates the accuracy of data analysis and turn-around-time of testing. Phenotypic susceptibility of 12 clinical, and one ATCC strain of C. glabrata was determined through antifungal susceptibility testing. These included three isolate pairs, from three patients, that developed rise in drug minimum inhibitory concentrations. In two pairs, the second isolate of each pair developed resistance to echinocandins. The second isolate of the third pair developed resistance to 5-flucytosine. The remaining comprised of susceptible and azole resistant isolates. Single nucleotide polymorphisms (SNPs) in genes linked to echinocandin, azole and 5-flucytosine resistance were confirmed in resistant isolates through WGS using the next generation sequencing. Non-synonymous SNPs in antifungal resistance genes such as FKS1, FKS2, CgPDR1, CgCDR1 and FCY2 were identified. Overall, an average of 98% of the WGS reads of C. glabrata isolates mapped to the reference genome with about 75-fold read depth coverage. The turnaround time and cost were comparable to Sanger sequencing. In conclusion, WGS of C. glabrata was feasible in revealing clinically significant gene mutations involved in resistance to different antifungal drug classes without the need for multiple PCR/DNA sequencing reactions. This represents a positive step towards establishing WGS capability in the clinical laboratory for simultaneous detection of antifungal resistance conferring substitutions.

High aspect-ratio elastomeric micropillars play important roles as the platform for microscale sensing and actuation. Many soft-lithographic techniques have been developed for their facile realization but most of the techniques are limited to build the micropillars only on totally flat, widely accessible substrate areas with the micropillar's structural characteristics completely predetermined, leaving little room for in situ control. Here we demonstrate a new technique which overcomes these limitations by directly drawing micropillars from pipette-dispensed PDMS microdroplets using vacuum-chucked microspheres. The combined utilization of PDMS microdroplets and microspheres not only enables the realization of microsphere-tipped PDMS micropillars on non-flat, highly space-constrained substrate areas at in situ controllable heights but also allows arraying of micropillars with dissimilar heights at a close proximity. To validate the new technique's utility and versatility, we realize PDMS micropillars on various unconventional substrate areas in various configurations. We also convert one of them, the optical fiber/micropillar hybrid, into a soft optical contact sensor. Both the fabrication technique and the resulting sensing scheme will be useful for future biomedical microsystems.

Histoplasma capsulatum is a dimorphic fungus that most often causes asymptomatic infection in the immunocompetent population. In immunocompromised patients, including solid organ transplant (SOT) and hematopoietic cell transplant (HCT) recipients, however, it is likely to cause severe life-threatening infection. Post-transplant histoplasmosis (PTH) in SOT is uncommon with an incidence of ≤1% and is even rarer in HCT patients. The majority of PTH in SOT is diagnosed in the first 2 years following transplantation. Histoplasmosis may result from endogenous reactivation of latent infection, de novo post-transplant acquisition, and donor-derived infection. Disseminated infection is common. Fever is the most common symptom and clinical features are often nonspecific, but patients with disseminated infection may present with a septic picture. Other features, including pancytopenia and hepatosplenomegaly, may not be prominent early in the course of illness. Contemporary histoplasma antigen assays are the most sensitive tests but cross-reactivity with antigens of other fungi, including with Aspergillus galactomannan, is not uncommon. Treatment should be continued for at least a year. Histoplasma antigen levels have prognostic value and can be used to monitor the response to therapy. The attributable mortality is approximately 10%. Routine screening of donors and recipients is not currently recommended., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Aflatoxin, produced by Aspergillus flavus , is hazardous to health of humans and livestock. The lack of information about large effect QTL for resistance to aflatoxin accumulation is a major obstacle to employ marker-assisted selection for maize improvement. The understanding of resistance mechanisms of the host plant and the associated genes is necessary for improving resistance to A. flavus infection. A suppression subtraction hybridization (SSH) cDNA library was made using the developing kernels of Mp715 (resistant inbred) and B73 (susceptible inbred) and 480 randomly selected cDNA clones were sequenced to identify differentially expressed genes (DEGs) in response to A. flavus infection and map these clones onto the corn genome by in-silico mapping. A total of 267 unigenes were identified and majority of genes were related to metabolism, stress response, and disease resistance. Based on the reverse northern hybridization experiment, 26 DEGs were selected for semi-quantitative RT-PCR analysis in seven inbreds with variable resistance to aflatoxin accumulation at two time points after A. flavus inoculation. Most of these genes were highly expressed in resistant inbreds. Quantitative RT-PCR analysis validated upregulation of PR-4, DEAD-box RNA helicase, and leucine rich repeat family protein in resistant inbreds. Fifty-six unigenes, which were placed on linkage map through in-silico mapping, overlapped the QTL regions for resistance to aflatoxin accumulation identified in a mapping population derived from the cross between B73 and Mp715. Since majority of these mapped genes were related to disease resistance, stress response, and metabolism, these should be ideal candidates to investigate host pathogen interaction and to reduce aflatoxin accumulation in maize.

Background Colonoscopy is a simple, safe and well tolerated procedure, the visualization of the mucosa of the entire colon and terminal ileum to detect intestinal abnormalities and obtain biopsy leads to the early detection of the pathologic process and institution of appropriate therapy. Objective To find out the correlation between clinical and histopathological diagnosis of colorectal diseases. Method A cross-sectional study was conducted at Department of Pathology, Dhulikhel Hospital, Kathmandu University Hospital from Jan. 2015 - Jan. 2016. Altogether, 95 colonoscopic biopsies were examined and recorded clinical data using pre-designed pro forma. The specimens were grossed, processed and embedded using standard procedures, stained with Hematoxylin and Eosin stain and were analyzed using light microscope. Special stains Ziehl Neelsen, and Periodic Acid-Schiff were used whenever necessary. Result Analyses of 95 cases of colonoscopic biopsies were done. The most common clinical diagnosis was polyp in 49 cases (51.57%) and the common histopathological diagnosis was non-neoplastic polyps 31(32.63%). There was no correlation in cases for suspected infectious colitis, microscopic colitis and hemorrhoids. Conclusion Colonoscopy is incomplete without biopsy and histopathology is the gold standard for the diagnosis of colorectal lesions. The clinico-pathological correlation for neoplastic lesions was excellent. However, correlation was poor in non-neoplastic lesion.

We present a concept for the characterization of micro-fabricated based resonator incorporating air-bridge metal-insulator-semiconductor (MIS) capacitor to continuously monitor an individual's state of glucose levels based on frequency variation. The investigation revealed that, the micro-resonator based on MIS capacitor holds considerable promise for implementation and recognition as a glucose sensor for human serum. The discrepancy in complex permittivity as a result of enhanced capacitor was achieved for the detection and determination of random glucose concentration levels using a unique variation of capacitor that indeed results in an adequate variation of the resonance frequency. Moreover, the design and development of micro-resonator with enhanced MIS capacitor generate a resolution of 112.38 × 10 -3 pF/mg/dl, minimum detectable glucose level of 7.45mg/dl, and a limit of quantification of 22.58mg/dl. Additionally, this unique approach offers long-term reliability for mediator-free glucose sensing with a relative standard deviation of less than 0.5%., (Copyright © 2017 IPEM. Published by Elsevier Ltd. All rights reserved.)

Leishmaniasis occurs in <1% of solid organ and hematopoietic stem cell transplant recipients in endemic countries in which transplants are performed. Visceral leishmaniasis (VL) makes up the bulk of reported cases. The onset generally occurs months after transplantation and the mode of acquisition is often impossible to determine, but de novo vector-borne infection and reactivation of inapparent infection are thought to be the principal means. The potential role of clinically inapparent donor infection is uncertain and screening is not currently recommended, nor is it recommended for recipients from endemic areas, some of whom may have detectable circulating protozoan nucleic acid. While transplant recipients with VL often present with the non-specific findings of fever and cytopenia, the additional presence of hepatosplenomegaly in patients from endemic areas should lead to a directed diagnostic evaluation with bone marrow examination and PCR testing of marrow and peripheral blood having a high yield. Management may often be complicated by the presence of concomitant infections. A lipid formulation of amphotericin B is the preferred treatment, especially for VL, but the relapse rate in transplant recipients is approximately 25%. PCR monitoring of blood for either secondary prophylaxis or preemptive therapy requires further study., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

We compare the rates of drug release from nanopatterned and flat biodegradable polymer surfaces, and observe significantly lower release rates from the nanopatterned surfaces. Specifically, we nanopattern poly(l-lactic acid) (PLLA), a biodegradable polymer frequently used for fabricating drug-eluting coronary stents, through microtransfer molding and solvent casting and investigate the nanopattern's impact on the release of sirolimus, an immunosuppressant agent, coated on the PLLA surface using high performance liquid chromatography/mass spectrometry. We find that PLLA surfaces nanopatterned with 750 nm-pitch nanocup or nanocone arrays exhibit drug release rates significantly lower (25-30%) than that of the flat surface, which is counter-intuitive given the nanopattern-induced increase in their surface areas. Based on diffusion and meniscus curvature minimization analyses, we attribute the decreased drug release rate to the incomplete wetting of the nanopatterned surface. These results provide new insights on how the surface nanopatterning of biomaterials can functionalize the surface and tailor the release kinetics of therapeutic agents coated on it for controlled drug elution.

Background Ovarian cysts, which present as neoplastic and non-neoplastic lesions, are the most common gynaecological cause of hospital admissions. Early diagnosis is difficult due to asymptomatic nature. Clinical, radiological and gross examination alone cannot distinguish benign from malignant lesions, hence, histopathological examination is important for diagnostic, therapeutic and prognostic approach. Objective The objective of the study is to analyze the spectrum of ovarian cystic lesions with their clinico-morphorgical features. Method This is a prospective study done in between July 2014 and July 2015 in Dhulikhel Hospital-Kathmandu University Hospital. Clinical data of patients were obtained from hospital records and requisition submitted along with the tissue specimens received in the department. Result A total 84 cases of ovarian cystic lesions were studied. Among these, 47 (55.9%) were non-neoplastic lesions, 33 (39.3%) were benign neoplasms, two (2.4%) were borderline and two (2.4%) were malignant neoplasms. The most common nonneoplastic lesions were follicular cysts, 26 (55.3%) followed by simple cysts 14 (29.8%), hemorrhagic cysts five (10.6%) and corpus luteal cysts two (4.3%). Among all neoplasms, 19 (51.4%) were mature cystic teratoma followed by 10 (27.0%) cases of mucinous cystadenoma and four (10.8%) cases of serous cystadenoma. Between two (5.4%) malignant cases, one was immature cystic teratoma and the other was mucinous cystadenocarcinoma. Besides these, two (5.4%) cases of borderline mucinous cystadenoma were also present. Conclusion Ovarian cystic lesions are difficult to categorize on the basis of clinical and radiological findings. Histopathological examination plays a significant role to differentiate benign lesion from malignant as well as for the proper management.

A positive Toxoplasma immunoglobulin M (IgM) result is often interpreted as a marker of an acute infection. However, IgM can persist for several years, and Toxoplasma commercial IgM diagnostic test kits can yield a number of false-positive results. For these reasons, a chronic Toxoplasma infection can be erroneously classified as an acute infection, resulting in serious adverse consequences, especially in pregnant women, leading to emotional distress and unnecessary interventions, including termination of pregnancy. Interpretation of Toxoplasma serology at a reference laboratory can help differentiate a recently acquired infection from a chronic infection. Serological test results for 451 patients with positive Toxoplasma IgM and IgG test results obtained at nonreference laboratories (NRLs) that were referred to Palo Alto Medical Foundation Toxoplasma Serology Laboratory (PAMF-TSL) to determine whether the patient was acutely or chronically infected were retrospectively reviewed. PAMF-TSL results established that of the 451 patients, 335 (74%) had a chronic infection, 100 (22%) had an acute infection, and 7 (2%) were not infected, and for 9 (2%), results were indeterminate. Positive Toxoplasma IgM and IgG test results obtained at NRLs cannot accurately distinguish between acute and chronic infections. To do so, testing at reference laboratories is required, as mandated in 1997 in a letter from the Food and Drug Administration (FDA) to clinicians and laboratories in the United States., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background Spontaneous abortion refers to a pregnancy that ends spontaneously before the fetus has reached a viable gestational age or expulsion or extraction of an embryo or fetus weighing 500 g or less from its mother. The Maternal Mortality Morbidity Survey of Nepal 2008/09 reported that 7% of maternal deaths in Nepal were due to complications related to abortion. Objective The main objective of this study was to examine the histopathological changes in the chorionic villi and endometrial decidual tissue in products of conception obtained from women with spontaneous abortion. Method This is a retrospective study of 111 patients admitted in the Department of Obstetrics and Gynecology at Dhulikhel Hospital, Kathmandu University Hospital (DH-KUH) with the diagnosis of spontaneous abortion during the period of January 2013 to January 2014. Result Among 111 cases of spontaneous abortions, products of conception was seen in 73 (65.77%) and with only one cases of choriocarcinoma. Majority of cases belongs to age group 21-30 years. The most common decidual changes were inflammation (41.4%) followed by fibrin deposition 29.7%. Majority of the cases shows hydropic changes as histopathological changes in chorionic villi. In the present study, minimum age of lady was 15 years and the maximum age was 45 years and the mean age was 25.09±5.58 years at the time of abortion. Among the cases, maximum 69 (62.2%) of them belonged to age group 21-30 years. Correlating the age group with number of abortions was found to be significantly different (Chi-square= 92.35, df= 3, p < 0.001) among four different age groups. Conclusion The histopathological diagnosis of spontaneous abortion will help in further management of the patient. Further study is required to know the cause of different histopathlogical changes in villi as well as in the decidua.

Purpose of Review: Toxoplasmosis in haematopoietic cell transplant (HCT) recipients is associated with high morbidity and mortality rates. Prophylaxis following HCT is recommended for high-risk pre-HCT toxoplasma-seropositive (pre-HCTSP) recipients. However, there is no agreement or consistency among programmes on whether to adopt prophylaxis or not, or if used, on the chosen antitoxoplasma prophylactic regimen. This review discusses the role of prophylaxis, and preemptive treatment, for toxoplasmosis in the setting of HCT., Recent Findings: Approximately two-thirds of toxoplasmosis cases following HCT are reported in allogeneic pre-HCTSP (allo pre-HCTSP) patients. This finding confirms a major role of reactivation of latent infection in the pathogenesis of toxoplasmosis in this patient population. Toxoplasma disease-related mortality in allo pre-HCTSP patients was reported at 62%, but it can be significantly decreased with early detection and treatment of toxoplasma infection. There are no randomized trials comparing the efficacy of different prophylactic agents to prevent toxoplasmosis after HCT. Several observational studies have demonstrated the efficacy of trimethoprim-sulfamethoxazole (TMP/SMX) in decreasing the incidence of toxoplasmosis following HCT. There is limited information regarding efficacy of other prophylactic agents. Preemptive treatment using routine blood PCR monitoring seems to be beneficial in detecting infection early and preventing disease in several observational studies and has been adopted for allo pre-HCTSP HCT patients when universal prophylaxis is not possible., Summary: Universal prophylaxis with TMP/SMX in allo pre-HCTSP patients should be implemented by all transplant programmes. Preemptive treatment with routine blood PCR monitoring is an option if prophylaxis cannot be used.

The mouth cavity hosts many types of anaerobic bacteria, including Streptococcus mutans and Porphyromonas gingivalis, which cause periodontal inflammatory diseases and dental caries. The present study was conducted to evaluate the antibacterial potential of extracts of Robinia pseudoacacia and its different fractions, as well as some of its natural compounds against oral pathogens and a nonpathogenic reference bacteria, Escherichia coli. The antibacterial activity of the crude extract and the solvent fractions (hexane, chloroform, ethyl acetate and butanol) of R. pseudoacacia were evaluated against S. mutans, P. gingivalis and E. coli DH5α by standard micro-assay procedure using conventional sterile polystyrene microplates. The results showed that the crude extract was more active against P. gingivalis (100% growth inhibition) than against S. mutans (73% growth inhibition) at 1.8 mg/mL. The chloroform and hexane fractions were active against P. gingivalis, with 91 and 97% growth inhibition, respectively, at 0.2 mg/mL. None of seven natural compounds found in R. pseudoacacia exerted an antibacterial effect on P. gingivalis; however, fisetin and myricetin at 8 µg/mL inhibited the growth of S. mutans by 81% and 86%, respectively. The crude extract of R. pseudoacacia possesses bioactive compounds that could completely control the growth of P. gingivalis. The antibiotic activities of the hexane and chloroform fractions suggest that the active compounds are hydrophobic in nature. The results indicate the effectiveness of the plant in clinical applications for the treatment of dental plaque and periodontal inflammatory diseases and its potential use as disinfectant for various surgical and orthodontic appliances.

Key Message: We report the significant increase of the content of free amino acids in Nicotiana benthamiana by the co-suppression of the ClpC1 and ClpC2 genes, which are translated to be the chaperonic part in the Clp protease at plastids. Clp protease with ClpC1 and ClpC2 proteins as the chaperonic part degrades denatured or improperly folded protein in plastids. Nicotiana benthamiana ClpC1 and ClpC2 genes (NbClpC1 and NbClpC2: NbClpC1/C2) share 93% similarities; therefore, co-suppression of the NbClpC1/C2 was possible using a single virus-induced silencing vector. Co-suppression of NbClpC1/C2 resulted in a pleiotropic phenotype including disappearance of apical dominance and formation of chlorotic leaves. NbClpC1/C2 co-suppressed leaves accumulated 11.9-fold more free amino acids than the GFP-silenced leaves. The co-suppression of NbClpC1/C2 did not change the expression levels of some selected genes in the biosynthetic pathways for the free amino acids, but reduced the total protein amounts to 32.5%, indicating that co-suppression affected the incorporation of free amino acids in proteins during translation. The loosely packed mesophyll cells and abnormal vascular bundles in the leaves suggested structural problems associated with translocation of free amino acids to sink tissues. NbClpC1/C2 co-suppression can offer a novel strategy for accumulation of free amino acids though it results in stunted growth.

Specific wavelengths of light can exert various physiological changes in plants, including effects on responses to disease incidence. To determine whether specific light wavelength had effects on rotting disease caused by Pseudomonas putida 229, soybean sprouts were germinated under a narrow range of wavelengths from light emitting diodes (LEDs), including red (650-660), far red (720-730) and blue (440-450 nm) or broad range of wavelength from daylight fluorescence bulbs. The controls were composed of soybean sprouts germinated in darkness. After germination under different conditions for 5 days, the soybean sprouts were inoculated with P. putida 229 and the disease incidence was observed for 5 days. The sprouts exposed to red light showed increased resistance against P. putida 229 relative to those grown under other conditions. Soybean sprouts germinated under red light accumulated high levels of salicylic acid (SA) accompanied with up-regulation of the biosynthetic gene ICS and the pathogenesis- related (PR) gene PR-1, indicating that the resistance was induced by the action of SA via de novo synthesis of SA in the soybean sprouts by red light irradiation. Taken together, these data suggest that only the narrow range of red light can induce disease resistance in soybean sprouts, regulated by the SA-dependent pathway via the de novo synthesis of SA and up-regulation of PR genes.

Tremendous demands for sensitive and reliable label-free biosensors have stimulated intensive research into developing miniaturized radiofrequency resonators for a wide range of biomedical applications. Here, we report the development of a robust, reusable radiofrequency resonator based integrated passive device biosensor chip fabricated on a gallium arsenide substrate for the detection of glucose in water-glucose solutions and sera. As a result of the highly concentrated electromagnetic energy between the two divisions of an intertwined spiral inductor coupled with an interdigital capacitor, the proposed glucose biosensor chip exhibits linear detection ranges with high sensitivity at center frequency. This biosensor, which has a sensitivity of up to 199 MHz/mgmL(-1) and a short response time of less than 2 sec, exhibited an ultralow detection limit of 0.033 μM and a reproducibility of 0.61% relative standard deviation. In addition, the quantities derived from the measured S-parameters, such as the propagation constant (γ), impedance (Z), resistance (R), inductance (L), conductance (G) and capacitance (C), enabled the effective multi-dimensional detection of glucose.

Random inductor–capacitor (LC) networks can exhibit percolative superconductor-insulator transitions (SITs). We use a simple and efficient algorithm to compute the dynamicalconductivity σ(ω,p) of one type of LC network on large (4000 × 4000) square lattices, where δ = p − p(c) is the tuning parameter for the SIT. We confirm that the conductivity obeys a scaling form, so that the characteristic frequency scales as∝|δ|(νz) with νz ≈ 1.91, the superfluid stiffness scales as ϒ∝|δ|(t) with t ≈ 1.3, and the electric susceptibility scales as χE∝|δ|(−s) with s = 2νz − t ≈ 2.52. In the insulating state, the low-frequency dissipative conductivity is exponentially small, whereas in the superconductor, it is linear in frequency.The sign of Im σ(ω) at small ω changes across the SIT. Most importantly, we find that right at the SIT Re σ(ω) ∝ ω(t/νz−1)∝ω(−0.32), so that the conductivity diverges in the DC limit, in contrast with most other classical and quantum models of SITs.

We present an autonomous, self-powered solar light controller based on functional integration of a flexible cantilever light guide and a paraffin wax-based optothermal actuator. The controller utilizes the optothermally induced volume increase in the elastomer-encapsulated paraffin wax to produce pneumatic force, which subsequently actuates the cantilever light guide to control the level of frustrated total internal reflection. In its linear response regime, it demonstrated 33% reduction in light intensity fluctuation in terms of the root-mean-square value.

The spore-forming bacterium Bacillus licheniformis is a common contaminant of milk and milk products. Strains of this species isolated from dairy products can be differentiated into three major groups, namely, G, F1, and F2, using random amplification of polymorphic DNA (RAPD) analysis; however, little is known about the genomic differences between these groups and the identity of the fragments that make up their RAPD profiles. In this work we obtained high-quality draft genomes of representative strains from each of the three RAPD groups (designated strain G-1, strain F1-1, and strain F2-1) and compared them to each other and to B. licheniformis ATCC 14580 and Bacillus subtilis 168. Whole-genome comparison and multilocus sequence typing revealed that strain G-1 contains significant sequence variability and belongs to a lineage distinct from the group F strains. Strain G-1 was found to contain genes coding for a type I restriction modification system, urease production, and bacitracin synthesis, as well as the 8-kbp plasmid pFL7, and these genes were not present in strains F1-1 and F2-1. In agreement with this, all isolates of group G, but no group F isolates, were found to possess urease activity and antimicrobial activity against Micrococcus. Identification of RAPD band sequences revealed that differences in the RAPD profiles were due to differences in gene lengths, 3' ends of predicted primer binding sites, or gene presence or absence. This work provides a greater understanding of the phylogenetic and phenotypic differences observed within the B. licheniformis species.

We present a novel thin and flat solar concentrator design, inspired by the structure and optical functionality of the ommatidium in the compound eye of insects. By combining a microlens with a curved light guide, rather than the conventionally employed dielectric or metallic reflectors, we could simultaneously achieve low-loss light redirection and wide acceptance angle without compromising the overall thinness or flatness of the concentrator. Through design optimizations, we could achieve optical concentration factors up to 39 and acceptance angle up to ±15° while maintaining the thickness of the concentrator under 1.1 cm for a length of 20 cm. We also showed that the optical concentration factor can be further increased to 81 through tapering of the geometry.

This paper presents a symmetric-type microstrip triple-band bandstop filter incorporating a tri-section meandered-line stepped impedance resonator (SIR). The length of each section of the meandered line is 0.16, 0.15, and 0.83 times the guided wavelength (λ g ), so that the filter features three stop bands at 2.59 GHz, 6.88 GHz, and 10.67 GHz, respectively. Two symmetric SIRs are employed with a microstrip transmission line to obtain wide bandwidths of 1.12, 1.34, and 0.89 GHz at the corresponding stop bands. Furthermore, an equivalent circuit model of the proposed filter is developed, and the model matches the electromagnetic simulations well. The return losses of the fabricated filter are measured to be -29.90 dB, -28.29 dB, and -26.66 dB while the insertion losses are 0.40 dB, 0.90 dB, and 1.10 dB at the respective stop bands. A drastic reduction in the size of the filter was achieved by using a simplified architecture based on a meandered-line SIR.

Due to their ubiquity in the environment and ability to survive heating processes, sporeforming bacteria are commonly found in foods. This can lead to product spoilage if spores are present in sufficient numbers and where storage conditions favour spore germination and growth. A rapid method to identify the major aerobic sporeforming groups in dairy products, including Bacillus licheniformis group, Bacillus subtilis group, Bacillus pumilus group, Bacillus megaterium, Bacillus cereus group, Geobacillus species and Anoxybacillus flavithermus was devised. This method involves real-time PCR and high resolution melt analysis (HRMA) of V3 (~70 bp) and V6 (~100 bp) variable regions in the 16S rDNA. Comparisons of HRMA curves from 194 isolates of the above listed sporeforming bacteria obtained from dairy products which were identified using partial 16S rDNA sequencing, allowed the establishment of criteria for differentiating them from each other and several non-sporeforming bacteria found in samples. A blinded validation trial on 28 bacterial isolates demonstrated complete accuracy in unambiguous identification of the 7 different aerobic sporeformers. The reliability of HRMA method was also verified using boiled extractions of crude DNA, thereby shortening the time needed for identification. The HRMA method described in this study provides a new and rapid approach to identify the dominant mesophilic and thermophilic aerobic sporeforming bacteria found in a wide variety of dairy products., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In dairy foods, the sporeformer Bacillus licheniformis can be the cause of spoilage or specification compliance issues. Currently used methods for genotyping B. licheniformis have limited discrimination with only 2 or 3 different subgroups being identified. Here, we have developed a multi-locus variable number tandem repeat analysis (MLVA) method and combined it with high resolution melt analysis (MLV-HRMA) for genotyping B. licheniformis. Five repetitive loci were identified and used as markers for genotyping 52 isolates from two milk powder processing plants and retail samples. Nineteen genotypes could be identified using both MLVA and MLV-HRMA leading to Hunter-Gaston discrimination indices (D-value) of 0.93 each. It was found that all 5 MLVA loci were stable following 10 days of sub-culturing of 8 representative isolates. All isolates were also genotyped using previously used methods including randomly amplified polymorphic DNA-PCR (RAPD) and partial rpoB sequencing. Five different RAPD profiles and 5 different partial rpoB sequence types were identified resulting in corresponding D-values of 0.6 and 0.46, respectively. Analysis of the genotypes from dairy samples revealed that dairy B. licheniformis isolates are more heterogeneous than previously thought and that this new method can potentially allow for more discriminatory tracking and monitoring of specific genotypes., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

GABA (γ-aminobutyric acid) is a four carbon non-protein amino acid that is widely distributed in plants, animals and microorganisms. As a metabolic product of plants and microorganisms produced by the decarboxylation of glutamic acid, GABA functions as an inhibitory neurotransmitter in the brain that directly affects the personality and the stress management. A wide range of traditional foods produced by microbial fermentation contain GABA, in which GABA is safe and eco-friendly, and also has the possibility of providing new health-benefited products enriched with GABA. Synthesis of GABA is catalyzed by glutamate decarboxylase, therefore, the optimal fermentation condition is mainly based on the biochemical properties of the enzyme. Major GABA producing microorganisms are lactic acid bacteria (LAB), which make food spoilage pathogens unable to grow and act as probiotics in the gastrointestinal tract. The major factors affecting the production of GABA by microbial fermentation are temperature, pH, fermentation time and different media additives, therefore, these factors are summarized to provide the most up-dated information for effective GABA synthesis. There has been a huge accumulation of knowledge on GABA application for human health accompanying with a demand on natural GABA supply. Only the GABA production by microorganisms can fulfill the demand with GABA-enriched health beneficial foods.

Spores of thermophilic Geobacillus species are a common contaminant of milk powder worldwide due to their ability to form biofilms within processing plants. Genotyping methods can provide information regarding the source and monitoring of contamination. A new genotyping method was developed based on multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) in conjunction with high-resolution melt analysis (MLV-HRMA) and compared to the currently used method, randomized amplified polymorphic DNA PCR (RAPD-PCR). Four VNTR loci were identified and used to genotype 46 Geobacillus isolates obtained from retailed powder and samples from 2 different milk powder processing plants. These 46 isolates were differentiated into 16 different groups using MLV-HRMA (D = 0.89). In contrast, only 13 RAPD-PCR genotypes were identified among the 46 isolates (D = 0.79). This new method was then used to analyze 35 isolates obtained from powders with high spore counts (>10(4) spores · g(-1)) from a single processing plant together with 27 historical isolates obtained from powder samples processed in the same region of Australia 17 years ago. Results showed that three genotypes can coexist in a single processing run, while the same genotypes observed 17 years ago are present today. While certain genotypes could be responsible for powders with high spore counts, there was no correlation to specific genotypes being present in powder plants and retailed samples. In conclusion, the MLV-HRMA method is useful for genotyping Geobacillus spp. to provide insight into the prevalence and persistence of certain genotypes within milk powder processing plants.

A study was conducted to analyze the status of the multidrug resistant (MDR) isolates producing Extended Spectrum of beta-lactamase (ESBL) among the uropathogens infecting children less than 15 years from November 2010 to April 2011 in the Bacteriology laboratory, Kathmandu Model Hospital. Urine samples received in the laboratory were processed for routine culture. The antimicrobial susceptibility of bacterial isolates was determined following Clinical and Laboratory Standard Institute (CLSI) recommended Kirby-Bauer Disc Diffusion method. The defining criterion in this study for an isolate to be multidrug resistant was resistance to two or more drugs of different structural classes. Isolates were confirmed for ESBL-production by performing the Inhibitor Potentiated Disk Diffusion (IPDD) Test/ Combined Disk Assay for ESBL confirmation. Out of 252 urine samples received in the laboratory, 59(23.41%) showed significant growth of which 54.23% (32/59) were MDR isolates. Additionally, 25 isolates (21 Escherichia coli and 3 Citrobacter freundii and single Enterobacter aerogenes) among them were ESBL producers. Among the first line drugs used against gram negative isolates, Nitrofurantoin was drug of choice; meanwhile among the second line drugs Cefoperazone/Sulbactum was drug of choice, whereas, Cephotaxime, Ciprofloxacin, Norfloxacin and Gentamicin were the drug of choice for Gram positive isolates. Significant association was found between ESBL production and spectrum of drug resistance (p < 0.05).

Background: Adequate nutritional support is important for the comprehensive management of patients in intensive care units (ICUs)., Aim: The study was aimed to survey prevalent enteral nutrition practices in the trauma intensive care unit, nurses' perception, and their knowledge of enteral feeding., Study Design: The study was conducted in the ICU of a level 1 trauma center, Jai Prakash Narayan Apex Trauma Centre, AIIMS, New Delhi, India. The study design used an audit., Materials and Methods: Sixty questionnaires were distributed and the results analyzed. A database was prepared and the audit was done., Results: Forty-two (70%) questionnaires were filled and returned. A majority (38) of staff nurses expressed awareness of nutrition guidelines. A large number (32) of staff nurses knew about nutrition protocols of the ICU. Almost all (40) opined enteral nutrition to be the preferred route of nutrition unless contraindicated. All staff nurses were of opinion that enteral nutrition is to be started at the earliest (within 24-48 h of the ICU stay). Everyone opined that the absence of bowel sounds is an absolute contraindication to initiate enteral feeding. Passage of flatus was considered mandatory before starting enteral nutrition by 86% of the respondents. Everyone knew that the method of Ryle's tube feeding in their ICU is intermittent boluses. Only 4 staff nurses were unaware of any method to confirm Ryle's tube position. The backrest elevation rate was 100%. Gastric residual volumes were always checked, but the amount of the gastric residual volume for the next feed to be withheld varied. The majority said that the unused Ryle's tube feed is to be discarded after 6 h. The most preferred (48%) method to upgrade their knowledge of enteral nutrition was from the ICU protocol manual., Conclusion: Information generated from this study can be helpful in identifying nutrition practices that are lacking and may be used to review and revise enteral feeding practices where necessary.

Objective: To screen for asymptomatic respiratory carriage of S. pneumoniae, H. influenzae and Group A Streptococcus (GAS) in children attending JIPMER, correlate carriage rate with different socio-demographic factors and to detect antimicrobial resistance among the isolates., Methods: Throat swabs were collected from both in patients and out patients (1 organism. Antibiotic resistance was highest in S. pneumoniae with 66.7% of strains resistant to penicillin. MDR strains were also encountered. Erythromycin resistance was observed in both H. influenzae (28.4%) and GAS (22%). No statistically significant association was found between the carriage rate of these organisms and different socio-demographic factors., Conclusions: S. pneumoniae carriage rate was comparatively higher in the Community and its antimicrobial resistance is an issue to address.

The objective of the study was to analyze the status and explore the challenges to decentralization policy implementation in Nepal. Thirty seven key informants rich in experience and knowledge, seven focus group discussions, observation of six health facilities and analysis of about 25 key policy documents provided the data for this study. The study identified the challenges to the implementation of decentralization reforms in the public health sector as: (i) centralised and weak management and programming practices of the government; (ii) weak legal and institutional framework; (iii) conflicting policy objectives; (iv) lack of implementation strategy; (v) poor financial and human resource management system; (vi) lack of adequate preparation for managing the reform; (vii) weak capacity at all levels; (viii) political instability. It was revealed that the implementation of the policy in Nepal was extremely poor as many of the important policy measures were either never initiated or they were only partially implemented. The challenges lie both at - policy design and implementation phase. Clear policy objectives, appropriate structure, sound planning, financing and human resources policy, adequate capacity, responsive information system, defined service packages, active participation of stakeholders and a conducive socio-political environment are considered imperative for successful implementation of the policy. Preparation for managing reform implementation at national and district levels is prerequisite for decentralization to work. Pushing for decentralization in a politically fragile environment may rather lead to further fragmentation, instead of strengthening government legitimacy.

Purpose: The purpose of this paper is to describe and discuss policy analysis in Nepal and review the wide range of choices feasible in decentralisation decision making., Design/methodology/approach: In this paper an iterative qualitative method was developed and used in the research, which consisted of focus group interviews, key informant interviews, document analysis, including descriptive statistics, and analysis of the policy context. Participants in the research reflected the urban/rural mix of districts and the geography of Nepal. Analysis combined transcribed interviews with findings from document searches and analysis of the policy context. Coding was pre-determined during the training workshop and further codes were generated during and after the fieldwork., Findings: The paper finds that Nepal is in the process of decentralising public services from the central level to the local level, particularly to local bodies: District Development Committees (DDCs), Village Development Committees (VDCs) and Municipalities. Key contextual factors referred to are the overall structure of decentralisation, the social context of poverty and the political instability leading to a fluid political situation characterised by political tension, armed conflict, controversies and agreements while carrying out the research. The key issues identified and discussed in the paper are the policy process leading to decentralisation, the organisational structure and tension in the proposed system, the systems of resource generation, allocation, planning and management and lastly the forms of accountability, participation, public-private relations and collaborative strategies., Originality/value: The paper discusses the challenges faced in conducting such a policy analysis, the broad ranging and unremitting nature of the decentralisation process, and the contextual setting of the process of change.

Objectives: to evaluate the diagnostic value of clinical symptoms and signs in enteric fever and to propose a clinical diagnostic criterion., Design: Prospective observational study., Setting: Kathmandu Medical College, Teaching Hospital, Kathmandu, Nepal., Materials and Methods: febrile patients with clinical diagnosis of enteric fever were included in the study with the aim of confirming diagnosis with blood culture, or bone marrow culture and evaluating the diagnostic accuracy of various clinical signs and symptoms., Results: 64% of the clinically diagnosed cases had blood/ bone marrow culture positive. The diagnostic accuracy of the various symptoms and signs excluding fever was between 42%-75.5%. Majority of the symptom and sign did not have very high diagnostic accuracy. Hence a diagnostic criterion was proposed and clinical features with diagnostic accuracy more than 50% were taken into consideration. Major criteria included fever with diagnostic accuracy of 64%, headache with accuracy of 75.5% and relative bradycardia with an accuracy of 66%. Minor criteria included vomiting, diarrhoea, Splenomegaly, chills and abdominal pain /discomfort with diagnostic accuracy of 57%, 55%, 55%, 53% and 51% respectively. Finally after combination of various major and minor criteria a final diagnostic criterion was proposed having an accuracy of 66% and including both major and minor clinical symptom and sign., Conclusion: clinical diagnosis of enteric fever will be very helpful in a country like ours. Though none of the clinical symptoms and sign have very high diagnostic accuracy a diagnostic criteria may be helpful. Criteria including both major and minor signs and symptoms would be the most appropriate diagnostic tool as it includes the important abdominal symptoms and signs of enteric fever.