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The aim of this paper is to motion of the smallest body in the 5-body problem where 4 bodies construct the cyclic kite configuration. Out of five bodies four bodies are placed at the vertices of a cyclic kite and the shapes of these bodies are considered as oblate heterogeneous with the supposition that these bodies are spinning around their own axes. It is also supposed that the smallest and fifth body is varying its mass according to Jeans law. The motion of smallest body is also affected by the coriolis and centrifugal forces. The equations of motion are determined under the above said perturbations. And then we numerically illustrated the locations of fixed points, their stability, the zero-velocity surfaces with projections and Poincaré surfaces of section. [ABSTRACT FROM AUTHOR]

A variety of methods and materials have been used for the treatment of the problems relating to the canalicular system. An insight into the rail roading technique for intubation of the canaliculi with sutupak in cases of common canalicular duct obstruction is presented here. About 30 patients with block at the common canalicular duct, which was detected by dacryocystography were operated for dacryocystorhinostomy with intubation of both the canaliculi with sutupak No. 0 by rail roading technique with good results.

Melanoma patients vaccinated with tumor-associated antigens frequently develop measurable peptide-specific CD8+ T cell responses; however, such responses often do not confer clinical benefit. Understanding why vaccine-elicited responses are beneficial in some patients but not in others will be important to improve targeted cancer immunotherapies.We analyzed peptide-specific CD8+ T cell responses in detail, by generating and characterizing over 200 cytotoxic T lymphocyte clones derived from T cell responses to heteroclitic peptide vaccination, and compared these responses to endogenous anti-tumor T cell responses elicited naturally (a heteroclitic peptide is a modification of a native peptide sequence involving substitution of an amino acid at an anchor residue to enhance the immunogenicity of the peptide). We found that vaccine-elicited T cells are diverse in T cell receptor variable chain beta expression and exhibit a different recognition profile for heteroclitic versus native peptide. In particular, vaccine-elicited T cells respond to native peptide with predominantly low recognition efficiency--a measure of the sensitivity of a T cell to different cognate peptide concentrations for stimulation--and, as a result, are inefficient in tumor lysis. In contrast, endogenous tumor-associated-antigen-specific T cells show a predominantly high recognition efficiency for native peptide and efficiently lyse tumor targets.These results suggest that factors that shape the peptide-specific T cell repertoire after vaccination may be different from those that affect the endogenous response. Furthermore, our findings suggest that current heteroclitic peptide vaccination protocols drive expansion of peptide-specific T cells with a diverse range of recognition efficiencies, a significant proportion of which are unable to respond to melanoma cells. Therefore, it is critical that the recognition efficiency of vaccine-elicited T cells be measured, with the goal of advancing those modalities that elicit T cells with the greatest potential of tumor reactivity.

A letter to the editor is presented regarding a case of ocular dirofilariasis in a three-year-old male child in Mumbai, India.

Exposure to nickel, an environmental respiratory toxicant, is associated with lung diseases including asthma, pulmonary fibrosis, bronchitis and cancers. Our previous studies have shown that a majority of the nickel-induced transcriptional changes are persistent and do not reverse even after the termination of exposure. This suggested transcriptional memory, wherein the cell 'remembers' past nickel exposure. Transcriptional memory, due to which the cells respond more robustly to a previously encountered stimulus has been identified in a number of organisms. Therefore, transcriptional memory has been described as an adaptive mechanism. However, transcriptional memory caused by environmental toxicant exposures has not been well investigated. Moreover, how the transcriptional memory caused by an environmental toxicant might influence the outcome of exposure to a second toxicant has not been explored. In this study, we investigated whether nickel-induced transcriptional memory influences the outcome of the cell's response to a second respiratory toxicant, nicotine. Nicotine, an addictive compound in tobacco, is associated with the development of chronic lung diseases including chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis. Our results show that nicotine exposure upregulated a subset of genes only in the cells previously exposed to nickel. Furthermore, our analyses indicate robust activation of interferon (IFN) signaling in these cells. IFN signaling is a driver of inflammation, which is associated with many chronic lung diseases. Therefore, our results suggest that nicotine exposure of lung cells that retain the transcriptional memory of previous nickel exposure could result in increased susceptibility to developing chronic inflammatory lung diseases., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Introduction: The coronavirus disease 2019 (COVID-19) wave has fluctuated erratically around the globe over the past three years of the pandemic, sometimes declining and at other times surging. The cases of infection in India have remained low, despite the continued surge of Omicron sub-lineages reported in a few countries. In this study, we determined the presence of the circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains in the population of Kangra District, Himachal Pradesh, India., Methodology: In vitro diagnostic real-time reverse transcriptase polymerase chain reaction (RT-qPCR) was performed using Tata MD CHECK RT-PCR Omisure kit (Tata Medical and Diagnostics Limited, Maharashtra, India), to detect the presence of Omicron in target samples. A total of 400 samples were analyzed in this study; 200 each for the second and third waves, respectively. The S gene target failure (SG-TF) and S gene mutation amplification (SG-MA) primer-probe sets were used., Results: Our results corroborated that during the third wave, SG-MA amplification was noted, while amplification of SG-TF was not, and vice versa in the case of the second wave, indicating that all the tested patients were infected with the Omicron variant during the third wave, while Omicron was absent during the second wave., Conclusions: This study added more information about the prevalence of Omicron variants during the third wave in the chosen area, and it projected a use of in vitro RT-qPCR method for rapid prospective determination of the prevalence of the variant of concern (VOC) in developing countries with limited sequencing facility., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2023 Suresh Kumar, Sahdev Choudhary, Sharad Thakur, Arbind Kumar, Sanjay Kumar.)

Objective: This study aims to describe the epidemiology, predisposing factors, clinical manifestations, management, and outcome of post-COVID rhino-cerebralorbital mucormycosis., Methods: This is a prospective observational study of patients with post-COVID RCOM conducted tertiary care hospital during May-June 2021., Results: The mean age of patients was 49.58±15.12 years and majority (64.80%) were male. The majority of patients were rural, Hindu and illiterate. Diabetes was present 78.10% patients, glucocorticoids were required in 66.30%, and supplemental oxygen was used in 27.60% of patients. Most of the patients developed symptoms of RCOM within 15 days of COVID-19. Majority of patients (46.67%) had stage 3 disease and orbit was involved in 60% of patients. All patients received intravenous antifungal drugs and combined antifungal drugs and surgical debridement was performed in 77.10% patients. Predictor associated with poor outcome were RCOM stage 3c or above and qSOFA score ≥2 at presentation., Conclusion: Diabetes and glucocorticoids are the most important risk factors for post-COVID RCOM. COVID-19 patients must be followed closely for 2-4 weeks to detect mucormycosis as earlier as possible. Antifungal drugs should be started immediately if clinico-radiological feature suggest RCOM before microbiological confirmation. Combined medical and surgical treatment significantly reduces mortality., (© Journal of the Association of Physicians of India 2011.)

Urotensin II (U-II) has been found to be one of the most potent vasoconstrictor (Ames et al., 1999; Bohm et al., 2002) reported till date. U-II exerts its response via activation of a G-protein coupled receptor, Urotensin II receptor(UT). Binding of U-II to UT leads to an instant increase in the inositol phosphate turnover and intracellular Ca 2+ . Such an instant Ca 2+ release and potent vasoconstriction exerted by U-II is expected to have an important role in the progression of cardiac diseases. We have previously shown that UT antagonist DS37001789 prevents U-II induced blood pressure elevation in mice (Nishi et al., 2019) in a dose dependent manner, with potent efficacy at 30 and 100 mg/kg. Further to this, we have also shown that DS37001789 ameliorates mortality in pressure-overload mice with heart failure (Nishi et al., 2020). We therefore conducted an extensive structure-activity relationship studies to identify molecules with superior efficacy. In the present manuscript, we report the identification of two potent, non-peptide small molecule antagonists of Urotensin II receptor (UT), RCI-0879 and RCI-0298 which blocked the action of U-II, both in vitro and in vivo. These molecules were found to be very potent in in vitro Ca 2+ and radioligand binding assays using human and mouse UT over-expressing CHO cells. RCI-0879 and RCI-0298 also exhibited superior efficacy in in vivo mouse pressor response model using C57BL/6 mice, compared to our initial molecules (Nishi et al., 2019) and demonstrated ED 50 values of 3.2 mg/kg and 6.8 mg/kg respectively. Our findings reported herewith, further strengthen our concept and belief in UT antagonization as a potential therapeutic approach for the management of chronic heart failure., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Hemophagocytic lymphohistiocytosis (HLH) is a rare but aggressive and potentially fatal condition characterized by excessive immune activation. It can occur as primary/ familial and secondary/sporadic/ acquired form. Infections can play a role as triggers in the secondary form of HLH. A case of Hemophagocytic lymphohistiocytosis (HLH) in a patient of Scrub typhus is being reported here. Such association of scrub typhus and HLH is rare., (© Journal of the Association of Physicians of India 2011.)

An ultrasonic phased array transmitter (PAT) consisting of 12×12 elements was designed and fabricated to evaluate its experimental characteristic performance in the medium range of 2m-10m for its directivity, pulse width affect and sound pressure level (SPL) measurements as a function of distance from its center in the open air. The SPL was measured at 2 m, 5m, and 10m distances as 135dB, 126dB and 68dB, respectively. The experimentally measured directivity patterns were found in good agreement with the theoretical results obtained by using MATLAB simulations. The SPL showed negligible change as a function of the ultrasonic pulse widths such as 0.5ms, 1ms and 2ms. The SPL and directivity of the PAT at 2m distance were measured for ultrasonic pulse width of 0.1ms only while as at 10m distance these were measured for the ultrasonic pulse widths of 0.5ms, 1ms and 2ms. The present investigations on the PAT characteristics are expected to be useful for its industrial, scientific and biomedical applications, e.g., robotics for 3D range imaging with improved and efficient object presence sensing as well as nondestructive health monitoring., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A long-range measurement system comprising an ultrasonic range sensor with a high-power ultrasonic transmitter array in air was investigated. The system is simple in construction and can be used under adverse conditions such as fog, rain, darkness, and smoke. However, due to ultrasonic waves are well absorbed by air molecules, the measurable range is limited to a few meters. Therefore, we developed a high-power ultrasonic transmitter array consisting of 144 transmitting elements. All elements are arranged in the form of a 12×12 array pattern. The sound pressure level at 5m from the transmitter array was >30dB higher than that of a single element. A measuring range of over 25m was achieved using this transmitter array in conjunction with a receiver array having 32 receiving elements. The characteristics of the transmitter array and range sensor system are discussed by comparing simulation and experimental results., (Copyright © 2016 Elsevier B.V. All rights reserved.)

p53 protein, the central molecule of the apoptosis pathway, is mutated in 50% of the human cancers. Of late, p53 homologues have been identified from different invertebrates including Drosophila melanogaster, Caenorhabditis elegans, Squid, and Clams. We report the identification of a p53-like protein in Spodoptera frugiperda (Sf9) insect cells, which is activated during oxidative stress, caused by exposure to UV-B or H(2) O(2) , and binds to p53 consensus DNA binding motifs as well as other p53 cognate motifs. Sf9 p53 motif-binding protein is similar to murine and Drosophila p53 in terms of molecular size, which is around 50-60 kDa, as evident from UV cross-linking, and displays DNA binding characteristics similar to both insect and vertebrate p53 as seen from electrophoretic mobility shift assays. The N-terminal sequencing of the purified Sf9 p53 motif-binding protein reveals extensive homology to the pro-apoptotic FK-506 binding protein (FKBP-46), earlier identified in Sf9 cells as a factor which interacts with murine casein kinase. FKBP, an evolutionarily conserved protein of mammalian origin functions as a pro-apoptotic factor. Identification of FKBP-46 as a novel p53 motif-binding protein in insect cells adds a new facet to our understanding of the mechanisms of apoptosis under oxidative stress in the absence of a typical p53 homologue., (Copyright © 2012 Wiley Periodicals, Inc.)

Malondialdehyde (MDA) is widely used as oxidative stress biomarker in biomedical research. Plasma is stored in deep freezers generally till analysis. Effect of such storage on MDA values, which may be variable and prolong, was incidentally observed in the ongoing study which is to estimate oxidative stress with oral iron. Plasma from blood samples of pregnant women (20-30 years age) in third trimester of singleton pregnancy (n = 139), consuming oral iron tablets was stored at -20 °C with intention of MDA estimation, as soon as possible. However logistic problems led this storage for prolonged and variable period (1-708 days). When values of MDA estimated using "Ohkawa" 79 method and readings were plotted against time to check the temporal effect, it showed a hyperbolic curve. Standard deviation (SD) was lowest when samples were tested within 3 weeks time. The samples analyzed within 3 weeks had mean ± SD value of 31.59 ± 26.11 μmol/L, while 123.7 ± 93.97 and 366.5 ± 189.8 μmol/L for samples stored for 1-3 and 4 months to 1 year respectively. Mean ± SD were 539.9 ± 196.8 in the samples store for more than a year. Rate of change in values was also lowest (0.0433 μmol/L/day) in the samples tested within first 3 weeks, which rose to 1.2 μmol/L/day during 3 month's storage. This rate peaked at storage of 120 days (1.87 μmol/L/day) and fell to 0.502 μmol/L/day in the second year of storage. It is concluded that at -20 °C, only 3 weeks of storage time should be considered valid for fairly acceptable stability in MDA values.

Baculovirus p35 belongs to the early class of genes of AcMNPV and requires viral factors like Immediate Early protein-1 for its transcription. To investigate the role of host factors in regulating p35 gene expression, the putative transcription factor binding sites were examined in silico and the role of these factors in influencing the transcription of p35 gene was assessed. We focused our studies on AP-1 and P53-like factors, which are activated under oxidative stress conditions. The AP-1 motif is located at -1401 while P53 motif is at -1912 relative to p35 translation start site. The predicted AP-1 and P53 elements formed specific complexes with Spodoptera frugiperda nuclear extracts. Both AP-1 and P53 motif binding proteins were down regulated as a function of AcMNPV infection in Spodoptera cells. To address the question whether during an oxidative outburst, the p35 transcription is enhanced; we investigated the role of these oxidative stress induced host transcription factors in influencing p35 gene transcription. Reporter assays revealed that AP-1 element enhances the transcription of p35 by a factor of two. Interestingly, P53 element appears to repress the transcription of p35 gene., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Clomiphene citrate is commonly used for ovulation induction in women with anovulatory infertility. However, pregnancy rates with this drug are not as good as ovulation rates. Tamoxifen may be a better choice in some patients who fail to either ovulate or conceive with clomiphene due to its favorable effect on the cervical mucus and endometrium. This study was conducted to evaluate the role of tamoxifen in women with anovulatory infertility and find out the optimum dose needed for achieving the best outcome., Materials and Methods: 160 women attending the infertility clinic and suffering from anovulatory infertility were recruited for the study. Tamoxifen was administered in the dose of 40 mg daily and ovulation monitored. In case of anovulation, the dose was increased to 80 mg daily. Ovulation and pregnancy rates were calculated., Results: Twenty-three out of 160 women who received 40 mg of tamoxifen conceived, giving a pregnancy rate of 14.38% and pregnancy rate per ovulatory cycle as 14.94%. 32 out of 80 women who received 80 mg of tamoxifen conceived, giving a pregnancy rate of 40% and pregnancy rate per cycle as 33.68%. This difference in the pregnancy rate between the two groups was statistically significant. 35 women out of 90 with polycystic ovary syndrome (PCOS) became pregnant with a pregnancy rate of 38.8% and 20 out of 70 women with clomiphene citrate failure conceived, giving a pregnancy rate of 28.5%., Conclusions: Tamoxifen is a good alternative to clomiphene in women with PCOS and clomiphene-resistant cases.

In this chapter, we concentrate on the production of high-quality protein samples for nuclear magnetic resonance (NMR) studies. In particular, we provide an in-depth description of recent advances in the production of NMR samples and their synergistic use with recent advancements in NMR hardware. We describe the protein production platform of the Northeast Structural Genomics Consortium and outline our high-throughput strategies for producing high-quality protein samples for NMR studies. Our strategy is based on the cloning, expression, and purification of 6×-His-tagged proteins using T7-based Escherichia coli systems and isotope enrichment in minimal media. We describe 96-well ligation-independent cloning and analytical expression systems, parallel preparative scale fermentation, and high-throughput purification protocols. The 6×-His affinity tag allows for a similar two-step purification procedure implemented in a parallel high-throughput fashion that routinely results in purity levels sufficient for NMR studies (>97% homogeneity). Using this platform, the protein open reading frames of over 17,500 different targeted proteins (or domains) have been cloned as over 28,000 constructs. Nearly 5000 of these proteins have been purified to homogeneity in tens of milligram quantities (see Summary Statistics, http://nesg.org/statistics.html), resulting in more than 950 new protein structures, including more than 400 NMR structures, deposited in the Protein Data Bank. The Northeast Structural Genomics Consortium pipeline has been effective in producing protein samples of both prokaryotic and eukaryotic origin. Although this chapter describes our entire pipeline for producing isotope-enriched protein samples, it focuses on the major updates introduced during the last 5 years (Phase 2 of the National Institute of General Medical Sciences Protein Structure Initiative). Our advanced automated and/or parallel cloning, expression, purification, and biophysical screening technologies are suitable for implementation in a large individual laboratory or by a small group of collaborating investigators for structural biology, functional proteomics, ligand screening, and structural genomics research., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We describe the core Protein Production Platform of the Northeast Structural Genomics Consortium (NESG) and outline the strategies used for producing high-quality protein samples. The platform is centered on the cloning, expression and purification of 6X-His-tagged proteins using T7-based Escherichia coli systems. The 6X-His tag allows for similar purification procedures for most targets and implementation of high-throughput (HTP) parallel methods. In most cases, the 6X-His-tagged proteins are sufficiently purified (>97% homogeneity) using a HTP two-step purification protocol for most structural studies. Using this platform, the open reading frames of over 16,000 different targeted proteins (or domains) have been cloned as>26,000 constructs. Over the past 10 years, more than 16,000 of these expressed protein, and more than 4400 proteins (or domains) have been purified to homogeneity in tens of milligram quantities (see Summary Statistics, http://nesg.org/statistics.html). Using these samples, the NESG has deposited more than 900 new protein structures to the Protein Data Bank (PDB). The methods described here are effective in producing eukaryotic and prokaryotic protein samples in E. coli. This paper summarizes some of the updates made to the protein production pipeline in the last 5 years, corresponding to phase 2 of the NIGMS Protein Structure Initiative (PSI-2) project. The NESG Protein Production Platform is suitable for implementation in a large individual laboratory or by a small group of collaborating investigators. These advanced automated and/or parallel cloning, expression, purification, and biophysical screening technologies are of broad value to the structural biology, functional proteomics, and structural genomics communities.

The baculovirus expression vector system (BEVS) has been widely used for over-expressing eukaryotic proteins due to a close resemblance in post-translational modification, processing, and transportation properties of the expressed protein, to that of the mammalian cells. In comparison to the bacterial expression system, protein yield from BEVS is relatively low, resulting in higher cost of production. To improve the existing recombinant protein expression levels, baculovirus homologous region1 (hr1) was strategically integrated into the bacmid-based transfer vectors. Luciferase reporter, human Protein Kinase B-alpha (PKB-A), and N-terminal-modified CYP-1A2 genes were independently cloned in non-hr1 and hr1 constructs for generating respective bacmids and baculoviruses. These recombinant baculoviruses were utilized for comparing the expression levels at varying multiplicity of infections (MOI) and time intervals in Spodoptera frugiperda (Sf21) or Trichoplusia ni (Tni) insect cell lines. Targeted insertion of hr1 upstream to CYP-1A2, PKB-A, and Luciferase genes, compared to the non-hr1 sets, led to 3-, 3.5-, and 4.5-fold increase in the resultant protein levels, respectively. Moreover, at equal protein concentration, the corresponding activity and inhibition characteristics of these high expression hr1 sets were comparable to that of the respective non-hr1 sets. Utilization of this modified baculovirus expression construct offers significant advantage of producing recombinant proteins in a cost-effective manner for various biotechnological and therapeutic applications.

Protein tyrosine phosphatases (PTPs) play multiple roles in many physiological processes. Over-expression of the PTPs has been shown to be associated with cellular toxicity, which may also lead to the deletion of the respective gene from stable cell clones. We also observed that PTP-1B over-expression in CHO and HEK293 stable cell clones led to cytotoxicity and low revival rates during clone generation and maintenance. To address these issues, bacmid transposition technology was utilized to generate recombinant PTP-1B baculovirus, and Spodoptera frugiperda (Sf9 and Sf21) insect cell lines were infected with the virus. The data obtained on expression and activity of the PTP-1B highlights clear advantage of the recombinant baculovirus-insect cell expression system over the mammalian cell line technique due to increase in enzyme activity, strongly inhibited by phosphatase specific inhibitor RK682. Possible application of the expression system for producing active enzymes in bulk quantity for a new drug discovery is also discussed.

Baculovirus immediate early P35 protein is well known for its anti-apoptotic as well as anti-oxidant properties. Mechanism of action of P35 involves inhibition of a vast range of initiator to executioner class of caspases. In addition, P35's role in inhibiting oxidant-induced mitochondrial damage, primarily in the apoptotic pathway, has also been extensively investigated. Elucidation of P35's functions during regulation of programmed cell death (PCD) has led to a renewed focus on exploiting this basic knowledge for clinical and other related applications. This review outlines specific biochemical and genetic pathways where P35 intervenes and regulates rate-limiting steps in the apoptotic signaling cascade. Research efforts are underway to utilize P35 as an agent in regulating apoptosis and under certain circumstances, also explore the therapeutic potential of its anti-oxidant features. One of the major outcomes of recent studies include significantly improved effectiveness of cytochrome P450 directed enzyme pro-drug delivery tools when used in conjunction with P35, which may help in alleviating drug resistance in tumor cells and simultaneously prolonging the cytotoxic effects of anti-cancer drugs. Moreover, applied research carried out recently in the fields of diabetes, ischemia-induced neuronal cell death, experimental autoimmune encephalomyelitis (EAE), multiple sclerosis (MS), inflammatory arthritis, cardiovascular and ocular disorders illustrate P35's utilization across diverse therapeutic areas and will certainly make it an attractive biomolecule for the discovery research.

Sarcoidosis is a multisystemic inflammatory disorder of unknown etiology. It can present in various clinical forms. Involvement of muscles is common but isolated muscle mass, the only initial presenting complaint has never been reported. We report a 55-year-old white female who presented with a muscle mass, with no other clinical features of sarcoidosis. She was later found to have hilar lymphadenopathy and muscle biopsy confirmed the diagnosis of sarcoidosis. During clinical follow up patient remained stable without any medical intervention.

Among the various expression systems employed for the over-production of proteins, bacteria still remains the favorite choice of a Protein Biochemist. However, even today, due to the lack of post-translational modification machinery in bacteria, recombinant eukaryotic protein production poses an immense challenge, which invariably leads to the production of biologically in-active protein in this host. A number of techniques are cited in the literature, which describe the conversion of inactive protein, expressed as an insoluble fraction, into a soluble and active form. Overall, we have divided these methods into three major groups: Group-I, where the factors influencing the formation of insoluble fraction are modified through a stringent control of the cellular milieu, thereby leading to the expression of recombinant protein as soluble moiety; Group-II, where protein is refolded from the inclusion bodies and thereby target protein modification is avoided; Group-III, where the target protein is engineered to achieve soluble expression through fusion protein technology. Even within the same family of proteins (e.g., tyrosine kinases), optimization of standard operating protocol (SOP) may still be required for each protein's over-production at a pilot-scale in Escherichia coli. However, once standardized, this procedure can be made amenable to the industrial production for that particular protein with minimum alterations.

PCR amplification failure from cDNA libraries or RNA templates, under the optimal conditions is generally attributed to high GC content. Utilization of various additives without thorough analysis of secondary structures of the template as well as primers and subsequent PCR cycle conditions, generally leads to inadequate yields and/or truncated products. To address these concerns, we have examined two highly GC-rich human genes namely insulin receptor (IR) and cSRC kinase. In silico analysis of these genes revealed that their -5' and -3' sequences have > 80% GC content. Primers designed through these GC-rich regions had high self-dimer free energy values (DeltaG). Null mutations were introduced to bring down these DeltaG levels below -5.0 kcal/mol. Oligo(dT)18 primed cDNA was synthesized from HepG2 and HT29 total RNA to amplify IR and cSRC kinase ORFs, respectively. A multi-prong strategy including primer modifications, various DMSO-betaine combinations and high denaturing temperature conditions was pursued during cDNA synthesis to achieve optimal PCR amplification. The reported approach can be utilized to improve the amplification of templates with high GC content, which are otherwise relatively difficult to resolve.

Objectives and Methods: In 100 anovulatory infertile women with polycystic ovary syndrome attending the infertility clinic of this institute, transvaginal ultrasound findings of ovaries were correlated with both clinical and hormonal parameters using Chi-square test., Results: All women in the study had oligomenorrhea or amenorrhea, 70% had hirsutism and more than half were obese. On transvaginal ultrasound, ovarian volume was more than 10 cm3 in all, 90% had more than 10 follicles in each ovary, 75% had stromal thickness more than 1 cm and more than half had increased stromal echogenicity. Seventy percent had high testosterone levels and 60% were detected to have insulin resistance. Ovarian volume correlated positively with body mass index, waist/hip ratio and menstrual cycle irregularity (p < 0.05). The correlation between ovarian size, LH/FSH ratio and hyperinsulinemia was highly significant (p < 0.005), but was low for serum androgens and also hirsutism. Number of follicles per ovary correlated positively with body mass index, menstrual irregularity (p < 0.01), insulin resistance as well as androgens (p < 0.005). Positive predictive value of ovarian follicle number was 100% for insulin resistance as well as D4 androstenedione. Increased stromal thickness also showed 70% positive prediction for clinical parameters, 66% for insulin resistance and 82% for serum D4 androstenedione. Contrary to the other ultrasound parameters of polycystic ovary, stromal echogenicity did not significantly correlate with any of the clinical or hormonal parameters except serum testosterone., Conclusion: Transvaginal ultrasonography of the ovaries confirms the clinical profile and also gives an insight to the hormonal milieu of the women with PCOS.

Selaginella bryopteris is a lithophyte with remarkable ressurection capabilities. It is full of medicinal properties, hence also known as 'Sanjeevani' (one that infuses life). For lack of credible scientific evidence the plant is not in active use as a medicinal herb. We provide scientific evidence for why S. bryopteris is known as 'Sanjeevani'. The aqueous extract of S. bryopteris possesses growth-promoting activity as well as protective action against stress-induced cell death in a number of experimental cell systems including mammalian cells. Treatment of the cells in culture with 10% aqueous extract enhanced cell growth by about 41% in Sf9 cells and 78% in mammalian cells. Pre-treatment of cells with the Selaginella extract (SE) (1-2.5%) protected against oxidative stress (H2O2) -induced cell death. The killing potential of ultra violet (UV) was also significantly reduced when the cells were pre-treated with SE for 1 h. Thermal radiation suppressed cell growth by about 50%. Pre-treatment of cells with SE for 1 h afforded complete protection against heat-induced growth suppression. SE may possess anti-stress and antioxidant activities that could be responsible for the observed effects. Chemical analysis shows that SE contains hexoses and proteins. Taken together, S. bryopteris extract may help in stress-induced complications including those due to heat shock.

Mutations in the beta-glucocerebrosidase gene cause Gaucher disease with the type 1 variant generally presenting later in life with mild disease and type 2 in infancy with severe neuronopathic symptoms. We describe a neonate homozygous for the D409 H mutation with thrombocytopenia, splenomegaly and cholestasis at birth as the major features.

Background: Melanoma patients vaccinated with tumor-associated antigens frequently develop measurable peptide-specific CD8+ T cell responses; however, such responses often do not confer clinical benefit. Understanding why vaccine-elicited responses are beneficial in some patients but not in others will be important to improve targeted cancer immunotherapies., Methods and Findings: We analyzed peptide-specific CD8+ T cell responses in detail, by generating and characterizing over 200 cytotoxic T lymphocyte clones derived from T cell responses to heteroclitic peptide vaccination, and compared these responses to endogenous anti-tumor T cell responses elicited naturally (a heteroclitic peptide is a modification of a native peptide sequence involving substitution of an amino acid at an anchor residue to enhance the immunogenicity of the peptide). We found that vaccine-elicited T cells are diverse in T cell receptor variable chain beta expression and exhibit a different recognition profile for heteroclitic versus native peptide. In particular, vaccine-elicited T cells respond to native peptide with predominantly low recognition efficiency--a measure of the sensitivity of a T cell to different cognate peptide concentrations for stimulation--and, as a result, are inefficient in tumor lysis. In contrast, endogenous tumor-associated-antigen-specific T cells show a predominantly high recognition efficiency for native peptide and efficiently lyse tumor targets., Conclusions: These results suggest that factors that shape the peptide-specific T cell repertoire after vaccination may be different from those that affect the endogenous response. Furthermore, our findings suggest that current heteroclitic peptide vaccination protocols drive expansion of peptide-specific T cells with a diverse range of recognition efficiencies, a significant proportion of which are unable to respond to melanoma cells. Therefore, it is critical that the recognition efficiency of vaccine-elicited T cells be measured, with the goal of advancing those modalities that elicit T cells with the greatest potential of tumor reactivity.

Spodoptera frugiperda (Sf9) ovarian cells, natural hosts for baculovirus, are good model systems to study apoptosis and also heterologous gene expression. We report that uninfected Sf9 cells readily undergo apoptosis and show increased phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) in the presence of agents such as UVB light, etoposide, high concentrations of cycloheximide, and EGTA. In contrast, tunicamycin, A23187, and low concentrations of cycloheximide promoted eIF2alpha phosphorylation in Sf9 cells but without apoptosis. These findings therefore suggest that increased eIF2alpha phosphorylation does not always necessarily lead to apoptosis, but it is a characteristic hallmark of stressed cells and also of cells undergoing apoptosis. Cell death induced by the above agents was abrogated by infection of Sf9 cells with wild-type (wt) AcNPV. In contrast, Sf9 cells when infected with vAcdelta35, a virus carrying deletion of the antiapoptotic p35 gene, showed increased apoptosis and enhanced eIF2alpha phosphorylation. Further, a recombinant wt virus vAcS51D expressing human S51D, a phosphomimetic form of eIF2alpha, induced apoptosis in UVB pretreated Sf9 cells. However, infection with vAcS51A expressing a nonphosphorylatable form (S51A) of human eIF2alpha partially reduced apoptosis. Consistent with these findings, it has been observed here that caspase activation has led to increased eIF2alpha phosphorylation, while caspase inhibition by z-VAD-fmk reduced eIF2alpha phosphorylation selectively in cells exposed to proapoptotic agents. These findings therefore suggest that the stress signaling pathway determines apoptosis, and caspase activation is a prerequisite for increased eIF2alpha phosphorylation in Sf9 cells undergoing apoptosis. The findings also reinforce the conclusion for the first time that the "pancaspase inhibitor" baculovirus p35 mitigates eIF2alpha phosphorylation.

In this study we report that the baculovirus p35 anti-apoptotic protein prevents cell death by quenching free radicals at a very upstream step in the apoptotic pathway. Mitochondria of activated rat peritoneal macrophages as well as Spodoptera frugiperda (Sf9) insect cells, following treatment with oxidants, H(2)O(2)/UVB irradiation, release cytochrome c followed by activation of caspase-3. Transfection of macrophages/Sf9 cells with a construct carrying the p35 gene under the CMV/HSP promoters resulted in p35 expression and consequent arrest of oxidative stress-induced apoptosis. p35 expression also inhibited cytochrome c release from the mitochondria of oxidant-exposed cells and blocked caspase-3 activation.

Host pathogen interaction results in a variety of responses, which include phagocytosis of the pathogen, release of cytokines, secretion of toxins, as well as production of reactive oxygen species (ROS). Recent studies have shown that many pathogens exert control on the processes that regulate apoptosis in the host. The induction of apoptosis upon infection results from a complex interaction of parasite proteins with cellular host proteins. Abrogation of host cell apoptosis is often beneficial for the pathogen and results in a successful host invasion. However, in some cases, it has been shown that induction of apoptosis in the infected cells significantly imparts protection to the host from the pathogen. There is a strong correlation between apoptosis and the host protein translation machinery: the pathogen makes all possible efforts to modify this process so as to inhibit cell suicide and ensure that it can survive and, in some cases, establish latent infection. This review discusses the significance of various pathways/steps during virus-mediated modulation of host cell apoptosis.

Oxidative stress has been shown to be associated with apoptosis (programmed cell death) in a number of cell systems. We earlier reported in vitro cultured Spodoptera frugiperda (Sf9) cells as a model system to study oxidative stress induced apoptosis (J Biosciences 24 (1999) 13) and the inhibition of UV-induced apoptosis by the baculovirus antiapoptotic p35 protein that acts as a sink to sequester reactive oxygen species (Proc Natl Acad Sci USA 96 (1999) 4838). We now show that UV-induced apoptosis in Sf9 cells, is preceded by the release of mitochondrial cytochrome c into the cytosol and consequent activation of Sf-caspase-1. The inhibitory effect of different antioxidants including scavengers of oxygen radicals such as butylated hydroxyanisole (BHA), alpha tocopherol acetate, benzoate and reduced-glutathione (GSH) on ultra violet B (UVB)-induced apoptosis in cultured Sf9 cells was assessed. Both, cytochrome c release as well as Sf-caspase-1 activation was inhibited by pre-treatment with antioxidants such as BHA and alpha tocopherol acetate, suggesting that these antioxidants inhibit apoptosis by acting quite upstream in the apoptosis cascade at the mitochondrial level, as well as downstream at the caspase level.

Cell death is a highly regulated process that is ubiquitous in all eukaryotes. Programmed cell death (PCD) is an integral part of both animal and plant development. Studies on apoptosis, the well characterized form of programmed cell death led to the identification of a central tripartite death switch i.e. apoptosome consisting of Apaf-1, Apaf-2 and Apaf-3. The caspases, a family of cysteine-dependent aspartate directed-proteases, constitute the central executioners of apoptosis. Much of the attention on programmed cell death is focused on caspases, however, cell death can still occur even when the caspase cascade is blocked, revealing the existence of nonapoptotic alternative pathway(s) of cell death. The mitochondrial release of cytochrome C following a PCD inducing stimulus in both plants and animals suggests the evolutionary conservation of death pathways. Dysregulation of apoptosis may be related to the development of several disease states as well as ageing. Excessive apoptosis is associated with neurodegenerative disorders, AIDS etc., whereas deficient apoptosis is associated with cancer, auto-immunity, viral infections etc. Understanding the regulation of programmed cell death would throw light in designing drugs and gene therapies that can target specific molecules in the apoptotic pathway opening the vistas for new therapeutic endeavors in many areas of medicine.

To date, not much has been known regarding the role of CD80 and CD86 molecules in signaling of B cells. The CD28/CTLA4 ligands, CD80 (B7-1) and CD86 (B7-2), are expressed on the surface of freshly isolated splenic B cells, and their expression is up-regulated by lipopolysaccharides. In the present study, we have investigated whether signaling via CD80/CD86 could alter the proliferation and immunoglobulin synthesis of B cells. Splenic B cells were stimulated with lipopolysaccharides in the presence of anti-B7-1 (16-10A1) and anti-B7-2 (GL1) monoclonal antibodies (mAbs). Exciting features observed during the study were that cross-linking of CD86 with GL1 enhanced the proliferation and production of IgG1 and IgG2a isotypes. In contrast, anti-B7-1 (16-10A1) mAb could efficiently block the proliferation and production of IgG1 and IgG2a. Furthermore, GL1 mAb could also induce the secretion of IgG isotypes from B cell lymphomas. Importantly, 16-10A1 could retard the growth of lymphomas and favored the up-regulation of pro-apoptotic molecules caspase-3, caspase-8, Fas, FasL, Bak, and Bax and down-regulation of anti-apoptotic molecule Bcl-x(L). In contrast, GL1 augmented the level of anti-apoptotic molecules Bcl-w and Bcl-x(L) and decreased the levels of pro-apoptotic molecule caspase-8, thereby providing a novel insight into the mechanism whereby triggering through CD80 and CD86 could deliver regulatory signals. Thus, this study is the first demonstration of a distinct signaling event induced by CD80 and CD86 molecules in B cell lymphoma. Finally, the significance of the finding is that CD80 provided negative signal for the proliferation and IgG secretion of normal B cells and B cell lymphomas. In contrast, CD86 encouraged the activity of B cells.

Neonatal urinary ascites caused by bladder perforation is rare, with fewer than 20 cases reported in earlier literature. Congenital bladder perforation can be associated with bladder outlet obstruction such as posterior urethral valves, urethral atresia, presacral mass, and neurogenic dysfunction of the bladder. The bladder perforation in these cases is most commonly intraperitoneal, which leads to congenital urinary ascites. However, intrauterine perforation of the bladder in a newborn infant with posterior urethral valves is extremely rare, as is evident from the three cases in previous literature. The present case report describes an unusual case of congenital bladder perforation and urinary ascites caused by posterior urethral valves.

Although cocaine abuse has declined in popularity in the United States, certain groups continue to use the drug at high rates. The teratogenicity of cocaine has been widely investigated in the newborn. We report a case of crossed renal ectopia in a term neonate whose mother practiced alkaloid cocaine abuse in the first trimester of pregnancy. We propose that this anomaly was caused by cocaine's direct pharmacologic effect, leading to vasoconstriction that affected the developing fetal kidney by compromising the blood vessels that supply the organ or by causing hemorrhages and infarctions with fibrosis, causing disruption during a crucial period of morphogenesis.

Oxidant injury and release of proteolytic enzymes in prematures with respiratory distress syndrome (RDS), who are treated with ventilators and oxygen, have been postulated as possible causes of bronchopulmonary dysplasia (BPD). The premature may be at particular risk due to low levels of antiproteases, such as alpha-1-proteinase inhibitor (alpha 1PI), and antioxidants, such as ceruloplasmin (CER). Both alpha 1PI and CER deficiencies have been correlated with the severity of RDS. We studied serial alpha 1PI activity as measured by trypsin inhibitory capacity (TIC) and CER in the serum 27 prematures who required ventilator therapy for RDS. Serum TIC values for day 1 were significantly lower (0.34 vs. 0.92 mg inhibited/ml of sample) in the 13 patients who developed BPD compared to the 14 who did not. No significant differences were seen on succeeding days. No significant differences in CER were seen, although both groups had levels 33-50% of adult normals (11.3 vs 9.3 mg/dl). Other significant variables included birthweight (p less than 0.005), severity of RDS (p less than 0.03), and gestational age (p less than 0.03). One way analysis of variances demonstrated day 1 TIC to be the most significant variable (p less than 0.0001), followed by weight (p less than 0.007), severity RDS (p less than 0.04), and gestational age (p less than 0.03). CER levels were not a significant variable. A formula utilizing unstandardized canonical discriminant function including day 1 TIC, birthweight, severity of RDS, and gestational age was 100% sensitive and 85% specific in the prediction of BPD for the original study group. In an additional 25 consecutive admissions with severe RDS of whom 18 survived, the formula was 100% sensitive (6/6) and 75% specific (9/12).(ABSTRACT TRUNCATED AT 250 WORDS)

A new case of ambiguous genitalia and immature tissue in the left gonad is presented. Cytogenetic findings with various techniques demonstrated that the distal two-thirds of the long arm of the Y chromosome is deleted. Q-banding showed a non-fluorescent Y; three positive bands were however noted when the DA/DAPI technique was applied. After a review of the literature, it was concluded that the non-fluorescent Y chromosome (Ynf) when inherited from generation to generation is a heteromorphism in normal males. However, in our case, where the proband's Y is lacking the fluorescent segment, a simple deletion does not appear to adequately explain the DA/DAPI positive bands. Possibly, a deletion followed by a structural rearrangement of the non-fluorescent segment had occurred de novo. The highly Y-specific DNA sequences present in the fluorescent segment are absent in these patients. The abnormal development in these cases is due to the presence of the 45,X cell line. The gene responsible for spermatogenesis has been localized to the non-fluorescent region in the long arm of the Y chromosome. Furthermore, it is concluded that two types of non-fluorescent Y chromosomes can be found in the population; one is a normal inherent heteromorphic variant, while the other appears to be an abnormality, especially in cases with azoospermia. Such distinctions should clearly be established prior to genetic counseling for patients with so called Ynf or del (Yd).