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Background: The US Food and Drug Administration recommends against feeding infants human milk from unscreened donors, but sharing milk via the Internet is growing in popularity. Recipient infants risk the possibility of consuming contaminated or adulterated milk. Our objective was to test milk advertised for sale online as human milk to verify its human origin and to rule out contamination with cow's milk., Methods: We anonymously purchased 102 samples advertised as human milk online. DNA was extracted from 200 μL of each sample. The presence of human or bovine mitochondrial DNA was assessed with a species-specific real-time polymerase chain reaction assay targeting the nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 5 gene. Four laboratory-created mixtures representing various dilutions of human milk with fluid cow's milk or reconstituted infant formula were compared with the Internet samples to semiquantitate the extent of contamination with cow's milk., Results: All Internet samples amplified human DNA. After 2 rounds of testing, 11 samples also contained bovine DNA. Ten of these samples had a level of bovine DNA consistent with human milk mixed with at least 10% fluid cow's milk., Conclusions: Ten Internet samples had bovine DNA concentrations high enough to rule out minor contamination, suggesting a cow's milk product was added. Cow's milk can be problematic for infants with allergy or intolerance. Because buyers cannot verify the composition of milk they purchase, all should be aware that it might be adulterated with cow's milk. Pediatricians should be aware of the online market for human milk and the potential risks., (Copyright © 2015 by the American Academy of Pediatrics.)

Antimicrobial peptides are increasingly being explored as alternative agents for therapy against the parasitic protozoan Leishmania. Previously, we reported that the synthetic magainin analog, pexiganan, induced apoptosis of surface protease-deficient Leishmania. Here, we report the development of an arginine-rich variant of this peptide which has reduced protease susceptibility and enhanced activity against wild type Leishmania in vitro. This peptide induces calcium delocalization and caspase 3/7 activity indicative of apoptosis, demonstrating that structural modification of pexiganan leads to drastic changes in biologic activity against Leishmania.

The digenetic protozoan Leishmania is dependent on ergosterol synthesis for growth and viability. We compared the in vitro activity of ergosterol synthesis inhibitor voriconazole with fluconazole and ketoconazole against cutaneous and visceral Leishmania species. We found the IC50 of voriconazole was comparable to ketoconazole and both were superior to fluconazole. Both ketoconazole and voriconazole were active against insect and mammalian stage parasites. This is the first report of the in vitro activity of voriconazole against leishmanial species.

The mechanisms by which Trypanosoma cruzi survives antimicrobial peptides and differentiates during its transit through the gastrointestinal tract of the reduviid vector are unknown. We show that cyclophilin, a peptidyl-prolyl isomerase secreted from T. cruzi epimastigotes, binds to and neutralizes the reduviid antimicrobial peptide trialysin promoting parasite survival. This is dependent on a singular proline residue in trialysin and is inhibited by the cyclophilin inhibitor cyclosporine A. In addition, cyclophilin-trialysin complexes enhance the production of ATP and reductase responses of parasites, which are inhibited by both calcineurin-specific inhibitors cyclosporine A and FK506. Calcineurin phosphatase activity of cyclophilin-trialysin-treated parasites was higher than in controls and was inhibited by preincubation by either inhibitor. Parasites exposed to cyclophilin-trialysin have enhanced binding and invasion of host cells leading to higher infectivity. Leishmanial cyclophilin also mediates trialysin protection and metabolic stimulation by T. cruzi, indicating that extracellular cyclophilin may be critical to adaptation in other insect-borne protozoa. This work demonstrates that cyclophilin serves as molecular sensor leading to the evasion and adaptive metabolic response to insect defense peptides.

Context: Globally, there has been an increase in incidence of paratyphoid fever, including paratyphoid fever caused by antimicrobial-resistant strains. Studying the clinical profile and antimicrobial sensitivity of paratyphoid fever would help in early diagnosis, appropriate treatment, rational use of antibiotics and prevent drug resistance., Aim: The aim of the study was to evaluate the clinical profile and sensitivity patterns of antibiotics used in the treatment of paratyphoid fever., Settings and Design: A record-based study was done in tertiary care hospital, South India., Materials and Methods: A retrospective analysis of culture-proven cases of paratyphoid fever was done in a tertiary care hospital. The socio-demographic characteristics, mode of presentation and the sensitivity pattern of isolates from blood culture were recorded. One hundred and ten case files of Salmonella paratyphi were reviewed from the medical records section and the required data (data regarding the clinical profile and antibiotic sensitivity) was collected and analyzed using SPSS version 11.5., Results: Fever was present in all patients. All the cases were sensitive for third-generation cephalosporins, and only 31.8% of the cases were sensitive for quinolones. Sensitivity towards other antibiotics in descending order was as follows: ampicillin 93.6%, chloramphenicol 91.8%, aminoglycosides 90.4% and sulphonamides 76.4%., Conclusions: Research shows that there is increasing resistance to fluoroquinolones and sensitivity to chloramphenicol. Considering the changing trend in the sensitivity pattern, the recommendations of treatment for enteric fever need to be rationalized and re-considered.

Hypertension is a chronic condition of concern and a major public health problem posing a challenge to population in socioeconomic and demographic transition. Prehypertension in adolescents and young adults is an important risk for developing hypertension in later years of life. Therefore this study was taken up among medicos of this institute to study about prehypertension and its associated factors. The objective of the study was to see the prevalence of prehypertension and its associated factors among medicos of SSIMS&RC, Devangere. The study duration was from 1st July 2009 to 31st August 2009. The sample size worked out to be 100 and this set of participants was selected by systematic random sampling. A predesigned and pretested proforma was used to collect the relevant data. Blood pressure (BP) was measured in sitting posture using a standard sphygmomanometer on two different occasions and the average was noted. BP was classified as per the Joint National Committee on prevention, detection, evaluation and treatment of blood pressure. Data was analysed by SPSS. Descriptive statistics was presented by, percentage, mean and SD. Groups are compared with the help of z-test, t-test and Chi-square test. All the participants were in the age group of 18 to 22 years and mean age was 20.29 +/- 0.86 years. Among 100 participants 56 were males and 44 were females. The mean systolic blood pressure was 117.68 +/- 7.16 mm Hg and that of diastolic blood pressure was 77.3 +/- 7.93 min Hg. The percentage of prehypertension and hypertension was 67%. Three participants were diagnosed to be hypertensive.

Cathelicidin-type antimicrobial peptides (CAMP) are important mediators of innate immunity against microbial pathogens acting through direct interaction with and disruption of microbial membranes and indirectly through modulation of host cell migration and activation. Using a mouse knock-out model in CAMP we studied the role of this host peptide in control of dissemination of cutaneous infection by the parasitic protozoan Leishmania. The presence of pronounced host inflammatory infiltration in lesions and lymph nodes of infected animals was CAMP-dependent. Lack of CAMP expression was associated with higher levels of IL-10 receptor expression in bone marrow, splenic and lymph node macrophages as well as higher anti-inflammatory IL-10 production by bone marrow macrophages and spleen cells but reduced production of the pro-inflammatory cytokines IL-12 and IFN-γ by lymph nodes. Unlike wild-type mice, local lesions were exacerbated and parasites were found largely disseminated in CAMP knockouts. Infection of CAMP knockouts with parasite mutants lacking the surface metalloprotease virulence determinant resulted in more robust disseminated infection than in control animals suggesting that CAMP activity is negatively regulated by parasite surface proteolytic activity. This correlated with the ability of the protease to degrade CAMP in vitro and co-localization of CAMP with parasites within macrophages. Our results highlight the interplay of antimicrobial peptides and Leishmania that influence the host immune response and the outcome of infection., (© 2011 Blackwell Publishing Ltd.)

Combining complementary nonviral gene delivery vehicles such as tissue engineering scaffolds and liposomes not only is a promising avenue for development of safe and effective gene delivery system but also provides an opportunity to design dynamic extended release systems with spatiotemporal control. However, the DNA loading capacity of scaffolds such as fibrin is limited. Fibrin microspheres carrying DNA complexes can be utilized to extend the capacity of fibrin scaffold. Here, in a proof of concept study, the feasibility of fibrin microspheres for extending gene delivery capacity is described. Toward this goal, fibrin microspheres encapsulating lipoplexes were fabricated. The structural and functional integrity of DNA was assessed respectively by gel electrophoresis and an in vivo pilot study, using endothelial nitric oxide synthase (eNOS) as a model therapeutic gene in a rabbit ear ulcer model of compromised wound healing. The results confirmed structural integrity and successful delivery and functional integrity, assessed qualitatively by angiogenic effect of eNOS. Finally, as a step toward development of a "fibrin in fibrin" temporal release system, fibrin microspheres were shown to degrade and release DNA differentially compared to fibrin scaffold. It can thus be concluded that fibrin microspheres can be utilized for gene delivery to extend the capacity of a fibrin scaffold and can form a component of a "fibrin in fibrin" temporal release system.

This unit presents the protocol for the NanoString nCounter Gene Expression Assay, a robust and highly reproducible method for detecting the expression of up to 800 genes in a single reaction with high sensitivity and linearity across a broad range of expression levels. The methodology serves to bridge the gap between genome-wide (microarrays) and targeted (real-time quantitative PCR) expression profiling. The nCounter assay is based on direct digital detection of mRNA molecules of interest using target-specific, color-coded probe pairs. It does not require the conversion of mRNA to cDNA by reverse transcription or the amplification of the resulting cDNA by PCR. Each target gene of interest is detected using a pair of reporter and capture probes carrying 35- to 50-base target-specific sequences. In addition, each reporter probe carries a unique color code at the 5' end that enables the molecular barcoding of the genes of interest, while the capture probes all carry a biotin label at the 3' end that provides a molecular handle for attachment of target genes to facilitate downstream digital detection. After solution-phase hybridization between target mRNA and reporter-capture probe pairs, excess probes are removed and the probe/target complexes are aligned and immobilized in the nCounter cartridge, which is then placed in a digital analyzer for image acquisition and data processing. Hundreds of thousands of color codes designating mRNA targets of interest are directly imaged on the surface of the cartridge. The expression level of a gene is measured by counting the number of times the color-coded barcode for that gene is detected, and the barcode counts are then tabulated.

Objective: To study the efficacy of the ultrafast Papanicolaou (UFP) staining technique for immediate cytologic diagnosis, to check specimen adequacy during radiologically guided fine needle aspiration cytology (FNAC) procedures and to objectively assess the quality of UFP stain and factors affecting it., Study Design: Serial and random samples of cytologic material from 347 patients during a 2-year period were divided in 3 groups. Group I included 238 outpatient FNACs, group II included 59 radiologically guided FNACs, and group III included 50 cases of intraoperative cytology. After staining air-dried smears with UFP stain, immediate diagnosis was given. In group II, material adequacy was also conveyed to the radiologist., Results: Overall diagnosis was possible in 297 (85.6%) cases. Only 8 (2.3%) cases could not be diagnosed due to staining difficulties. The overall concordance rate was 98%. Seventy-five percent of cases showed 'excellent' and 'good' morphology, allowing easy diagnosis. In 25% of 'fair' cases diagnosis was still possible, although with some difficulties. Factors affecting the quality of staining are discussed., Conclusion: The UFP staining technique is an accurate and reliable method for rapid cytology reporting. It significantly reduces total turn around time of the test result, thereby having the potential to be cost-effective, both for the patient and the hospital., (Copyright © 2011 S. Karger AG, Basel.)

Antimicrobial peptides (AMPs) are multifunctional components of the innate systems of both insect and mammalian hosts of the pathogenic trypanosomatids Leishmania and Trypanosoma species. Structurally diverse AMPs from a wide range of organisms have in vitro activity against these parasites acting mainly to disrupt surface-membranes. In some cases AMPs also localize intracellularly to affect calcium levels, mitochondrial function and induce autophagy, necrosis and apoptosis. In this review we discuss the work done in the area of AMP interactions with trypanosomatid protozoa, propose potential targets of AMP activity at the cellular level and discuss how AMPs might influence parasite growth and differentiation in their hosts to determine the outcome of natural infection., ((c) 2010 Elsevier Inc. All rights reserved.)

Vaginal hysterectomy was the first minimally invasive surgical approach for benign gynecologic conditions. Recently a number of other approaches for hysterectomy have been introduced, yet when compared with vaginal hysterectomy these approaches do not offer significant benefit for similar indications. The focus of this chapter is to provide comprehensive review of vaginal hysterectomy for the practicing clinician.

alpha- and -defensin-, magainin-, and cathelicidin-type antimicrobial peptides (AMPs) can kill the pathogenic protozoan Leishmania. Comparative studies of a panel of AMPs have defined two distinct groups: those that induce nonapoptotic (Class I) and apoptotic (Class II) parasite killing based on their differential ability to induce phosphatidyl serine exposure, loss of mitochondrial membrane potential and decreased ATP production, induction of caspase-3/7 and -12 activity, and DNA degradation. Class II AMPs cause rapid influx of the vital stain SYTOX and an increase in intracellular Ca2+, whereas Class I AMPs cause a slow accumulation of SYTOX and do not affect intracellular Ca2+ levels. Inhibitors of cysteine or caspase proteases diminished fast influx of SYTOX through the surface membrane and DNA degradation but do not ablate the annexin V staining or the induction of apoptosis by Class II AMPs. This suggests that the changes in surface permeability in AMP-mediated apoptosis are related to the downstream events of intracellular cysteine/caspase activation or the loss of ATP. The activation of caspase-12-like activity was Ca(2+)-dependent, and inhibitors of voltage-gated and nonspecific Ca2+ channels diminished this activity. Flufenamic acid, a nonspecific Ca2+ inhibitor, completely ablated AMP-induced mitochondrial dysfunction and cell death, indicating the importance of dysregulation of Ca2+ in antimicrobial peptide-induced apoptosis.

The protozoan Trypanosoma cruzi expresses multiple isoforms of the GP63 family of metalloproteases. Polyclonal antiserum against recombinant GP63 of T. cruzi (TcGP63) was used to study TcGP63 expression and localization in this organism. Western blot analysis revealed that TcGP63 is 61 kDa in epimastigotes, amastigotes, and tissue culture-derived trypomastigotes but 55 kDa in metacyclic trypomastigotes. Antiserum specific for Leishmania amazonensis GP63 specifically reacted with a 55-kDa TcGP63 form in metacyclic trypomastigotes, suggesting stage-specific expression of different isoforms. Surface biotinylation and endoglycosidase digestion experiments showed that TcGP63 is an ecto-glycoprotein in epimastigotes but is intracellular and lacking in N-linked glycans in metacyclic trypomastigotes. Immunofluorescence microscopy showed that TcGP63 is localized on the surfaces of epimastigotes but distributed intracellularly in metacyclic trypomastigotes. TcGP63 is soluble in cold Triton X-100, in contrast to Leishmania GP63, which is detergent resistant in this medium, suggesting that GP63 is not raft associated in T. cruzi. Western blot comparison of our antiserum to a previously described anti-peptide TcGP63 antiserum indicates that each antiserum recognizes distinct TcGP63 proteins. Preincubation of trypomastigotes with either TcGP63 antiserum or a purified TcGP63 C-terminal subfragment reduced infection of host myoblasts. These results show that TcGP63 is expressed at all life stages and that individual isoforms play a role in host cell infection.

Infection by vector-borne protozoa of the genus Leishmania occurs by the deposition of parasites within the skin of the mammalian host, where they eventually bind to and are phagocytized by Mphis. Our previous work supported the idea that parasites can interact with extracellular matrix and basement membrane proteins, such as fibronectin (FN), within the skin, leading to enhanced invasion. In this report, we extend these findings and show that both promastigotes and amastigotes of Leishmania species can bind directly to soluble FN and laminin (LM) and that promastigotes express a distinct surface protein of approximately 60 kDa that binds both FN and LM. Promastigotes of multiple Leishmania species can rapidly degrade FN by using surface-localized and secreted metalloprotease (leishmanolysin). FN degradation at the surfaces of amastigotes is leishmanolysin dependent, whereas both secreted leishmanolysin and cysteine protease B contribute to extracellular FN degradation. Leishmania-degraded FN decreased the production of reactive oxygen intermediates by parasite-infected macrophages and affected the accumulation of intracellular parasites. These findings show that both parasite stages of Leishmania species bind to and proteolytically degrade FN at the parasite surface and distantly through secreted proteases and that degraded forms of FN can influence the activation state of parasite-infected macrophages.

Human infection by the vector-borne protozoan Leishmania is responsible for substantial worldwide morbidity and mortality. The surface-metalloprotease (leishmanolysin) of Leishmania is a virulence factor which contributes to a variety of functions including evasion of complement-mediated parasite-killing and host intramacrophage survival. We tested the hypothesis that leishmanolysin serves to protect parasites from the cytolytic effects of various antimicrobial peptides (AMPs) which are important components of the innate immune system. We found that members of the alpha- and theta-defensins, magainins and cathelicidins had substantially higher leishmanicidal activity against leishmanolysin-knock out mutants of L. major. Using the magainin analogue, pexiganan, as a model peptide we show that AMP evasion is due to rapid and extensive peptide degradation by wild-type parasites. Pexiganan-treatment of knock out mutants induced disruption of surface-membrane permeability and expression of features of apoptosis including smaller cell size, loss of mitochondrial membrane potential, exposure of surface phosphatidyl serine as well as induction of caspase 3/7 activity. These results demonstrate leishmanolysin as a virulence factor preventing AMP-mediated apoptotic killing. This study serves as a platform for the dissection of the AMP-mediated death pathways of Leishmania and demonstrates the potential that AMP evasion plays during host infection by this parasite.

To evaluate the specificity of long dsRNAs used in high-throughput RNA interference (RNAi) screens performed at the Drosophila RNAi Screening Center (DRSC), we performed a global analysis of their activity in 30 genome-wide screens completed at our facility. Notably, our analysis predicts that dsRNAs containing > or = 19-nucleotide perfect matches identified in silico to unintended targets may contribute to a significant false positive error rate arising from off-target effects. We confirmed experimentally that such sequences in dsRNAs lead to false positives and to efficient knockdown of a cross-hybridizing transcript, raising a cautionary note about interpreting results based on the use of a single dsRNA per gene. Although a full appreciation of all causes of false positive errors remains to be determined, we suggest simple guidelines to help ensure high-quality information from RNAi high-throughput screens.

The experimental results of the studies on the transportation of water droplets on a superhydrophobic silica aerogel-powder-coated surface are reported. The superhydrophobic silica aerogels were prepared using sol-gel processing of methyltrimethoxysilane (MTMS) precursor, methanol (MeOH) solvent, and base (NH4OH)-catalyzed water followed by supercritical drying using methanol solvent. The molar ratio of NH4OH/MTMS, H2O/MTMS, and MeOH/MTMS were varied from 1.7x10(-1) to 3.5x10(-1), 2 to 8, and 1.7 to 14, respectively, to find out the best-quality aerogels in terms of higher hydrophobicity and high droplet velocity. A specially built device was used for the measurement of velocity of water droplet of size 2.8 mm (+/-0.2 mm) on an inclined surface coated with superhydrophobic aerogel powder. Liquid marbles were prepared by rolling water droplets on aerogel powder and the marble(s) velocities on a noncoated inclined surface were compared with that of the water droplets. It was observed that the microstructure of the aerogel affects the droplet as well as marble velocities considerably. For an aerogel with uniform and smaller particles, the water droplet and marble velocities were observed to be maximum, i.e., 144 and 123 cm/s, respectively, whereas for the aerogels with bigger and nonuniform particles, the water droplet and marble velocities were observed to be minimum, i.e., 92 and 82 cm/s, respectively. The results have been discussed by taking into account the contact angles and microstructural observations.

Bioinformatics analysis of transcriptional control is guided by knowledge of the characteristics of cis-regulatory regions or enhancers. Features such as clustering of binding sites and co-occurrence of binding sites have aided enhancer identification, but quantitative predictions of enhancer function are not yet generally feasible. To facilitate the analysis of regulatory sequences in Drosophila melanogaster, we identified quantitative parameters that affect the activity of short-range transcriptional repressors, proteins that play key roles in development. In addition to the previously noted distance dependence, repression is strongly influenced by the stoichiometry, affinity, spacing, and arrangement of activator binding sites. Repression is insensitive to the type of activation domain, suggesting that short-range repression may primarily affect activators at the level of DNA binding. The activity of several short-range, but not long-range, repressors is circumscribed by the same quantitative parameters. This cis-regulatory "grammar" may aid the identification of enhancers regulated by short-range repressors and facilitate bioinformatic prediction of the functional output of transcriptional regulatory sequences.

The increasing prevalence of obesity and other nutrition-related chronic diseases has prompted considerable efforts to understand their pathogenesis and treatment. One experimental approach is to overexpress, inactivate, or manipulate specific genes that regulate energy metabolism and fat storage. Many such techniques are fully established, routine tools in Drosophila and C. elegans, which provide elegant models for dissecting endocrine problems and metabolic pathways.

In higher eukaryotes, transcriptional enhancers play critical roles in the integration of cellular signaling information, but apart from a few well-studied model enhancers, we lack a general picture of transcriptional information processing by most enhancers. Here we discuss recent studies that have provided fresh insights on information processing that occurs on enhancers, and propose that in addition to the highly cooperative and coordinate action of "enhanceosomes", a less integrative, but more flexible form of information processing is mediated by information display or "billboard" enhancers. Application of these models has important ramifications not only for the biochemical analysis of transcription, but also for the wider fields of bioinformatics and evolutionary biology., ((c) 2005 Wiley-Liss, Inc.)

Transcriptional enhancers integrate positional and temporal information to regulate the complex expression of developmentally controlled genes. Current models suggest that enhancers act as computational devices, receiving multiple inputs from activators and repressors and resolving them into a single positive or a negative signal that is transmitted to the basal transcriptional machinery. We show that a simple, compact enhancer is capable of representing both repressed and activated states at the same time and in the same nucleus. This finding suggests that closely apposed factor binding sites, situated within compact cis-elements, can be independently interpreted by the transcriptional machinery, possibly through successive enhancer-promoter interactions. These results provide clear evidence that the computational functions usually ascribed to the enhancer itself are actually shared with the basal machinery. In contrast to the autonomous computer model of enhancer function, an information-display or 'billboard' model of enhancer activity may better describe many developmentally regulated transcriptional enhancers.

Although Fallopian tubes are frequently involved in benign gynaecological conditions, primary malignant involvement is rare, and because of its rarity, lack of diagnostic accuracy, nonpresenting symptoms and physical findings, primary fallopian tube carcinoma is seldom diagnosed before laparotomy. We are reporting one such rare case.