Search

We report a systematic investigation of caffeine-induced dissociation of ethidium (Et) cation, a potential mutagen. Time-resolved fluorescence studies are consistent with a mechanism where caffeine-Et complex formation in bulk solution drives the dissociation of DNA-bound Et. Temperature-dependent picosecond-resolved studies show the caffeine-Et complex to be stable over a wide range of temperature, within and beyond the normal physiological limit. A combination of NMR spectroscopy and dynamic light scattering experiments allowed us to propose a molecular model of the caffeine-Et complex. Caffeine-induced extraction of Et from whole cells was also performed on squamous epithelial cells collected from the inner lining of the human mouth, A549 (lung carcinoma), A375 (human skin), RAW (macrophage), and Vero (African green monkey kidney epithelium) cell lines. Interestingly the efficiency of caffeine in extracting Et has been found to be dependent on cell types. Our results both in vitro as well as ex vivo provide important clues about the efficiency and mechanism of caffeine as a potential antimutagenic therapeutic agent.

Previous work by us and others reported decreased expression of miR-199a-3p in hepatocellular carcinoma (HCC) tissues compared to adjacent benign tissue. We report here a significant reduction of miR-199a-3p expression in 7 HCC cell lines. To determine if miR-199a-3p has a tumor suppressive role, pre-miR-199a-3p oligonucleotides were transfected into the HCC cell lines. Pre-miR-199a-3p oligonucleotide reduced cell proliferation by approximately 60% compared to control oligonucleotide in only two cell lines (SNU449 and SNU423); the proliferation of the other 5 treated cell lines was similar to control oligonucleotide. A pre-miR-199a-3p oligonucleotide formulated with chemical modifications to enhance stability while preserving processing, reduced cell proliferation in SNU449 and SNU423 to the same extent as the commercially available pre-miR-199a-3p oligonucleotide. Furthermore, only the duplex miR-199a-3p oligonucleotide, and not the guide strand alone, was effective at reducing cell viability. Since a CD44 variant was essential for c-Met signaling [V. Orian-Rousseau, L. Chen, J.P. Sleeman, P. Herrlich, H. Ponta, CD44 is required for two consecutive steps in HGF/c-Met signaling, Genes Dev. 16 (2002) 3074-3086] and c-Met is a known miR-199a-3p target, we hypothesized that miR-199a-3p may also target CD44. Immunoblotting confirmed that only the two HCC lines that were sensitive to the effects of pre-miR-199a-3p were CD44+. Direct targeting of CD44 by miR-199a-3p was confirmed using luciferase reporter assays and immunoblotting. Transfection of miR-199a-3p into SNU449 cells reduced in vitro invasion and sensitized the cells to doxorubicin; both effects were enhanced when hyaluronic acid (HA) was added to the cell cultures. An inverse correlation between the expression of miR-199a-3p and CD44 protein was noted in primary HCC specimens. The ability of miR-199a-3p to selectively kill CD44+ HCC may be a useful targeted therapy for CD44+ HCC.

Stroke is a leading cause of death and disability. Globally, 15 million people suffer a stroke each year, of whom more than 5 million die, and a further 5 million are left permanently disabled. Current treatment options offer modest benefits, and there is a pressing need for new and effective treatments. Stem cell therapy is a well-established treatment modality for various haematological diseases, with its use now being explored in different disease processes, including various neurological diseases, as well as vascular conditions such as ischaemic heart disease and peripheral vascular disease. Promising results have been seen in animal models of stroke, with evidence of significant functional benefits. Translation to the bedside, however, is in its early stages. This review will discuss the scientific background to stem cell therapy in ischaemic stroke, including evidence from current clinical trials.

Objective: The objective was to study the evaluation of antidiabetic and antioxidant activity of different doses ethanolic extract of Mikania scandens leaves.Methods: The rats were divided into five groups of six animals each (non-diabetic control, diabetic control, standard drug-treated, and plant treated groups (250 and 500 mg/kg body weight)). Diabetes was induced in the healthy male Wistar rats (150–200 g body weight, 4–6 weeks old) by the administration of alloxan monohydrate (150 mg/kg, i.p.). On the 0th, 3th, 7th, 11th, and 15th days, the blood samples were analyzed for blood glucose. The antioxidant and antidiabetic parameters were evaluated by standard protocol. The liver tissue was used for histopathological assessment of liver damage.Results: The ethanolic extract of leaves of M. scandens showed a significant reduction (p

Glutathione acts as a protein disulphide reductant, which detoxifies herbicides by conjugation, either spontaneously or by the activity of one of a number of glutathione-S-transferases (GSTs), and regulates gene expression in response to environmental stress and pathogen attack. GSTs play roles in both normal cellular metabolisms as well as in the detoxification of a wide variety of xenobiotic compounds, and they have been intensively studied with regard to herbicide detoxification in plants. A newly discovered plant GST subclass has been implicated in numerous stress responses, including those arising from pathogen attack, oxidative stress and heavy-metal toxicity. In addition, plants GSTs play a role in the cellular response to auxins and during the normal metabolism of plant secondary products like anthocyanins and cinnamic acid. The present work involves two in silico analytical approaches-general secondary structure prediction studies of the proteins and detailed signature pattern studies of some selected GST classes in Arabdiopsis thaliana, mustard, maize and bread wheat by standard Bioinformatics tools; structure prediction tools; signature pattern tools; and the evolutionary trends were analyzed by ClustalW. For this purpose, sequences were obtained from standard databases. The work reveals that these proteins are mainly alpha helical in nature with specific signature pattern similar to phosphokinase C, tyrosine kinase and casein kinase II proteins, which are closely related to plant oxidative stress. This study aims to comprehend the relationship of GST gene family and plant oxidative stress with respect to certain specific conserved motifs, which may help in future studies for screening of biomodulators involved in plant stress metabolism.

Background Transient ischaemic attacks (TIAs) carry a significant early risk of stroke. New national guidelines state patients should be seen within 7 days of the incident, with higher-risk patients being seen within 24 h. Meeting these targets across the NHS poses a significant challenge. A novel approach to TIA assessment has been developed using a nurse-led rapid-access anterior circulation TIA clinic. Methods This was a prospective evaluation of all patients attending the FAST-TIA clinic between November 2003 and December 2006. Diagnostic yield of neurovascular events among patients seen through the TIA service and median time from referral to assessment and from event to assessment were measured. Results 282 patients were eligible for investigation, and seen through the clinic over a period of 38 months. A vascular event was diagnosed in 242 (86%). TIA was diagnosed in 133 (55%), minor ischaemic stroke in 77 (32%), haemorrhagic stroke in three (1%), and an ocular event in 29 (12%). Median time from referral to assessment was 3 days (interquartile range (IQR) 1–7), and from event to assessment it was 7 days (IQR 3–18). 34% of patients were seen within 24 h of referral. Conclusions This model has a high diagnostic rate of 86% vascular events, significantly higher than current national averages of ∼55%. Current national guidelines for early assessment of patients (published subsequent to this study) are achievable using this service. The FAST-TIA model is an easily reproducible and pragmatic method of improving the diagnostic yield of TIA services, while keeping within national targets.

This study explores the synergistic effect of aluminium phosphate (ALP) nanoparticles and tungstophosphoric acid (TPA) on the physicochemical properties of sulfonated poly ether ether ketone (SPEEK) nanocomposite membranes. SPEEK/TPA/ALP nano- composite containing optimum TPA (10 wt %) and varying ALP content (3-10 wt %) are fabricated to investigate the effect of ALP nanoparticles on membrane properties. Experimental results reveal that nanocomposite membrane containing 3 wt % ALP nanopar- ticles and 10 wt % TPA exhibits 3.3 and 18.8 times higher proton conductivity compared to 10 wt % TPA filled SPEEK composite membrane and reference SPEEK membrane. ALP nanoparticles help in retaining water within the membranes and thus 59.4% reduc- tion in water desorption rate is achieved compared to SPEEK/TPA membrane. The leaching of TPA is reduced by 34.5% which helps in retaining membrane properties. Membranes are thermally stable up to 2008C. Microstructure of the composite films is investigated by scanning electron microscope. V C 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 133, 42952.

Controversies about the origin of circulating miRNAs have encouraged us to identify organ specific circulating miRNAs as disease biomarkers. To identify liver-specific miRNAs for hepatocellular carcinoma (HCC), global expression profiling of miRNAs in liver tissue of HBV-HCC and HBV-control with no or mild fibrosis was evaluated. A total of 40 differentially expressed miRNAs were identified in HCC. Among ten highly altered miRNAs, six miRNAs were successfully validated in tissues, whereas only two miRNAs, miR-126 and miR-142-3p showed increased expression in plasma of HBV-HCC compared to HBV-non-HCC patients. Subsequently, ROC curve analysis revealed that neither miR-126 nor miR-142-3p performed better than AFP in discriminating HCC from non-HCC while combination of each with AFP showed significantly higher efficiency rather than AFP alone (AUC: 0.922, 0.908 vs. 0.88; sensitivity: 0.84, 0.86 vs. 0.82 and specificity: 0.92, 0.94 vs. 0.86 respectively). Interestingly, triple combination of markers (miR-126 + miR-142-3p + AFP) showed no additive effect on efficiency (AUC: 0.925) over the dual combination. Again, the expression of only miR-126 was noticed significantly higher in HBV-HCC patients with low-AFP [250 ng/ml] compared to either non-HCC or liver cirrhosis (AUC: 0.77, 0.64, respectively). Furthermore, no alteration in expression of mir-126 in HCV-HCC or non-viral-HCC revealed that miR-126 + AFP might be specific to HBV-HCC. To understand the physiological role of these two miRNAs in hepato-carcinogenesis, target genes related to cancer pathways (APAF1, APC2, CDKN2A, IRS1, CRKL, LIFR, EGR2) were verified. Thus, combination of circulating miR-126 + AFP is a promising noninvasive diagnostic biomarker for HBV-HCC and may be useful in the management of HCC patients.

The precise mechanism by which HBx protein of hepatitis B virus (HBV) impacts on hepato-carcinogenesis remain largely elusive despite strong evidences for its' involvement in the process. Here, we have investigated the role of HBx on expression of a novel gene hELG1/ATAD5, which is required for genome maintenance and its' importance in hepatocarcinogenesis. This study has for the first time showed that the expression of this gene was significantly higher in human cancer such as HBV-associated hepatocellular carcinoma (HCC) and in different HCC cell lines compared to normal liver. In addition, a significant elevation in ATAD5 expression was also found in HBx transfected HCC cell lines implicating HBx mediated transcriptional regulation on ATAD5. Using different deletion mutant constructs of putative promoter, the active promoter region was first identified here and subsequently the regulatory region of HBx was mapped by promoter-luciferase assay. But ChIP assay with anti-HBx antibody revealed that HBx was not physically present in ATAD5 transcription machinery whereas anti-E2F1 antibody showed the presence of E2F1 in the complex. Luciferase assay with E2F1 binding site mutant had further confirmed it. Moreover, both loss-and gain-of-function studies of ATAD5 showed that ATAD5 could enhance HBV production in transfected cells whereas knock down of ATAD5 increased the sensitivity of HCC cell line to chemotherapeutics 5-fluorouracil. Overall, this data suggests that a positive feedback loop regulation between ATAD5 and HBV contributed to both viral replication and chemo-resistance of HCC cells.

Objective: The aim of the present study was to characterize the predominant hepatitis B virus (HBV) strains and their molecular variants present in the HBV isolates of the different genotypes found among the chronic carriers of the virus in our community. Methods: Precore/core and core promoter regions of HBV DNA were amplified by polymerase chain reaction and then subjected to direct sequencing. Of the 64 hepatitis B surface antigen (HBsAg)-positive chronic HBV carriers investigated, 44 were HBeAg negative and 20 were HBeAg positive. Results: In addition to genotype D, which was the predominant genotype, 12 genotype C (18.7%) and 6 genotype A (9.4%) were also detected. Presence of T at nt 1858 has often been related to the development of precore stop mutation at nt 1896, while that of C has been related to the development of 1762–1764 double mutation. In our study group, 39 of the 44 HBeAg-negative samples have T1858. The precore stop codon mutation was found in only 8 (18%) of the HBeAg-negative samples. More than half of the HBeAg-negative samples had wild-type sequence in the precore region. The core promoter region could be sequenced from 40 samples, and 1762–1764 double mutation was detected in 13 (32.5%) of them. No significant changes could be detected in the core amino acid sequence of these isolates. Conclusion: The pattern of core promoter and precore mutation of HBV isolates in the present study is atypical and not in accordance with reports from other parts of the world, where genotype D and genotype C with T at codon 1858 are common.

Gross chromosomal rearrangements (GCRs) are frequently observed in cancer cells. Abnormalities in different DNA metabolism including DNA replication, cell cycle checkpoints, chromatin remodeling, telomere maintenance, and DNA recombination and repair cause GCRs in Saccharomyces cerevisiae . Recently, we used genome-wide screening to identify several genes the deletion of which increases GCRs in S. cerevisiae . Elg1, which was discovered during this screening, functions in DNA replication by participating in an alternative replication factor complex. Here we further characterize the GCR suppression mechanisms observed in the elg1 Δ mutant strain in conjunction with the telomere maintenance role of Elg1. The elg1 Δ mutation enhanced spontaneous DNA damage and resulted in GCR formation. However, DNA damage due to inactivation of Elg1 activates the intra-S checkpoints, which suppress further GCR formation. The intra-S checkpoints activated by the elg1 Δ mutation also suppress GCR formation in strains defective in the DNA replication checkpoint. Lastly, the elg1 Δ mutation increases telomere size independently of other previously known telomere maintenance proteins such as the telomerase inhibitor Pif1 or the telomere size regulator Rif1. The increase in telomere length caused by the elg1 Δ mutation was suppressed by a defect in the DNA replication checkpoint, which suggests that DNA replication surveillance by Dpb11-Mec1/Tel1-Dun1 also has an important role in telomere length regulation.

Different types of gross chromosomal rearrangements (GCRs), including translocations, interstitial deletions, terminal deletions with de novo telomere additions, and chromosome fusions, are observed in many cancers. Multiple pathways, such as S-phase checkpoints, DNA replication, recombination, chromatin remodeling, and telomere maintenance that suppress GCRs have been identified. To experimentally expand our knowledge of other pathway(s) that suppress GCRs, we developed a generally applicable genome-wide screening method. In this screen, we identified 10 genes ( ALO1, CDC50, CSM2, ELG1, ESC1, MMS4, RAD5, RAD18, TSA1 , and UFO1 ) that encode proteins functioning in the suppression of GCRs. Moreover, the breakpoint junctions of GCRs from these GCR mutator mutants were determined with modified breakpoint-mapping methods. We also identified nine genes ( AKR1, BFR1, HTZ1, IES6, NPL6, RPL13B, RPL27A, RPL35A , and SHU2 ) whose mutations generated growth defects with the pif1 Δ mutation. In addition, we found that some of these mutations changed the telomere size.

Increasing significance of tumor–stromal interaction in development and progression of cancer implies that signaling molecules in the tumor microenvironment (TME) might be the effective therapeutic targets for hepatocellular carcinoma (HCC). Here, the role of microRNA miR-199a-3p in the regulation of TME and development of HCC has been investigated by several in vitro and in vivo assays. Expression of miR-199a-3p was observed significantly low in HCC tissues and its overexpression remarkably inhibited in vivo tumor growth and metastasis to lung in NOD-SCID mice. In vitro restoration of miR-199a-3p expression either in endothelial cells (ECs) or in cancer cells (CACs) significantly diminished migration of ECs in co-culture assay. Again incubation of miR-199a-3p transfected ECs with either conditioned media (CM) of CACs or recombinant VEGF has reduced tube formation, in ECs and it was also dropped upon growth in CM of either anti-VEGF antibody-treated or miR-199a-3p-transfected CACs. In addition, bioinformatics and luciferase-reporter assays revealed that miR-199a-3p inhibited VEGF secretion from CACs and VEGFR1 and VEGFR2 expression on ECs and thus restricted cross talk between CACs and ECs. Again, restoration of miR-199a-3p in hepatic stellate cells (HSCs) reduced migration and invasion of CACs in co-culture assay, while it was enhanced by the overexpression of HGF suggesting miR-199a-3p has hindered HSC-CACs cross talk probably by inhibiting HGF and regulating matrix metalloproteinase MMP2, which were found as targets of miR-199a-3p subsequently by luciferase-reporter assay and gelatin zymography, respectively. Thus, these findings collectively highlight that miR-199a-3p restricts metastasis, invasion and angiogenesis in HCC and hence it may be considered as one of the powerful effective therapeutics for management of HCC patients.

Along with color loss, another severe problem of underwater optical imaging is Marine Snow effect which occurs because of back scattering from suspended organic detritus, solid particles or bubbles. Their appearance like tiny sparkling dots often reduces the scene perception and sometimes leads to spurious features on segmentation. This paper is concerned with removal of the marine snow effect from underwater images by a probabilistic approach considering the local statistics of luminance properties after a RGB to YCbCr transform.

Electron transfer (ET) reactions are important for their implications in both oxidative and reductive DNA damages. The current contribution investigates the efficacy of caffeine, a xanthine alkaloid in preventing UVA radiation induced ET from a carcinogen, benzo[a]pyrene (BP) to DNA by forming stable caffeine-BP complexes. While steady-state emission and absorption results emphasize the role of caffeine in hosting BP in aqueous medium, the molecular modeling studies propose the energetically favorable structure of caffeine-BP complex. The picosecond-resolved emission spectroscopic studies precisely explore the caffeine-mediated inhibition of ET from BP to DNA under UVA radiation. The potential therapeutic activity of caffeine in preventing DNA damage has been ensured by agarose gel electrophoresis. Furthermore, time-gated fluorescence microscopy has been used to monitor caffeine-mediated exclusion of BP from various cell lines including squamous epithelial cells, WI-38 (fibroblast), MCF-7 (breast cancer) and HeLa (cervical cancer) cells. Our in vitro and ex vivo experimental results provide imperative evidences about the role of caffeine in modified biomolecular recognition of a model carcinogen BP by DNA resulting dissociation of the carcinogen from various cell lines, implicating its potential medicinal applications in the prevention of other toxic organic molecule induced cellular damages.

Reactions involving electron transfer (ET) and reactive oxygen species (ROS) play a pivotal role in carcinogenesis and cancer biochemistry. Our present study emphasizes UVA radiation induced ET reaction as one of the key aspects of a potential carcinogen, benzo[a]pyrene (BP), in the presence of a wide variety of molecules covering organic p-benzoquinone (BQ), biological macromolecules like calf-thymus DNA (CT-DNA), human serum albumin (HSA) protein, and inorganic zinc oxide (ZnO) nanorods (NRs). Steady-state and picosecond-resolved fluorescence spectroscopy have been used to monitor such ET reactions. Physical consequences of BP association with CT-DNA have been investigated through temperature-dependent circular dichroism (CD) spectroscopy. The temperature-dependent steady-state, picosecond-resolved fluorescence lifetime and anisotropy studies reveal the effect of temperature on the perturbation of such ET reactions from BP to biological macromolecules, highlighting their temperature-dependent association. Furthermore, the electron-donating property of BP has been corroborated by measuring wavelength-dependent photocurrent in a BP-anchored ZnO NR-based photodevice, offering new physical insights for the carcinogenic study of BP.

We report a systematic investigation of caffeine induced dissociation of ethidium (Et) cation, a potential mutagen from nucleic acids and biomimetic systems. Time-resolved fluorescence studies are consistent with a mechanism where caffeine-Et complex formation in bulk solution drives the dissociation of DNA-bound Et. Caffeine induced extraction of Et from whole cells were also performed on squamous epithelial cells collected from the inner lining of the human mouth, A549 (lung carcinoma), A375 (human skin), RAW (macrophage) and Vero (African green monkey kidney epithelium) cell lines. Interestingly, the efficiency of caffeine in extracting Et has been found to be dependent on cell types. Our steady state and picosecond resolved spectroscopic studies on the detachment of Et from various biomimicking micelles of different charges reveal the specificity of caffeine molecule for carrying out such dissociation. The picosecond resolved Forster resonance energy transfer (FRET) studies between a DNA minor groove binder dye Hoeschst 33258 (H258, donor) and Et (acceptor) have been employed to investigate the alteration in their association in presence of caffeine in the molecular level. Finally, our fluorescence micrographs of epithelial cells validate the alteration of FRET efficiency between the donor and the acceptor due to the caffeine mediated release of the latter. Our results both in-vitro as well as ex-vivo provide important clues about efficiency and mechanism of caffeine as a potential anti-mutagenic therapeutic agent.

Picosecond-resolved Forster resonance energy transfer (FRET) from various vibronic bands in benzo[a] pyrene (BP) shows a strong dependency on the spectral overlap of an energy acceptor in a confined environment. Our study on the dipolar interactions between BP and different acceptors, including ethidium (Et), acridine orange (AO), and crystal violet (CV), at the surface of a model anionic micelle revealed that the Forster distance (R-0) and the rate of energy transfer is dependent on the individual spectral overlap of the vibronic bands of BP with the absorption spectra of the different energy acceptors. The differential behavior of the vibronic bands is compared with that of different dyes [quantum dots (QDs)] in a " dye-blend" (mixture) under FRET to an energy acceptor. Comparison of the FRET of the QDs with that of BP confirmed the independent nature of the dipolar interaction of the vibronic bands with other organic molecules, and the use of deconvolution techniques in the interpretation of the donor-acceptor (D-A) distance was also justified. We also showed that the consideration of differential FRET from the vibronic bands of BP and from the QDs in the dye-blend is equally acceptable in theoretical frameworks including the Infelta-Tachiya model and D-A distribution analysis in nanoenvironments. Refereed/Peer-reviewed

Genetic susceptibility is an important modifier of clinical outcome and natural history of progression in Alcoholic liver disease (ALD). While the significance of ethnicity in this evolution is very clear, subtle inter-individual genetic variant(s) might be important and thus we investigated those in an Indian population. Fourteen markers were genotyped within two alcohol metabolism genes [Alcohol dehydrogenase (ADH) gene clusters (ADH1B and ADH1C) and Aldehyde dehydrogenase (ALDH2)], one microsomal ethanol oxidizing enzyme cytochrome p450 (CYP2E1) and three oxidative stress response (OSR) genes (MnSOD, GSTT1 and GSTM1) among 490 Bengali individuals (322 ALD and 168 control) from Eastern and North-Eastern India and validation was performed in a new cohort of 150 Bengali patients including 100 ALD and 50 advanced non-alcoholic steatohepatitis (NASH). Out of 14 genetic variants, carriage of 5 genotypes (rs2066701CC in ADH1B, rs1693425TT in ADH1C, rs4880TT in MnSOD and GSTT1/GSTM1 null, p-value

Summary Genome diversity is a hallmark of hepatitis B virus (HBV), which allowed its classification into 10 genotypes (A–J) and numerous subgenotypes. Among them, Genotype D is currently segregated into eight subgenotypes (D1–D8). Here, we report the identification and characterization of a novel subgenotype within genotype D of HBV from chronic hepatitis B e antigen (HBeAg)-negative patients of Eastern India. Phylogenetic tree analysis based on complete genome sequences revealed that six of 39 HBV/D isolates formed a distinct cluster supported by high bootstrap value and had nucleotide divergence >4% relative to the known D subgenotypes (D1–D8), justifying their assignment into a new subgenotype (D9). By comparing the amino acid sequences of the four ORFs of HBV/D9 with D1–D8, 36 specific residues, including a unique one (E112 in the core region), were identified that could be considered as a signature of D9. Further analysis by Simplot, BootScan and jpHMM demonstrated that D9 resulted from a discrete recombination with genotype C over the precore–core region. This type of recombination has not been described previously as all C/D recombinants reported so far possessed genotype C backbones with mosaic fragments derived from HBV/D. Interestingly, compared to other subgenotypes of HBV/D, D9 isolates had a higher frequency of mutations (A1762T and G1764A) in the basal core promoter region that had been implicated in the development of hepatocellular carcinoma. Further investigations are needed to determine the overall prevalence and clinical significance of these newly characterized D9 strains and to assess the impact of inter-genotypic recombination on viral properties.

Video abstract Video, Stroke is a leading cause of death and disability worldwide. Stem cell therapy is an emerging therapeutic modality with evidence of significant benefits in preclinical stroke models. A number of phase I and II clinical trials have now been completed, with several more currently under way. Translation to the bedside, however, remains a long way off, and there are many questions that remain unanswered. This review will summarize the current evidence and ongoing clinical trials worldwide, and explore the challenges to making this a realistic treatment option for the future.

A 68-year-old man known to have non-insulin-dependant diabetes, hypertension and hypercholesterolemia presented with a 2-day history of abnormal sensation on the left side of his face and associated weakness of his right arm and leg. The symptoms presented with a sudden onset and had a stuttering course over the next 2 days at which point he presented to hospital. Initial examination revealed a left lower motor neurone VII nerve type weakness (Figure 1) with associated upper motor neurone type hemiparesis …

The self-assembly of non-toxic well-consumed small caffeine molecules into well-defined structures has important implications for future medical applications seeking to target the transport of small drugs in human body. Particularly, the solvation of the microenvironments of the self assembly ultimately dictates the interaction with the drug molecules and their therapeutic efficacy. We present femtosecond-resolved studies of the dynamics of aqueous solvation within self-assembled dimeric structure of caffeine molecules. We have placed small hydrophobic probes 4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl) 4H-pyran (DCM), coumarin 500 (C500) into the caffeine dimer to enable spectroscopic examinations of the interior. While molecular modeling and NMR studies of the probes in the caffeine dimers reveal a well-defined location (stacked in between two caffeine molecules), dynamical light scattering (DLS), Fourier transform infrared (FTIR) spectroscopy, densimetric and sonometric experiments explore the structural evolution of the dimer upon complexation with the probes. We have extended our studies in various temperatures in order to explore structural evolution of the self assembled structure and consequently the dynamics of solvation in the interior of the dimer. Picoseconds/femtosecond resolved dynamics and the polarization gated spectroscopic studies unravel the hydration and energetics associated with activated viscous flow of the confined probes. Our studies indicate that the interior of the caffeine dimer is well-solvated; however, the dynamics of solvation is retarted significantly compared to that in bulk water, clearly revealing the dimers maintain some ordered water molecules. We have also explored the consequence of the retarded dynamics of solvation on the photo-induced electron transfer (ET) reaction of a model probe, 2-(p-toluidino) naphthalene-6-sulfonate (TNS) encapsulated in the dimer.

Hepatitis B virus (HBV) strains isolated from members of the primitive Paharia ethnic community of Eastern India were studied to gain insight into the genetic diversity and evolution of the virus. The Paharia tribe has remained quite separate from the rest of the Indians and differs culturally, genetically, and linguistically from the mainstream East Indian population, whose HBV strains were previously characterized. Full-length HBV DNA was PCR amplified, cloned, and sequenced. Phylogenetic relationships between the tribal sequences and reference sequences from the mainstream population were assessed, and divergence times of subgenotypes of HBV genotype D were estimated. HBV was found in 2% of the Paharias participating in the study. A predominance of hepatitis B e antigen-negative infection (73%) was observed among the Paharias, and the genome sequences of the HBV strains exhibited relative homogeneity, with a very low prevalence of mutations. The novel feature of Paharia HBV was the exclusive presence of the D5 subgenotype, which was recently identified in Eastern India. Analysis of the four open reading frames (ORFs) of these tribal HBV D5 sequences and comparison with previously reported D1 to D7 sequences enabled the identification of 27 specific amino acid residues, including 6 unique ones, that could be considered D5 signatures. The estimated divergence times among subgenotypes D1 to D5 suggest that D5 was the first to diverge and hence is the most ancient of the D subgenotypes. The presence of a specific, ancient subgenotype of HBV within an ethnically primitive, endogamous population highlights the importance of studies of HBV genetics in well-separated human populations to understand viral transmission between communities and genome evolution.

Background: South Asians comprise the largest ethnic minority population in the UK. This subgroup is known to have an elevated risk of stroke. However, there is limited data on patterns of cerebrovascular disease and associated risk factors in this population. Aim: The aim of this study was to analyse differences in stroke subtype and risk factor profile between South Asian and White stroke patients admitted to a central London teaching hospital. Design : Prospective database of all admissions to the St Mary's Hospital stroke unit. Methods: We examined ethnicity, stroke subtype and risk factor profile of consecutive patients admitted to the stroke unit between 8 October 2003 and 14 February 2007. Results: A total of 811 patients were identified of whom 736 had strokes. Four hundred and ninety-six (67%) occurred in the White subgroup, and 72 (10%) in the Asian subgroup. The South Asian subgroup was significantly younger (65 vs. 73 years in the White subgroup; P < 0.001). They had higher rates of hypertension (age adjusted frequency 87% vs. 64%; P < 0.0001), diabetes (54% vs. 15%; P < 0.0001), and hyperlipidaemia (70% vs. 45%; P = 0.001). There were lower rates of smoking (15% vs. 33%; P < 0.0001).There was a trend towards more lacunar infarcts and less total anterior circulation infarcts in South Asians, although after age adjustment this was not significant at the 5% level. Conclusions: The South Asian subgroup has shown important differences in risk factor profile compared with the White population. The higher frequency of hypertension, diabetes and hyperlipidaemia seen in this subgroup are an important consideration in designing secondary prevention programmes tailored specifically to this community.

Gross chromosomal rearrangement (GCR) is a type of genomic instability associated with many cancers. In yeast, multiple pathways cooperate to suppress GCR. In a screen for genes that promote GCR, we identified MPH1, which encodes a 3′–5′ DNA helicase. Overexpression of Mph1p in yeast results in decreased efficiency of homologous recombination (HR) as well as delayed Rad51p recruitment to double-strand breaks (DSBs), which suggests that Mph1p promotes GCR by partially suppressing HR. A function for Mph1p in suppression of HR is further supported by the observation that deletion of both mph1 and srs2 synergistically sensitize cells to methyl methanesulfonate-induced DNA damage. The GCR-promoting activity of Mph1p appears to depend on its interaction with replication protein A (RPA). Consistent with this observation, excess Mph1p stabilizes RPA at DSBs. Furthermore, spontaneous RPA foci at DSBs are destabilized by the mph1Δ mutation. Therefore, Mph1p promotes GCR formation by partially suppressing HR, likely through its interaction with RPA.

Defects in DNA replication fidelity lead to genomic instability. Gross chromosomal rearrangement (GCR), a type of genomic instability, is highly enhanced by various initial mutations affecting DNA replication. Frequent observations of GCRs in many cancers strongly argue the importance of maintaining high fidelity of DNA replication to suppress carcinogenesis. Recent genome wide screens in Saccharomyces cerevisiae identified a new GCR suppressor gene, ELG1, enhanced level of genome instability gene 1. Its physical interaction with proliferating cell nuclear antigen (PCNA) and complex formation with Rfc2-5p proteins suggest that Elg1 functions to load/unload PCNA onto DNA during a certain DNA metabolism. High level of DNA damage accumulation and enhanced phenotypes with mutations in genes involved in cell cycle checkpoints, homologous recombination (HR), or chromatin assembly in the elg1 strain suggest that Elg1p-Rfc2-5p functions in a fundamental DNA metabolism to suppress genomic instability.

a) Absence of pathognomonic symptoms in patients with early phase of hepatocellular carcinoma (HCC) often leads to untreatable disease when diagnosed. Alpha-fetoprotein (AFP) with radiological images is the only potential HCC diagnosis option while disease prognosis remains dismal mandating necessity of biomarker identification with diagnostic, prognostic potentials as well as ability to assist in therapy. Thus this study aimed to identify non-invasive biomarker correlated with HCC pathogenesis by analyzing deregulated miRNAs in premalignant liver cirrhosis (LC) and in HCC. b) Among 148 study subjects, 117 were chronic Hepatitis B virus (HBV) (NAsymptomatic- control = 28, NLC = 30, NHCC = 59), 14 chronic Hepatitis C virus (HCV) infected (NLC = 7, NHCC = 7) and 17 were uninfected control. Differential expression profiling of miRNAs in each of 4 HBV infected LC and HCC tissues were performed by comparing with 8 asymptomatic controls using microarray and validated by qRT-PCR. R packages were used to determine area under receiver operating characteristics (AUROC) curve and other statistical analysis. Different softwares were used for prediction of target pathways associated with HCC development. c) Microarray analysis leads to identification of significantly altered [> or < 1.5 fold, p value d) In conclusion, combination of miR-126 with AFP may be a better predictor of HCC in high-risk chronic hepatitis patients and miR-126 alone can show better performance in detection of HCC with low AFP. Citation Format: Amit Ghosh, Alip Ghosh, Somenath Datta, Debanjali Dasgupta, Soumyajit Das, Sukanta Ray, Subash Gupta, Simanti Datta, Abhijit Chowdhury, Saroj Kant Mohapatra, Soma Banerjee. Hepatic microRNA as biomarker for detection of hepatocellular carcinoma in high-risk chronic Hepatitis B patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3969. doi:10.1158/1538-7445.AM2015-3969

Aim: To investigate the complex interplay between human papilloma virus (HPV) infection and p53 gene alteration in 92 head and neck squamous cell carcinoma (HNSCC) and 28 leukoplakia samples from eastern India. Methods: DNA isolated from the patient samples was subjected to HPV detection, loss of heterozygosity (LOH) analysis of the chromosome 17p region harbouring p53, genotyping at the p53 codon 72 locus and sequencing of the entire p53 gene to identify somatic mutations. Codon 72 heterozygotes carrying the p53 mutation were further cloned and resequenced to identify the allele harbouring the mutation. Results: HPV positivity in the HNSCC samples was 69%; 21% of the HNSCC were found to harbour p53 mutations in the coding region of the gene. The absence of the p53 mutation in HPV positive tumours was statistically significant compared to the HPV negative tumours (p = 0.01), but the same did not hold true for p53 LOH (p = 1.0). Among the germline p53 codon 72 heterozygotes, the Pro allele was preferentially lost (p = 0.02) while the Arg allele was mutated in the majority of cases. The risk of HPV mediated tumourigenesis increased with the increase in number of Arg alleles at the codon 72 locus. Conclusion: It is proposed that genetic and epigenetic alteration of p53 follow distinct pathways during the development of HNSCC from normal epithelium via dysplasia. The p53 mutation and HPV mediated p53 inactivation possibly constitute two independent pathways of tumourigenesis.

The development of effective clinical teams is vital to the delivery of quality clinical care and reducing clinical errors. Implementation of the European working-time directive will disrupt team dynamics and reduce the training opportunities for junior doctors. Funding is needed to increase further the number of doctors if the directive is not to be at the expense of patient care.

In our analysis, alterations in the P16 tumor suppressor gene were seen in 33% (15/46) of sampled uterine cervical lesions. Among the alterations, mutations in P16 were detected in 15% (7/46) of the samples. One mutation occurred at intron 1/exon 2 splice junction. All the other mutations were in exon 2 with three of them as silent mutations. The promoter hypermethylation and homozygous deletion of P16 gene were detected in 6.5% (3/46) and 8.7% (4/46) of the samples respectively. Loss of heterozygosity and microsatellite size alterations at the P16 locus were seen in 17% (8/46) of the samples. HPV16/18 infection was detected in 76% (35/46) of the samples. But no association was found between P16 alterations and HPV infection. Thus, it seems that P16 inactivation may be associated with the development of some uterine cervical carcinoma.

The aim of this study was to analyse the alterations of the genes in the CDKN2A/CCND1/CDK4/RB1 pathway in the G1-S phase of the cell cycle during development of head and neck squamous cell carcinoma (HNSCC). The alterations of these genes were analysed in 22 dysplastic lesions, 26 stage-I/II and 33 stage-III/IV HNSCC tumours of Indian patients. The alterations [mutation, hypermethylation, homozygous deletion and loss of heterozygosity/microsatellite size alteration (LOH/MA)] in the CDKN2A were found to be highest in 57% of the samples, followed by CCND1 amplification and LOH/MA at the RB1 locus in 14% and 8.5% of the samples, respectively. No dominant CDK4 Arg24Cys mutation was seen in our samples. Comparatively high frequency of CDKN2A alterations (except homozygous deletion) was found in dysplastic head and neck lesions and remained almost constant or increased during progression of the tumour, whereas the homozygous deletion of CDKN2A and the alterations in CCND1 and RB1 genes were seen mainly in the later stages of the tumour. Our study suggested that mutation/hypermethylation/allelic alterations (LOH/MA) of CDKN2A were associated with the development of dysplastic head and neck lesions. All the other alterations might provide some cumulative effect during progression of later stages of the tumour to have selective growth advantages.

The present study was designed to estimate zooplankton abundance qualitatively and quantitatively in different Fish-Livestock Integrated Farming Systems (FLIFS) over the Non Integrated Fish-Livestock Farming System (NIFLFS) in Terai region of West Bengal. Three treatments in triplicates for four consecutive years (2008–2011) were studied in Belacoba village of Jalpaiguri district involving nine pond of 0.01 hectare (ha) namely, NIFLFS (Control): The aquaculture was not integrated with the animal waste, FLIFS -I: Integration of cattle manure with aquaculture and FLIFS -II: FLIFS-I+ ducks grazing on the ponds. Zooplankton samples were collected bimonthly from the treated ponds for the analysis (qualitatively and quantitatively). Ponds under FLIFS-II and FLIFS-I were found to contain significantly higher concentration of zooplanktons (131±12 no l−1 and 128±11 no l−1, respectively) than NIFLFS (27 ± 2 no l−1). The identified zooplanktons were under 4 orders namely copepoda, rotifera, cladocera, and Diaptomus. Dominant groups of the zooplankton available in all the samples were observed to be Copepoda and Cladocera represented by Cyclops sp.and Daphnia sp., respectively. Total seven and six species were identified in the FLIFS-II and FLIFS-I, respectively in comparison to the four species in NIFLFS. In the present study the Daphnia was also significantly increased by 32.8% and 31.8% in FLIFS-I and FLIFS-II, respectively, where frequently manure was applied. Again Bosmina sp. was observed to be contributing in the FLIFS-II where ducks are grazing and the duck droppings are introduced in the ponds. Hence, it was concluded that utilization of cow dung and duck manure for aquaculture can successfully increase the availability and diversity of the natural food (zooplankton) to support the growing fishes under the integrated fish farming systems followed in the terai region of West Bengal.

The antineoplastic effect of Trigonella foenum graecum seed extract has been evaluated in the Ehrlich ascites carcinoma (EAC) model in Balb-C mice. Intra-peritoneal administration of the alcohol extract of the seed both before and after inoculation of EAC cell in mice produced more than 70% inhibition of tumour cell growth with respect to the control. Treatment with the extract was found to enhance both the peritoneal exudate cell and macrophage cell counts. The extract also produced a significant antiinflammatory effect. We report here the antiinflammatory and antineoplastic effects, of Trigonella foenum graecum seed extract.

The growth of a solid tumor depends on diffusion of nutrients from the tumor microenvironment through vascular system. Angiogenesis, the new blood vessels formation is the primary route by which tumor cells get nutrients as well as exit from primary site and enter into the blood circulation. The proliferation and migration of vascular endothelial cells are triggered by angiogenic growth factors secreted by tumor cells, such as VEGF, TGF-β etc. Recent evidences showed HGF also plays an important role in angiogenesis by enhancing the proliferative activity and intracellular signaling. HGF/its receptor, c-MET and VEGF/VEGFR1 are two independent pathways for angiogenesis and metastasis. Thus detection of a molecule that hits both pathways could be a potent therapeutic agent that could facilitate the inhibition of improved barrier function. Recently mir199a* has been shown to target both CD44 and cMET molecule to block the metastasis in cancer. The present study aim to show that mir199a* also target HGF, the key modulator of the HGF/c-Met signaling cascade in epithelial cells to shut off the mesenchymal transition and VEGFR1 to prevent angiogenesis more effectively. Luciferase 3’ UTR assay was performed to investigate HGF as a target of mir-199a*. HGF mRNA and protein expression was determined by real time RT-PCR, Western blot analysis in liver stellate cell line LX2 transfected with mir-199a* and HGF shRNA. The cultured media of mir-199a* transfected LX2 cells was used to verify the effect of mir-199a* on HGF induced migration and invasion ability on metastatic cell lines using Boyden chamber assay with or without matrigel coated membrane and angiogenesis by tube formation assay. The mir-199a* binding site was detected by pictar, target scan analysis of the 3’UTR of HGF and VEGFRs. The binding of mir-199a* to the 3′UTR of HGF and VEGFR1 were confirmed by luciferase assay, mutagenesis and western blotting. Down regulation of HGF protein expression in HGF producing hepatic stellate cell, LX2 was observed by mir-199a*. Addition of LX2 conditioned media transfected with mir199a* to metastatic cell reduces the migration and invasion and also block angiogenesis in endothelial cells. As Mir-199a* could target HGF/cMET, CD44-cMET and VEGF/VEGFR1 signaling cascade, it could be used as a potential therapeutic target of HCC. Citation Format: Alip Ghosh, Amit Ghosh, Debanjali Dasgupta, Shrabasti Roychowdhury, Shrabasti Roychowdhury, Simanti Datta, Abhijit Chowdhury, Soma Banerjee. MicroRNA 199a*: a potent suppressor of tumor metastasis and angiogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2306. doi:10.1158/1538-7445.AM2013-2306