Scopoletin, an active principle of tree tobacco (Nicotiana glauca) inhibits human tumor vascularization in xenograft models and modulates ERK1, VEGF-A, and FGF-2 in computer model.

We recently reported the antineovascularization effect of scopoletin on rat aorta and identified its potential anti-angiogenic activity. Scopoletin could be useful as a systemic chemotherapeutic agent against angiogenesis-dependent malignancies if its antitumorigenic activity is investigated and scientifically proven using a suitable human tumor xenograft model. In the present study, bioassay-guided (anti-angiogenesis) phytochemical investigation was conducted on Nicotiana glauca extract which led to the isolation of scopoletin. Further, anti-angiogenic activity of scopoletin was characterized using ex vivo, in vivo and in silico angiogenesis models. Finally, the antitumorigenic efficacy of scopoletin was studied in human colorectal tumor xenograft model using athymic nude mice. For the first time, an in vivo anticancer activity of scopoletin was reported and characterized using xenograft models. Scopoletin caused significant suppression of sprouting of microvessels in rat aortic explants with IC50 (median inhibitory concentration) 0.06µM. Scopoletin (100 and 200mg/kg) strongly inhibited (59.72 and 89.4%, respectively) vascularization in matrigel plugs implanted in nude mice. In the tumor xenograft model, scopoletin showed remarkable inhibition on tumor growth (34.2 and 94.7% at 100 and 200mg/kg, respectively). Tumor histology revealed drastic reduction of the extent of vascularization. Further, immunostaining of CD31 and NG2 receptors in the histological sections confirmed the antivascular effect of scopoletin in tumor vasculature. In computer modeling, scopoletin showed strong ligand affinity and binding energies toward the following angiogenic factors: protein kinase (ERK1), vascular endothelial growth factor A (VEGF-A), and fibroblast growth factor 2 (FGF-2). These results suggest that the antitumor activity of scopoletin may be due to its strong anti-angiogenic effect, which may be mediated by its effective inhibition of ERK1, VEGF-A, and FGF-2.

Isolation, identification, and phylogenetic analysis of reassortant low-pathogenic avian influenza virus H3N1 from Pakistan.

During routine avian influenza surveillance in Pakistan, a low-pathogenic avian influenza virus (LPAI) subtype H3N1 was isolated for the first time from domestic chickens. The higher seroprevalence of H3N1 was recorded in both commercial and domestic poultry in ecological zones of Pakistan where the geographical proximity with neighboring countries and attractive birding sites provide better opportunities for frequent movements of wild and migratory birds, and their intermingling with the local domestic and commercial poultry. Subsequent whole genome sequencing of this virus revealed a new introduction of a reassortant Eurasian avian strain, which was distinguishable from corresponding human and swine strains isolated elsewhere. Phylogenetically, the HA gene was mostly clustered with Nordic (Scandinavian) strains of influenza viruses, whereas the NA and PB1 genes showed a maximum nucleotide sequence homology with the Indian H11N1, and the PB2 gene was found to be closely related to the Altai H5N2. The Matrix and NP genes of H3N1 mostly clustered with the European avian influenza viruses (AIV), whereas its NS and PA genes showed maximum nucleotide homologies with the African (Egypt) AIV strains. A sequence and amino acid analysis revealed an LP motif, avian-like receptor specificity, potential glycosylation sites, and sensitivities to oseltamivir, zanamivir, and amantadine. Some point mutations possessed by this Pakistani AIV H3N1 were also found in human, equine, and swine H3 influenza viruses. This H3N1 isolate showed less nucleotide sequence homology with the previously known Pakistani AIV as compared with other Eurasian AIV strains.

Molecular Diagnostics of Ciliopathies and Insights Into Novel Developments in Diagnosing Rare Diseases.

The definition of a rare disease in the European Union describes genetic disorders that affect less than 1 in 2,000 people per individual disease; collectively these numbers amount to millions of individuals globally, who usually manifest a rare disease early on in life. At present, there are at least 8,000 known rare conditions, of which only some are clearly molecularly defined. Over the recent years, the use of genetic diagnosis is gaining ground into informing clinical practice, particularly in the field of rare diseases, where diagnosis is difficult. To demonstrate the complexity of genetic diagnosis for rare diseases, we focus on Ciliopathies as an example of a group of rare diseases where an accurate diagnosis has proven a challenge and novel practices driven by scientists are needed to help bridge the gap between clinical and molecular diagnosis. Current diagnostic difficulties lie with the vast multitude of genes associated with Ciliopathies and trouble in distinguishing between Ciliopathies presenting with similar phenotypes. Moreover, Ciliopathies such as Autosomal Recessive Polycystic Kidney Disease (ARPKD) and Meckel-Gruber syndrome (MKS) present with early phenotypes and may require the analysis of samples from foetuses with a suspected Ciliopathy. Advancements in Next Generation Sequencing (NGS) have now enabled assessing a larger number of target genes, to ensure an accurate diagnosis. The aim of this review is to provide an overview of current diagnostic techniques relevant to Ciliopathies and discuss the applications and limitations associated with these techniques.

Synchronization of Fractional Order Neurons in Presence of Noise.

The firing rate of some biological neurons such as neocortical pyramidal neurons is consistent with fractional order derivative, and the fractional-order neuron models depict the firing rate of neurons more accurately than other integer order neuron models do. For this reason, first, the dynamical characteristics of fractional order Hindmarsh Rose (HR) neuron are investigated, here and then a two coupled neuronal system based on Hindmarsh Rose neuron is presented. The results show several differences in the dynamical cha.racteristics of integer order and fractional order Hindmarsh Rose neuron model. The integer order model shows only one type of firing characteristics when the parameter of the model remained the same. The fractional-order model depicts several dynamical behaviors even for the same parameters as the order of the fractional operator is varied with the same parameter values. The firing frequency increases as the order of the fractional operator decreases. The fractional-order is therefore key in determining the firing characteristics of biological neuron models. A linearized model of HR neuron is also given for hardware resource minimizations and to implement this neuronal network on a large scale. A synchronized system of two fractional-order fractional Hindmarsh-Rose (HR) neurons in the presence of noise is also presented. The dynamical characteristics of the modified coupled neuron are determined by the parameters of the neuron model and the coupling function. The robustness of the network in the presence of noise is verified by both amplitude and phase synchronization techniques. A simplification of the coupling function is also presented to reduce the hardware cost. The synchronization results show that the model can produce the desired behavior with acceptable error.

HIV fusion inhibitors.

Drugs based on amino acid sequence of Heptad Repeats of gp41 of HIV have been explored in search of anti-HIV drugs acting by inhibition of the gp41 6-helix formation and subsequent cellular infection. These are classified under a distinct discipline called HIV fusion inhibitors. Resistance to HIV fusion inhibitors and their solutions have also been discussed in this review.

Methodology: a modified method for high-quality DNA extraction for molecular analysis in cereal plants.

Cereal crops that have rigid non-cellulose components in the cell wall tissues of leaves and high starch and protein content in grains face limitations in DNA extraction. Advanced molecular genetic techniques such as mapping and marker-assisted selection programs require pure and quick DNA extraction. In this study, we developed methods for isolating high-quality genomic DNA from leaves and seeds of major cereal crops with minor modifications. DNA yields ranged from 300 to 1800 ng for 0.01 g seed or leaf tissue.

Isolation of genomic DNA from defatted oil seed residue of rapeseed (Brassica napus).

A simple protocol for obtaining pure, restrictable and amplifiable megabase genomic DNA from oil-free seed residue of Brassica napus, an important oil seed plant, has been developed. Oil from the dry seeds was completely recovered in an organic solvent and quantified gravimetrically followed by processing of the residual biomass (defatted seed residue) for genomic DNA isolation. The isolated DNA can be cut by a range of restriction enzymes. The method enables simultaneous isolation and recovery of lipids and genomic DNA from the same test sample, thus allowing two independent analyses from a single sample. Multiple micro-scale oil extraction from the commercial seeds gave approximately 39% oil, which is close to the usual oil recovery from standard oil seed. Most of the amplified fragments were scored in the range of 2.5 to 0.5 kb, best suited for scoring as molecular diagnostics.

Serum sialic acid changes in type 2 diabetic patients on metformin or rosiglitazone treatment.

WHAT IS KNOWN AND OBJECTIVE: Serum sialic acid is a recently investigated potential risk-marker for cardiovascular complications. There is a known association between sialic acid and cardiovascular complications in diabetes mellitus. We aimed to investigate the effect of antidiabetic drugs on the serum concentration of sialic acid. METHODS: We investigated the effect of metformin and rosiglitazone on the concentration of sialic acid in 120 type 2 diabetic patients, divided into a group (n = 60) receiving metformin and a group (n = 60) receiving rosiglitazone treatment. RESULTS: Serum sialic acid was significantly higher in patients on rosiglitazone (66·90 ± 8·80 mg/dL vs. 57·6 ± 8·46 mg/dL, P < 0·01) and metformin (61·95 ± 10·49 mg/dL vs. 57·6 ± 8·46 mg/dL, P < 0·04) when compared with control subjects. In addition, rosiglitazone-treated patients showed a significant increase in cardiovascular risk factors, notably total cholesterol (246·45 ± 20·2 mg/dL vs. 170·6 ± 15·1 mg/dL, P = 0·01), triglyceride (178 ± 9·20 mg/dL vs. 149·35 ± 6·31 mg/dL, P < 0·04) and glycohemoglobin (HbA1-c) concentration (8·17 ± 1·43% vs. 4·38 ± 0·96%, P < 0·02) compared with normal control subjects. The patients on metformin also showed significantly higher levels of serum glucose (133·7 ± 9·63 mg/dL vs. 88·35 ± 6·31 mg/dL, P < 0·04) and glycohemoglobin (HbA1-c) (8·23 ± 1·75% vs. 4·38 ± 0·96%, P < 0·02) when compared with control subjects. Comparison of the two groups of patients revealed a significantly higher serum sialic acid (66·90 ± 8·80 mg/dL vs. 61·95 ± 10·49 mg/dL, P < 0·05), total cholesterol (246·45 ± 20·2 mg/dL vs. 192 ± 14·23 mg/dL, P < 0·02) and triglyceride (178 ± 9·20 mg/dL vs. 158 ± 14·51mg/dL, P < 0·05) concentrations in the rosiglitazone-treated patients. WHAT IS NEW AND CONCLUSIONS: This study suggests significantly higher levels of serum sialic acid and other cardiovascular risk factors in rosiglitazone-treated patients than in metformin-treated patients. The lower sialic acid concentration may explain a better metformin antidiabetic effect than with rosiglitazone.

Multiplex polymerase chain reaction for the detection and differentiation of avian influenza viruses and other poultry respiratory pathogens.

A multiplex reverse transcription-PCR (mRT-PCR) was developed and standardized for the detection of type A influenza viruses, avian influenza virus (AIV) subtype H7, H9, and H5 hemagglutinin gene with simultaneous detection of 3 other poultry respiratory pathogens, Newcastle disease virus (NDV), infectious bronchitis virus (IBV), and infectious laryngotracheitis virus (ILTV). Seven sets of specific oligonucleotide primers were used in this study for the M gene of AIV and hemagglutinin gene of subtypes H7, H9, and H5 of AIV. Three sets of other specific oligonucleotide primers were used for the detection of avian respiratory pathogens other than AIV. The mRT-PCR DNA products were visualized by agarose gel electrophoresis and consisted of DNA fragments of 1,023 bp for M gene of AIV, 149 bp for IBV, 320 bp for NDV, and 647 bp for ILTV. The second set of primers used for m-RT-PCR of H7N3, H9N2, and H5N1 provided DNA products of 300 bp for H7, 456 bp for H5, and 808 bp for H9. The mRT-PCR products for the third format consisted of DNA fragments of 149 bp for IBV, 320 bp for NDV, 647 bp for ILTV, 300 bp for H7, 456 bp for H5, and 808 bp for H9. The sensitivity and specificity of mRT-PCR was determined and the test was found to be sensitive and specific for the detection of AIV and other poultry respiratory pathogens. In this present study, multiplex PCR technique has been developed to simultaneously detect and differentiate the 3 most important subtypes of AIV along with the 3 most common avian respiratory pathogens prevalent in poultry in Pakistan. Therefore, a mRT-PCR that can rapidly differentiate between these pathogens will be very important for the control of disease transmission in poultry and in humans, along with the identification of 3 of the most common respiratory pathogens often seen as mixed infections in poultry, and hence economic losses will be reduced in poultry.

Differential response of antioxidant defense in HepG2 cells on exposure of Livotrit®, in a concentration dependent manner.

Livotrit®, a polyherbal formulation (Zandu, India) is commonly prescribed for liver health. The present study was undertaken to elucidate possible mechanism of antioxidant potential of Livotrit®. Livotrit® exhibited concentration dependent radical scavenging activity, inhibition of lipid peroxidation as well as activation and gene expression of antioxidant enzymes. Interestingly, lower concentration of Livotrit® (0.05%) significantly increased activities and gene expression of catalase, Glutathione reductase (GR) and Gluthathione peroxidase (GPx), while higher concentration of Livotrit® (0.5%) significantly increased antioxidant enzyme Heme-oxygenase 1(HO-1) and not catalase (CAT), GR and GPx. Transcription factor, Nuclear factor erythroid 2-related factor 2 (Nrf2) required for expression of catalase, GR, GPx and HO-1 was efficiently translocated into the nucleus at both concentrations. Inspite of this, concentration dependent activation of these enzymes was found to be mediated through miRNAs involved in regulation of their gene expression.

Evaluation of morbidity, mortality and outcome following cervical spine injuries in elderly patients.

We analysed the morbidity, mortality and outcome of cervical spine injuries in patients over the age of 65 years. This study was a retrospective review of 107 elderly patients admitted to our tertiary referral spinal injuries unit with cervical spine injuries between 1994 and 2002. The data was acquired by analysis of the national spinal unit database, hospital inpatient enquiry system, chart and radiographic review. Mean age was 74 years (range 66-93 years). The male to female ratio was 2.1:1 (M = 72, F = 35). The mean follow-up was 4.4 years (1-9 years) and mean in-hospital stay was 10 days (2-90 days). The mechanism of injury was a fall in 75 and road traffic accident in the remaining 32 patients. The level involved was atlanto-axial in 44 cases, sub-axial in 52 cases and the remaining 11 had no bony injury. Multilevel involvement occurred in 48 patients. C2 dominated the single level injury and most of them were type II odontoid fractures. Four patients had complete neurology, 27 had incomplete neurology, and the remaining 76 had no neurological deficit. Treatment included cervical orthosis in 67 cases, halo immobilization in 25, posterior stabilization in 12 patients and anterior cervical fusion in three patients. The overall complication rate was 18.6% with an associated in-hospital mortality of 11.2%. The complications included loss of reduction due to halo and Minerva loosening, non-union and delayed union among conservatively treated patients, pin site and wound infection, gastrointestinal bleeding and complication due to associated injuries. Among the 28.9% patients with neurological involvement, 37.7% had significant neurological recovery. Outcome was assessed using a cervical spine outcome questionnaire from Johns Hopkins School of Medicine. Sixty-seven patients (70%) completed the form, 20 patients (19%) were deceased at review and 8 patients (7%) were uncontactable. Functional disability was more marked in the patients with neurologically deficit at time of injury. Outcome of the injury was related to increasing age, co-morbidity and the severity of neurological deficit. Injuries of the cervical spine are not infrequent occurrence in the elderly and occur with relatively minor trauma. Neck pain in the elderly patients should be thoroughly evaluated to exclude C2 injuries. Most patients can be managed in an orthosis but unstable injuries require rigid external immobilization or surgical stabilization.

Plasma lipid profile in gynecologic cancers.

BACKGROUND: Lipids are associated with cancer because they play a key role in the maintenance of cell integrity. We studied the relationship of plasma lipids with gynecologic cancer. METHODS: A total of 196 female individuals were included in the study. Of these 50 were normal subjects. The remaining were cancer patients: 80 breast cancer, 40 ovarian cancer and 26 patients with other gynecologic cancers. Plasma levels of triglycerides, total cholesterol, LDL-cholesterol and HDL-cholesterol were estimated by using spectrophotometer. RESULTS: In breast cancer patients there is moderate increase in the plasma levels of triglycerides (18%) and cholesterol (21%), and a high increase in LDL-cholesterol (43%), while there is a moderate decrease in HDL-cholesterol levels (30%) when compared with normal subjects. In ovarian cancer patients, there is a high decrease in the plasma levels of triglycerides (31%) and HDL-cholesterol (39%), while a moderate decrease in cholesterol (28%) and LDL-cholesterol levels (11%) when compared with normal subjects. In gynecologic cancers other than breast and ovarian cancer, there is a moderate decrease in plasma levels of the triglycerides (25%), cholesterol (21%), and HDL-cholesterol levels (27%), while a non-significant decrease in LDL-cholesterol (6.2%) when compared with normal subjects. CONCLUSIONS: Plasma lipid levels, except HDL-cholesterol, are raised in breast cancer and are decreased in other gynecologic cancers. HDL-cholesterol is decreased in all gynecologic cancers. As there is an alteration in the plasma lipid profile during gynecologic cancers, it may be helpful for diagnosis of the disease.

Physical interaction of estrogen receptor with MnSOD: implication in mitochondrial O2.- upregulation and mTORC2 potentiation in estrogen-responsive breast cancer cells.

Augmented reactive oxygen species levels consequential to functional alteration of key mitochondrial attributes contribute to carcinogenesis, either directly via oxidative DNA damage infliction or indirectly via activation of oncogenic signaling cascades. We previously reported activation of a key oncogenic signaling cascade via mammalian target of rapamycin (mTOR) signaling complex-2 (mTORC2) owing to estrogen receptor (ER-α)-dependent augmentation of O2.- within the mitochondria of 17-ß-estradiol (E2)-stimulated breast cancer cells. Manganese superoxide dismutase (MnSOD) is the principal mitochondrial attribute governing mitochondrial O2.- homeostasis, raising the possibility that its functional alteration could be instrumental in augmenting mitochondrial O2.- levels in breast cancer cells. Here we show ER-dependent transient inhibition of MnSOD catalytic function in breast cancer cells. Catalytic function of MnSOD is tightly regulated at the post-translational level. Post-translational modifications such as phosphorylation, nitration and acetylation represent key regulatory means governing the catalytic function of MnSOD. Acetylation at lysine-68 (K68) inhibits MnSOD catalytic activity and thus represents an important post-translational regulatory mechanism in human cells. Using reciprocal immunoprecipitation and proximity ligation assay, we demonstrate the occurrence of direct physical interaction between ER-α and MnSOD in human breast cancer cells, which in turn was associated with potentiated acetylation of MnSOD at K68. In addition, we also observed diminished interaction of MnSOD with sirtuin-3, the key mitochondrial deacetylase that deacetylates MnSOD at critical K68 and thereby activates it for scavenging O2.-. Consequently, compromised deacetylation of MnSOD at K68 leading to its inhibition and a resultant buildup of O2.- within the mitochondria culminated in the activation of mTORC2. In agreement with this, human breast cancer tissue specimen exhibited a positive correlation between acetyl-MnSODK68 levels and phospho-Ser2481 mTOR levels. In addition to exposing the crosstalk of ER-α with MnSOD post-translational regulatory mechanisms, these data also unravel a regulatory role of ER/MnSOD interaction as an important control switch for redox regulation of ER-α-responsive oncogenic signaling cascades. Furthermore, our study provides a mechanistic link for ER-α-dependent O2.- potentiation and resultant mTORC2 activation in breast cancer cells.

Prevalence of chronic airflow limitation in Kashmir, North India: results from the BOLD study.

BACKGROUND: Data on spirometrically defined chronic airflow limitation (CAL) are scarce in developing countries. OBJECTIVE: To estimate the prevalence of spirometrically defined CAL in Kashmir, North India. METHODS: Using Burden of Obstructive Lung Disease survey methods, we administered questionnaires to randomly selected adults aged ⩾40 years. Post-bronchodilator spirometry was performed to estimate the prevalence of CAL and its relation to potential risk factors. RESULTS: Of 1100 participants initially recruited, 953 (86.9%) responded and 757 completed acceptable spirometry and questionnaires. The prevalence of a forced expiratory volume in 1 s/forced vital capacity (FEV1/FVC) ratio less than the lower limit of normal was 17.3% (4.5) in males and 14.8% (2.1) in females. Risk factors for CAL included higher age, cooking with wood and lower educational status. The prevalence of current smoking was 61% in males and 22% in females; most smoked hookahs. CAL was found equally in non-smoking males and females, and was independently associated with the use of the hookah, family history of respiratory disease and poor education. A self-reported doctor's diagnosis of chronic obstructive pulmonary disease was reported in 8.4/1000 (0.9% of females and 0.8% of males). CONCLUSION: Spirometrically confirmed CAL is highly prevalent in Indian Kashmir, and seems to be related to the high prevalence of smoking, predominantly in the form of hookah smoking.

Relation of serum sialic acid with serum lipids in cardiac patients.

Elevated serum sialic acid concentration is a risk factor for coronary heart disease in general population along with dyslipidemia. Sialic acid and lipid profile was determined in healthy subjects (Group A1 and A2) and sixty cardiac patients, divided into two groups, group B comprising of thirty male patients and group C comprising of thirty female patients. A highly significant increase was observed in the level of sialic acid and different variables of lipid profile except HDL-C in all patients. HDL-C showed a significant decrease in patients. An increase in the level of sialic acid in cardiac patients showing the implications of raised level of sialic acid in the development of cardiovascular complications.

Determinants of hospital charges for coronary artery bypass surgery: the economic consequences of postoperative complications.

This is a prospective study of 500 consecutive patients having coronary artery bypass surgery; mean hospital charge from time of surgery to discharge was +11,900 +/- 12,700. Multiple regression analysis was performed using preoperative variables and postoperative complications. No preoperative clinical feature was a significant predictor of higher average charge. Sternal wound infection (p = 0.0001), respiratory failure (p = 0.0001) and left ventricular failure (p = 0.017) were associated with higher average hospital charge. The absence of any complication predicted a lower average charge, and postoperative death (4.4 +/- 4.5 days after surgery) was also associated with lower average charge. A cost equation was developed: hospital charge equalled $11,217 + $41,559 of sternal wound infection, + $28,756 for respiratory failure, + $5,186 for left ventricular failure, - $1,798 for no complication and - $6,019 for death. Recognition of the influence of complications on charges suggests that low average charges can only be achieved by surgical programs with a low complication rate.

Evaluation of pathogenic potential of avian influenza virus serotype H9N2 in chickens.

Recently seven isolates of avian influenza virus (AIV) serotype H9N2 recovered from an outbreak of AI were analyzed on the basis of their biological and molecular characteristics. All the isolates belonged to the low-pathogenicity group of AIV. To further evaluate their pathogenic potential in association with other organisms, an isolate was inoculated experimentally in chickens using different routes and subsequently challenged with infectious bronchitis virus, Ornithobacterium rhinotracheale or Escherichia coli. The virus isolation and seromonitoring data revealed a significant role of Escherichia coli in aggravating the clinical condition of the birds earlier infected with AIV (H9N2). The AIV-antigen was detected in lung, trachea, kidney, and cloacal bursa among the infected birds, using immunofluorescent antibody technique. In another experiment, chickens that were immunosuppressed chemically showed high mortality when challenged with AIV H9N2. The results indicated that this low pathogenicity AIV (H9N2) isolate could produce severe infection depending on the type of secondary opportunistic pathogens present under field conditions. This may explain the severity of infection with the present H9N2 outbreak in the field. A prolonged antibacterial therapy in flocks infected with AIV H9N2 and use of oil-based vaccine at an early age in new flocks has helped to control this infection and the disease.

Immunosuppressive potential and pathogenicity of an avian adenovirus isolate involved in hydropericardium syndrome in broilers.

The role of avian adenovirus isolate PARC-1 as an immunosuppressive agent was investigated using a Newcastle disease virus (NDV) vaccine immune response procedure. The immunosuppressive effect on the humoral immune response was investigated up to 21 days after inoculation with adenovirus. Infected chickens showed a serologic response to NDV that was reduced compared with that of the controls. To further investigate the effect of the virus on major lymphoid organs, the pattern of virus dissemination in various organs was studied at various time intervals after inoculation. Spleen, thymus, bursa of Fabricius, and cecal tonsils of broilers were examined using a dot-immunobinding assay. The virus was found to have a predilection for lymphoid organs, and virus from lymphoid organs was capable of producing disease when inoculated into healthy chickens. The relationship of virus predilection to its immunosuppressive effect also was studied.

Cervicofacial sinuses.

One hundred consecutive cases of cervicofacial sinuses are reviewed in terms of aetiology, diagnosis and subsequent management. One hundred and thirty-eight cases recorded in the world literature are reviewed for comparison.