In vitro evaluation of growth reticence and anticancer potential of 5α,8α-epidioxy-24ᶓ-methylcholesta-6,22-dien-3ß-ol and ergosta-5,7,22-trien-3ß-ol bioactive isolated from an edible mushroom Lentinus tuberregium (fr.).

The focus point of this current work is to evaluate the anticancer and growth inhibitory efficacy of compounds 5α,8α-epidioxy-24ᶓ-methylcholesta-6,22-dien-3ß-ol (LT1), and Ergosta-5,7,22-trien-3ß-ol (LT2) of Lentinus tuberregium (Fr.) on three cell lines such as A673 (Rhabdomyosarcoma), MCF7 (breast cancer), and HCT116 (colorectal carcinoma) by MTT assay. LT1 and LT2 exerted maximal growth inhibition in the order as A673 > HCT116 > MCF7. Comparatively, LT1 was more potent in causing cell growth inhibition than LT2 in the A673 cancer cell line. Based on the MTT assay, A673 cells alone proceeded further as a model to evaluate the anticancer potential of LT1 and LT2 at three different semilogarithmic concentrations (3, 10, 30 µM). The cells exposed with compounds at 24 and 48 h were analyzed by flow cytometry. Exposure of LT1 at 3 and 10 µM concentrations for 24 h caused a G2-M arrest. At 10 µM concentration, cells also accumulated in the G0-G1 phase, indicating a G1 block. These effects were only transient as prolonged exposure (48 h) of LT1 treatment brought back the cell population to normalcy. Both the compounds only at 30 µM concentration have the potential to induce a hypodiploid peak (sub G0), indicating an induction of apoptosis which was explicit by nuclear condensation and fragmentation of nuclei in cells. The dose-dependent and compound-specific apoptotic induction was further confirmed by caspase activity higher in LT1 than LT2. The results highlight the significant growth inhibitory activity and anticancer potential of LT1 and LT2 which are recommended for further in-depth analysis.

17ß-Estradiol Concentration and Direct ß2-Adrenoceptor Inhibition Determine Estrogen-Mediated Reversal of Adrenergic Immunosuppression.

Background: Sympathetic innervation of lymphoid organs, and the presence of 17ß-estradiol (estrogen or E2) and adrenergic receptors (ARs) on lymphocytes, suggests that sympathetic stimulation and hormonal activation may influence immune functions. Purpose: Modeling and simulating these pathways may help to understand the dynamics of neuroendocrine-immune modulation at the cellular and molecular levels. Methods: Dose- and receptor-dependent effects of E2 and AR subtype-specific agonists were established in vitro on lymphocytes from young male Sprague-Dawley rats and were modeled in silico using the MATLAB Simbiology toolbox. Kinetic principles were assigned to define receptor-ligand dynamics, and concentration/time plots were obtained using Ode15s solvers at different time intervals for key regulatory molecules. Comparisons were drawn between in silico and in vitro data for validating the constructed model with sensitivity analysis of key regulatory molecules to assess their individual impacts on the dynamics of the system. Finally, docking studies were conducted with key ligands E2 and norepinephrine (NE) to understand the mechanistic principles underlying their interactions. Results: Adrenergic activation triggered proapoptotic signals, while E2 enhanced survival signals, showing opposing effects as observed in vitro. Treatment of lymphocytes with E2 shows a 10-fold increase in survival signals in a dose-dependent manner. Cyclic adenosine monophosphate (cAMP) activation is crucial for the activation of survival signals through extracellular signal-regulated kinase (p-ERK) and cAMP responsive element binding (p-CREB) protein. Docking studies showed the direct inhibition of ERK by NE and ß2-AR by E2 explaining how estrogen signaling overrides NE-mediated immunosuppression in vitro. Conclusion: The cross-talk between E2 and adrenergic signaling pathways determines lymphocyte functions in a receptor subtype and coactivation-dependent manner in health and disease.

GC-MS and Surface Characteristics of Polyvinyl Siloxane-An In Vitro Analysis.

OBJECTIVE: To assess the surface characteristics, composition and release of components of Polyvinyl siloxane (PVS) impression material and explore the use of the material other than for impressions. METHODS: Forty samples of 0.5-mm thickness and 8-mm diameter PVS discs, with few perforations, were prepared and divided into four groups. Group 1 in methanol was analyzed by GC-MS immediately and on 1st, 7th and 14th day. Group 2 and 3 placed in 10 mL of human saliva and in artificial nasal fluid, respectively, were analyzed by GC-MS at 24 hours. Group 4 was subjected to surface characteristics analysis by placing five PVS discs each in 1 mL of human saliva and 1 mL of artificial nasal fluid, respectively. RESULTS: On Day 1, Dodecanoic acid, methyl ester, Cyclononasiloxane, octadecamethyl, Cyclodecasiloxane and eicosamethyl (four peaks) were observed in Group 1. At 24 hours, Group 2 had additional compounds of 2-Decene,7-methyl-(Z)-, 2-Undecene,8-methyl-(Z)- and Lauryl acetate. Group 3 showed release of 32 compounds, but their retention time was less. Surface characteristics on Day 14 revealed no noticeable changes. CONCLUSIONS: The study revealed that there was a lack of any adverse component release from PVS for a period of 2 weeks based on GC-MS analysis.

Inhibition of MMP2-PEX by a novel ester of dihydroxy cinnamic and linoleic acid from the seagrass Cymodocea serrulata.

Matrix metalloproteinases (MMPs) are pivotal for cancer cell migration and metastasis which are generally over-expressed in such cell types. Many drugs targeting MMPs do so by binding to the conserved catalytic domains and thus exhibit poor selectivity due to domain-similarities with other proteases. We report herein the binding of a novel compound [3-(E-3,4-dihydroxycinnamaoyloxyl)-2-hydroxypropyl 9Z, 12Z-octadeca-9, 12-dienoate; Mol. wt: 516.67 Da], (C1), isolated from a seagrass, Cymodocea serrulata to the unconserved hemopexin-like (PEX) domain of MMP2 (- 9.258 kcal/mol). MD simulations for 25 ns, suggest stable ligand-target binding. In addition, C1 killed an ovarian cancer cell line, PA1 at IC50: 5.8 µM (lesser than Doxorubicin: 8.6 µM) and formed micronuclei, apoptotic bodies and nucleoplasmic bridges whilst causing DNA laddering, S and G2/M phase dual arrests and MMP disturbance, suggesting intrinsic apoptosis. The molecule increased mRNA transcripts of BAX and BAD and down-regulated cell survival genes, Bcl-xL, Bcl-2, MMP2 and MMP9. The chemical and structural details of C1 were deduced through FT-IR, GC-MS, ESI-MS, 1H and 13C NMR [both 1D and 2D] spectra.

De novo transcriptome analysis unravels tissue-specific expression of candidate genes involved in major secondary metabolite biosynthetic pathways of Plumbago zeylanica: implication for pharmacological potential.

KEY MESSAGE: The present study provides comparative transcriptome analysis, besides identifying functional secondary metabolite genes of Plumbago zeylanica with pharmacological potential for future functional genomics, and metabolomic engineering of secondary metabolites from this plant towards diversified biomedical applications. ABSTRACT: Plumbago zeylanica is a widely used medicinal plant of the traditional Indian system of medicine with wide pharmacological potential to treat several disorders. The present study aimed to carry out comparative transcriptome analysis in leaf and root tissue of P. zeylanica using Illumina paired end sequencing to identify tissue-specific functional genes involved in the biosynthesis of secondary metabolites, contributing to its therapeutic efficacy. De novo sequencing assembly resulted in the identification of 62,321 "Unigenes" transcripts with an average size of 1325 bp. Functional annotation using BLAST2GO resulted in the identification of 50,301 annotated transcripts (80.71%) and GO assigned to 18,814 transcripts. KEGG pathway annotation of the "Unigenes" revealed that 2465 transcripts could be assigned to 242 KEGG pathway maps wherein the number of transcripts involved in secondary metabolism was distinct in root and leaf transcriptome. Among the secondary metabolite biosynthesis pathways, the cluster of "Unigenes" encoding enzymes of 'Phenylpropanoid biosynthesis pathway' represents the largest group (84 transcripts) followed by 'Terpenoid Backbone biosynthesis' (48 transcripts). The transcript levels of the candidate unigenes encoding key enzymes of phenylpropanoid (PAL, TAL) and flavanoid biosynthesis (CHS, ANS, FLS) pathways were up-regulated in root, while the expression levels of candidate "Unigenes" transcript for monoterpenoid (DXS, ISPF), diterpenoid biosynthesis (SPS, SDS) and indole alkaloid pathways (STR) were significantly higher in leaf of P. zeylanica. Interestingly, validation of differential gene expression profile by qRT-PCR also confirmed that candidate "Unigenes" enzymes of phenylpropanoid and flavonoid biosynthesis were highly expressed in the root, while the key regulatory enzymes of terpenoid and indole alkaloid compounds were up-regulated in the leaf, suggesting that (differences in) the levels of these functional genes could be attributed to the (differential) pharmacological activity (between root and leaf) in tissues of P. zeylanica.

Synchronization of Autonomic and Cerebral Rhythms During Listening to Music: Effects of Tempo and Cognition of Songs.

A large number of studies document cardiorespiratory changes occurring while listening to music. Less is known, however, about the interaction between cardiorespiratory and cerebral electrical rhythms during listening to music and how cognition and acoustic structural aspects of songs influence that interaction. We focused on tempo as a structural feature of songs, since tempo is a major determinant of physiological responses to music, and on familiarity and randomization of phase of local spectra of known and unknown songs for cognition. Our results indicated an overall increase in the degree of synchronization among cardiorespiratory variables (Heart rate (RR), systolic and diastolic blood pressure (SBP, DBP), respiration) and between cardiorespiratory and cerebral (EEG) oscillations during all songs. We also observed a marked decrease in respiratory frequency bandwidth and increase in respiratory rate while listening to songs, and slow song produced the most periodic breathing. Compared with slow tempo, during fast song, DBP and cerebral oscillations became less synchronized with high frequency components of RR suggesting that the processes causing the previously known reduction in vagal activity with increase in tempo also may have caused the decrease in these synchronizations. Cognition of songs affected the SBP coherencies the most. DBP was synchronized with respiration more than all other measured variables in response to auditory stimuli. Results indicate an overall increase in the degree of synchronization among a variety of cerebral electrical and autonomically driven cardiovascular rhythms. It is possible that this significant increase in synchronizations underlies the widely reported pleasurable and palliative effects of listening to music.

Establishment of T-lymphocyte subset reference intervals in a healthy adult population in Chennai, India.

Estimation of CD4+ T-lymphocytes continues to be an important aspect for monitoring HIV disease progression and response to antiretroviral therapy. Most of the diagnostic laboratories often rely on western text books for CD4+ T-lymphocyte reference values, which could, often be unreliable for usage in local settings. Therefore, we attempted to establish the reference values for T-lymphocyte subsets among healthy adults in a cross-sectional study carried out at the YRG Centre for AIDS Research and Education (YRG CARE) in Chennai, south India, in 213 (84 female and 129 male) healthy, HIV-1/2 seronegative adults as volunteers. Whole blood specimens were processed for CD4+, CD8+ T-lymphocyte estimation and haematological parameters. The established range of CD4+ T-lymphocyte counts for men and women were 383-1347 cells/microl (mean 865 and median 845 cells/microl) and 448-1593 cells/microl (mean 1021 and median 954 cells/microl), respectively. Women had significantly higher absolute CD4+ Tlymphocyte counts (P<0.001) and CD4+:CD8+ T-lymphocyte ratio as compared to men. The established normal range of CD4+ T-lymphocyte % was 21-59 (mean 40.2 and median 40.1). The influence of age was not observed in any of the parameters except CD4+/CD8+ T-lymphocyte ratio with the >45 yr age group. Further studies with greater sample size may be required to define the staging of HIV disease in relation to the normal CD4 T-lymphocyte count in the general population.

Possible glutamatergic and lipid signalling mechanisms in ECT-induced retrograde amnesia: experimental evidence for involvement of COX-2, and review of literature.

We sought to explore nonselective vs. selective COX mechanisms in ECS-induced retrograde amnesia using indomethacin and celecoxib as in vivo probes. Adult Wistar rats (n=72) which showed adequate learning on a passive avoidance task received 5 once-daily 30 mC true or sham ECS. During the learning and ECS periods, indomethacin (4 mg/kg/day), celecoxib (15 mg/kg/day), or vehicle were orally administered. One day after the fifth ECS, recall of pre-ECS learning was tested. There were no baseline or pre-ECS differences in learning between groups. ECS seizure duration did not differ across groups. ECS-treated rats showed impaired recall in the vehicle but not indomethacin and celecoxib groups. Celecoxib but not indomethacin significantly protected against ECS-induced retrograde amnesia. We interpret these results as follows: ECS may impair cognition by pathologically upregulating glutmatergic signalling, thereby causing cation and water influx, oxidative stress, and saturation of hippocampal LTP. These may result from glutamatergic disinhibition through COX-2-mediated removal of endogenous cannabinoids, and by ECS-activated, NMDA-mediated upregulation of platelet activating factor and COX-2 signalling pathways. Thus, indomethacin and celecoxib, by inhibiting COX-2, may protect against ECS-induced amnesia. Furthermore, COX-2 mediated increase in hippocampal kynurenic acid may impair glutamate-dependent learning and memory processes at ionotropic glutamatergic receptor sites; the inhibition of kynurenic acid synthesis by celecoxib and its induction by indomethacin may explain the greater benefits with celecoxib. These findings suggest new avenues for the study of the neurobiology of ECT-induced amnesia and the attenuation thereof.

High proportion of isosporiasis among HIV-infected patients with diarrhea in southern India.

We investigated 245 diarrheal stool specimens from HIV-positive subjects between January 2003 and December 2006 to determine the etiological role of coproparasites. Parasitic etiology was observed in 91 (37.1%) cases. Isospora belli (26.1%) was the most common parasite followed by Entameba histolytica/dispar (3.3%), Cryptosporidium spp. (2.9%), Giardia intestinalis (1.6%), and Strongyloides stercoralis (1.2%). Interesting trends of significant increase in the number of cases of I. belli and decline in Cryptosporidium spp. were observed during the study period.

Prevalence of aflatoxin B1 in liver biopsies of proven hepatocellular carcinoma in India determined by an in-house immunoperoxidase test.

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death in the world. The incidence of HCC in India is reportedly low and varies from 0.2 to 1.9 %. Aflatoxins, secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus, are potent human carcinogens implicated in HCC. The prevalence of aflatoxin B1 (AFB1) as co-carcinogen was analysed using an in-house immunoperoxidase test in 31 liver biopsies and 7 liver-resection specimens from histopathologically proven HCC, and in 15 liver biopsies from cirrhosis patients (control group). Serum was tested for hepatitis B and C serological markers using commercial assays, and for AFB1 using an in-house ELISA with a sensitivity of approximately 1 ng ml(-1) for AFB1. In spite of positive AFB1 immunostaining in HCC cases, all serum specimens, from both HCC and the control groups, were AFB1-negative. There were 18 (58.1 %) HCC cases that revealed AFB1 in liver biopsies; 68.8 % (n=11) of non-B non-C hepatitis cases with HCC and 46.1 % (n=6) of the hepatitis B surface-antigen-positive subjects were positive for AFB1. Out of the two hepatitis B/hepatitis C virus co-infected cases, one was positive for AFB1. Of seven tumour-resection samples, six were positive for AFB1. Only one case revealed AFB1 in the non-tumour area of the resected material. Thus AFB1 staining was significantly associated with tumour tissue (P=0.03). Aflatoxins proved to have a significant association with HCC in this peninsular part of the subcontinent. The impact seems to be a cumulative process, as revealed by the AFB1 deposits in HCC liver tissue, even though the serum levels were undetectable.

Diagnostic utility of serologic markers for genital chlamydial infection in STD patients in Chennai, India.

OBJECTIVES: To evaluate the diagnostic utility of serological markers for C. trachomatis in different clinical groups of STD patients. METHODS: Blood and genital swab specimens were collected from symptomatic STD patients (n=143) attending the STD out patient clinic at the Institute of STDs, Government General hospital, Chennai who enrolled for the study. Serological determination for IgM, IgA and IgG antibodies to C. trachomatis was done using commercial kits. PCR analysis was performed on genital swab samples by using plasmid and major outer membrane protein (MOMP) based primers and patients who were positive by both PCR assays were considered as proven cases of C. trachomatis infection. The serological marker positivity was analysed with PCR positivity. RESULTS: Serologic positivity by IgM, IgA and IgG was 22.4%, 28.7% and 58.7% respectively. The PCR analysis showed 44 (30.8%) cases with confirmed C. trachomatis infection. Seropositivity for IgM (34.1% (15/44) vs. 17.2% (17/99); P<0.05) as well as for IgA (40.9% (18/44) vs. 23.2% (23/99); P<0.05) significantly correlated to PCR positivity, while significant correlation was not seen with IgG positivity. The overall seropositivity (IgM/IgA/IgG) in the study population was 68.5%. CONCLUSIONS: The observations of the present study indicate a high exposure rate to chlamydial infection in STD clinic patients in India. The study also suggests the usefulness of serology instead of PCR to trace chlamydial etiology, especially in deep-seated upper genital tract diseases and to facilitate better clinical management as there was good correlation between serology and PCR positivity.

An audit of corneal abrasion management following the introduction of local guidelines in an accident and emergency department.

BACKGROUND AND OBJECTIVES: Corneal abrasions are a common presentation to accident and emergency (A&E) departments. Patients can be treated and discharged by A&E staff without the need for an ophthalmologist's attention; complicated cases, however, should be recognised and referred. Inexperience and limited training in ophthalmology may lead to suboptimal patient care and inappropriate use of ophthalmology outpatient clinics. Issues of poor documentation may also arise. The purpose of this audit was to assess the effect of guidelines on the management of corneal abrasion by A&E staff. METHODS: A retrospective case note audit was performed to assess current management of corneal abrasions. Guidelines for management of corneal abrasions were formulated following a literature search and collaboration between A&E and ophthalmology staff. A prospective case note audit was undertaken to assess management after introduction of the guidelines. RESULTS: A total of 51 cases were audited before the introduction of the guidelines and 57 cases after. Following the introduction of the guidelines documentation of visual acuity increased to 93% and specific enquiry into contact lens wear rose from 35.3% to 71.9%. A&E staff stopped giving out local anaesthetic eye drops. The follow up profile also improved; appropriate patient discharges increased by 40% whereas inappropriate referrals to eye clinic dropped by 75%. More patients were given abrasion advice (a 101% increase). CONCLUSIONS: A&E staff members are capable of managing corneal abrasions if they are given guidance and some training. This audit identified shortcomings in current management and showed that guidelines can significantly improve clinical practice.

HIV-1 western blot assay: What determines an indeterminate status?

BACKGROUND: The Western blot assay is the gold standard for the detection of antibodies to human immunodeficiency virus type 1 (HIV-1). However, indeterminate Western blot reactivity to HIV-1 proteins may occur in individuals, who may not be infected with HIV. AIM: This retrospective study was aimed to determine the diagnostic value of the interpretation criteria in relation to commercial kits for HIV-1 diagnosis. METHODS AND MATERIALS: A total of 556 serum/plasma specimens collected from high-risk population attending our HIV clinic from 2000-2004 were tested by three different western blot kits: NEW LAV BLOT I (n=244), HIV BLOT 2.2; (n=112), Genetic Systems HIV-1 (n=237). And the results of western blot strips were analyzed using the various interpretation criteria: WHO/NACO, CDC/ ASTPHLD, ARC, FDA, CRSS and JHU. Some specimens were run on more than one kit. RT-PCR assay was performed on 5 specimens, which were indeterminate with LAV BLOT I. RESULTS: The discrepancy in LAV BLOT I positive results were between 157(64)-176(72), and indeterminate results were between 44(18) to 63(25). No such variations were observed in genetic systems. There are some HIV negative (by PCR) specimens were indeterminate in LAV BLOT I revealing the kit more sensitive and less effective for diagnostic purpose. CONCLUSION: The genetic systems kit is superior to other kits we analyzed and its results are concordant with HIV-1 PCR results. To report, the choice of western blot commercial kit is paramount important than the use of particular interpretation criteria for the diagnosis of HIV-1.

Community prevalence of hepatitis B infection and modes of transmission in Tamil Nadu, India.

BACKGROUND AND OBJECTIVE: There have been very few community based studies on prevalence of hepatitis B virus (HBV) infection in India. We undertook this study to determine the prevalence of HBV infection in a southern State of India, Tamil Nadu and to describe the important factors related to transmission of the virus in the community. METHODS: Analysis of stored blood samples from a representative population of Tamil Nadu from an earlier community cluster survey on sexually transmitted diseases (STD) prevalence using proportionate to population size (PPS) technique was done. Serum markers of HBV viz., hapatitis B surface antigen (HBsAg), hepatitis B e antigen (HBe Ag) and antibody to surface antigen (anti-HBs) were performed. RESULTS: 1981 subjects were screened in the study. HBsAg prevalence was 5.7 per cent (CI 4.6- 6.8) with 23.5 per cent (25/106) of these having positive HBe-antigen. Community seroprevalence (HbsAg + anti-HBs) of hepatitis B infection was 27.4 per cent (CI: 25.3-29.5) with the highest prevalence of 32.7 per cent (CI: 30.2-35.2) noted in the 15-20 yr age group. Significant independent association (OR 1.4; P=0.006) was detected with family history of exposure to HBV infection by logistic modeling. Other risk factors noted to have significant association were use of disposable needles during injection (OR 0.5; P=0.02) in men, smoking (OR 3; P=0.04) and use of condom (OR 0.6; P=0.08) in women. INTERPRETATION AND CONCLUSION: This community based study shows a high prevalence of hepatitis B infection in the state of Tamil Nadu with the highest prevalence being in the younger (15-20 yr) age group. High prevalence rate in childhood with e-antigenemia seen in 23.5 per cent of HBsAg positive subjects suggest childhood transmission. Poor injection practices and high-risk sexual behavior were found to be additional risk factors for transmission of the disease in the community.

Genital chlamydial infection in STD patients: its relation to HIV infection.

In the present report, we have analysed C.trachomatis infection and HIV positivity among patients (n-143) who attended the STD clinic at the Institute of STDs, Government General Hospital, Chennai. HIV positivity rate was significantly high among those with chlamydial infection than in those without chlamydial infection (29.5% (13/44) vs. 11.1% (11/99); p<0.05). The results of the present study suggest the association between C.trachomatis and HIV infections and reinforce the need for routine screening for C.trachomatis as a necessary intervention to reduce the burden of chlamydial diseases and to reduce the risk of HIV and its spread in India.