The present study evaluated the ability of methanolic extract of Centella asiatica (Linn) Urban (Umbelliferae) to induce apoptosis in different cancer cell lines. MCF-7 cells emerged as the most sensitive cell line for in vitro growth inhibitory activity. C. asiatica extract induced apoptosis in MCF-7 cells as indicated by nuclear condensation, increased annexin staining, loss of mitochondrial membrane potential and induction of DNA breaks identified by TUNEL reactivity. It is possible that the use of C. asiatica extract as a component in herbal medicines could be justifiable.
Apoptosis; Cancer; Centella asiatica
Current guidelines for the conduct of cost-effectiveness analysis (CEA) are mainly applicable to facility-based interventions in high-income settings. Differences in the unit of analysis and the high cost of data collection can make these guidelines challenging to follow within public health trials in low- and middle- income settings.
This paper reflects on the challenges experienced within our own work and proposes solutions that may be useful to others attempting to collect, analyse, and compare cost data between public health research sites in low- and middle-income countries.
We describe the generally accepted methods (norms) for collecting and analysing cost data in a single-site trial from the provider perspective. We then describe our own experience applying these methods within eight comparable cluster randomised, controlled, trials. We describe the strategies used to maximise adherence to the norm, highlight ways in which we deviated from the norm, and reflect on the learning and limitations that resulted.
When the expenses incurred by a number of small research sites are used to estimate the cost-effectiveness of delivering an intervention on a national scale, then deciding which expenses constitute ‘start-up’ costs will be a nontrivial decision that may differ among sites. Similarly, the decision to include or exclude research or monitoring and evaluation costs can have a significant impact on the findings. We separated out research costs and argued that monitoring and evaluation costs should be reported as part of the total trial cost. The human resource constraints that we experienced are also likely to be common to other trials. As we did not have an economist in each site, we collaborated with key personnel at each site who were trained to use a standardised cost collection tool. This approach both accommodated our resource constraints and served as a knowledge sharing and capacity building process within the research teams.
Given the practical reality of conducting randomised, controlled trials of public health interventions in low- and middle- income countries, it is not always possible to adhere to prescribed guidelines for the analysis of cost effectiveness. Compromises are frequently required as researchers seek a pragmatic balance between rigor and feasibility. There is no single solution to this tension but researchers are encouraged to be mindful of the limitations that accompany compromise, whilst being reassured that meaningful analyses can still be conducted with the resulting data.
cost-effectiveness analysis; cost data; LMIC; multisite; randomised control trials
Compensatory endocytosis of exocytosed membrane and recycling of synaptic vesicle components is essential for sustained synaptic transmission at nerve terminals. At the ribbon-type synapse of retinal bipolar cells, manipulations expected to inhibit the interactions of the clathrin adaptor protein complex (AP2) affect only the slow phase of endocytosis (τ = 10–15 s), leading to the conclusion that fast endocytosis (τ = 1–2 s) occurs by a mechanism that differs from the classical pathway of clathrin-coated vesicle retrieval from the plasma membrane. Here we investigate the role of endophilin in endocytosis at this ribbon synapse. Endophilin A1 is a synaptically enriched N-BAR domain-containing protein, suggested to function in clathrin-mediated endocytosis. Internal dialysis of the synaptic terminal with dominant-negative endophilin A1 lacking its linker and Src homology 3 (SH3) domain inhibited the fast mode of endocytosis, while slow endocytosis continued. Dialysis of a peptide that binds endophilin SH3 domain also decreased fast retrieval. Electron microscopy indicated that fast endocytosis occurred by retrieval of small vesicles in most instances. These results indicate that endophilin is involved in fast retrieval of synaptic vesicles occurring by a mechanism that can be distinguished from the classical pathway involving clathrin-AP2 interactions.
We previously reported a 90% decreased risk in major depression, assessed prospectively, in adult offspring of depressed probands who reported that religion or spirituality was highly important to them. Frequency of church attendance was not significantly related to depression risk. Our previous brain imaging findings in adult offspring in these high-risk families also revealed large expanses of cortical thinning across the lateral surface of the right cerebral hemisphere.
To determine whether high-risk adults who reported high importance of religion or spirituality had thicker cortices than those who reported moderate or low importance of religion or spirituality and whether this effect varied by family risk status.
DESIGN, SETTING, AND PARTICIPANTS
Longitudinal, retrospective cohort, familial study of 103 adults (aged 18–54 years) who were the second- or third-generation offspring of depressed (high familial risk) or nondepressed (low familiar risk) probands (first generation). Religious or spiritual importance and church attendance were assessed at 2 time points during 5 years, and cortical thickness was measured on anatomical images of the brain acquired with magnetic resonance imaging at the second time point.
MAIN OUTCOMES AND MEASURES
Cortical thickness in the parietal regions by risk status.
Importance of religion or spirituality, but not frequency of attendance, was associated with thicker cortices in the left and right parietal and occipital regions, the mesial frontal lobe of the right hemisphere, and the cuneus and precuneus in the left hemisphere, independent of familial risk. In addition, the effects of importance on cortical thickness were significantly stronger in the high-risk than in the low-risk group, particularly along the mesial wall of the left hemisphere, in the same region where we previously reported a significant thinner cortex associated with a familial risk of developing depressive illness. We note that these findings are correlational and therefore do not prove a causal association between importance and cortical thickness.
CONCLUSIONS AND RELEVANCE
A thicker cortex associated with a high importance of religion or spirituality may confer resilience to the development of depressive illness in individuals at high familial risk for major depression, possibly by expanding a cortical reserve that counters to some extent the vulnerability that cortical thinning poses for developing familial depressive illness.
The prevalence of diarrheagenic Escherichia coli (DEC) in children under 5 years was studied in children with diarrhea and controls in South India. Four polymerase chain reaction (PCR) “schemes” were used to detect genes of the 6 pathotypes of DEC. In 394 children with diarrhea, 203 (52%) DEC infections were found. Among the 198 controls, 126 (63%) DEC infections were found. Enteroaggregative E. coli was the most common pathotype by multiplex PCR both in cases (58, 14.7%) and controls (47, 23.7%), followed by enteropathogenic E. coli seen in 10% cases and 8% of controls. Enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC), and diffusely adherent E. coli (DAEC) were found in 4.1%, 2.0%, 1.0%, and 0.5% of cases, respectively. ETEC was found in 2.5% of controls, but EHEC, EIEC, and DAEC were not detected. Overall, no single assay worked well, but by discounting genes with a pathogenicity index of less than 1, it was possible to use the PCR assays to identify DEC in 75/394 (19%) cases and 12/198 (6.1%) controls, while mixed infection could be identified in 8/394 (2%) cases and 2/198 (1%) controls.
Diarrheagenic E. coli; Multiplex PCR; Virulence genes; Adherence assays
Currently, the discovery of effective chemotherapeutic agents poses a major challenge to the field of cancer biology. The present study focuses on enhancing the therapeutic and anti cancer properties of atorvastatin calcium loaded BSA (ATV-BSA) nanoparticles in vitro.
BSA-ATV nanoparticles were prepared using desolvation technique. The process parameters were optimized based on the amount of desolvating agent, stabilization conditions as well as the concentration of the cross linker. The anti cancer properties of the protein coated ATV nanoparticles were tested on MiaPaCa-2 cell lines. In vitro release behavior of the drug from the carrier suggests that about 85% of the drug gets released after 72 hrs. Our studies show that ATV-BSA nanoparticles showed specific targeting and enhanced cytotoxicity to MiaPaCa-2 cells when compared to the bare ATV.
We hereby propose that the possible mechanism of cellular uptake of albumin bound ATV could be through caveolin mediated endocytosis. Hence our studies open up new facet for an existing cholesterol drug as a potent anti-cancer agent.
The majority of patients with acute myeloid leukemia (AML) are elderly and have a poor prognosis despite induction therapy. Decitabine, a DNA-hypomethylating agent that induces differentiation and apoptosis of leukemic cells, is a well-tolerated alternative to aggressive chemotherapy. It is currently FDA-approved for myelodysplastic syndrome, including patients with 20%–30% bone marrow blasts. Recent clinical attention has focused on evaluating decitabine as frontline therapy for untreated high-risk elderly AML patients. A large randomized international phase III study comparing decitabine to supportive care and cytarabine in elderly AML patients demonstrated significantly improved complete remission rates, but the survival difference did not reach significance. Due to this, decitabine did not achieve FDA approval for AML, but continues to be used off-label. Current research is focused on further defining subgroups of elderly AML patients who may derive greater benefit from decitabine therapy and combining it with other low-intensity active agents for AML.
decitabine; elderly; AML
Saturated fatty acids, such as palmitic and stearic acid, cause detrimental effects in endothelial cells (ECs) and have been suggested to contribute to macrophage accumulation in adipose tissue and the vascular wall in states of obesity and insulin resistance. Long-chain fatty acids are believed to require conversion into acyl-CoA derivatives to exert most of their detrimental effects, a reaction catalyzed by acyl-CoA synthetases (ACSL). The objective of this study was to investigate the role of ACSL1, an ACSL isoform previously shown to mediate inflammatory effects in myeloid cells, in regulating EC responses to a saturated fatty acid-rich environment in vitro and in vivo.
Methods and Results
Saturated fatty acids caused increased inflammatory activation, ER stress, and apoptosis in mouse microvascular ECs. Forced ACSL1 overexpression exacerbated the effects of saturated fatty acids on apoptosis and ER stress. However, endothelial ACSL1-deficiency did not protect against the effects of saturated fatty acids in vitro, nor did it protect insulin resistant mice fed a saturated fatty acid-rich diet from macrophage adipose tissue accumulation or increased aortic adhesion molecule expression.
Endothelial ACSL1 is not required for inflammatory and apoptotic effects of a saturated fatty acid-rich environment.
Acyl-CoA synthetase; Apoptosis; Endothelium; Inflammation
Human T lymphotropic virus type 1 (HTLV-1) mainly causes adult T cell leukemia and predominantly immortalizes/transforms CD4+ T cells in culture. HTLV-2 is aleukemic and predominantly immortalizes/transforms CD8+ T cells in culture. We have shown previously that the viral envelope is the genetic determinant of the differential T cell tropism in culture. The surface component (SU) of the HTLV-1 envelope is responsible for binding to the cellular receptors for entry. Here, we dissect the HTLV-1 SU further to identify key domains that are involved in determining the immortalization tropism. We generated HTLV-1 envelope recombinant virus containing the HTLV-2 SU domain. HTLV-1/SU2 was capable of infecting and immortalizing freshly isolated peripheral blood mononuclear cells in culture. HTLV-1/SU2 shifted the CD4+ T cell immortalization tropism of wild-type HTLV-1 (wtHTLV-1) to a CD8+ T cell preference. Furthermore, a single amino acid substitution, N195D, in HTLV-1 SU (Ach.195) resulted in a shift to a CD8+ T cell immortalization tropism preference. Longitudinal phenotyping analyses of the in vitro transformation process revealed that CD4+ T cells emerged as the predominant population by week 5 in wtHTLV-1 cultures, while CD8+ T cells emerged as the predominant population by weeks 4 and 7 in wtHTLV-2 and Ach.195 cultures, respectively. Our results indicate that SU domain independently influences the preferential T cell immortalization tropism irrespective of the envelope counterpart transmembrane (TM) domain. We further showed that asparagine at position 195 in HTLV-1 SU is involved in determining this CD4+ T cell immortalization tropism. The slower emergence of the CD8+ T cell predominance in Ach.195-infected cultures suggests that other residues/domains contribute to this tropism preference.
Various neuroimaging measures are being evaluated for tracking Alzheimer’s disease (AD) progression in therapeutic trials, including measures of structural brain change based on repeated scanning of patients with magnetic resonance imaging (MRI). Methods to compute brain change must be robust to scan quality. Biases may arise if any scans are thrown out, as this can lead to the true changes being overestimated or underestimated. Here we analyzed the full MRI dataset from the first phase of Alzheimer’s Disease Neuroimaging Initiative (ADNI-1) from the first phase of Alzheimer’s Disease Neuroimaging Initiative (ADNI-1) and assessed several sources of bias that can arise when tracking brain changes with structural brain imaging methods, as part of a pipeline for tensor-based morphometry (TBM). In all healthy subjects who completed MRI scanning at screening, 6, 12, and 24 months, brain atrophy was essentially linear with no detectable bias in longitudinal measures. In power analyses for clinical trials based on these change measures, only 39 AD patients and 95 mild cognitive impairment (MCI) subjects were needed for a 24-month trial to detect a 25% reduction in the average rate of change using a two-sided test (α=0.05, power=80%). Further sample size reductions were achieved by stratifying the data into Apolipoprotein E (ApoE) ε4 carriers versus non-carriers. We show how selective data exclusion affects sample size estimates, motivating an objective comparison of different analysis techniques based on statistical power and robustness. TBM is an unbiased, robust, high-throughput imaging surrogate marker for large, multi-site neuroimaging studies and clinical trials of AD and MCI.
Alzheimer’s disease; Mild cognitive impairment; Aging; ADNI; Tensor-based morphometry; Drug trial
The objective of this in-vitro study was to evaluate the effect of grape seed extract (oligomericproanthocyanidin complexes [OPCs]) on the bond strength of composite resin to bleached enamel using 5th and 7th generations of bonding agents.
Materials & Methods:
Eighty maxillary central incisors were randomly assigned to four groups as follows: Group I (n=20): no bleaching (control); Group II (n=20): bleaching with 38% hydrogen peroxide gel, without the use of an antioxidant; Group III (n=20): bleaching followed by the use of 10% sodium ascorbate; Group IV (n=20) : bleaching followed by the use of 5% proanthocyanidin solution. All the four groups were further subdivided into two groups of ten teeth each depending on the adhesive system used in order to bond the resin composite to enamel surfaces. Subgroup A: 5th generation bonding agent and subgroup B : 7th generation bonding agent. Shear bond strength of the specimens was tested under universal testing machine.
The shear bond strength values were observed to be significantly higher in teeth treated with 10% sodium ascorbate and 5% proanthocyanidin as compared to the group in which no antioxidant was used. Also, bonding with 5th generation bonding agent resulted in significantly higher shear bond strength when compared to the 7th generation bonding agent.
The use of grape seed extract as an antioxidant after bleaching significantly improves the bond strength of composite resin to bleached enamel. 5th generation bonding agents have higher shear bond strength of composite resin to enamel.
How to cite this article:
Abraham S, Ghonmode WN, Saujanya KP, Jaju N, Tambe VH, Yawalikar PP. Effect of grape seed extract on bond strength of bleached enamel using fifth and seventh generation bonding agents.
J Int Oral Health 2013; 5(6):101-7
Antioxidants; bond strength; bonding agents
Background: Abnormal uterine bleeding is the Common presenting complaint in Gynaecology Outpatient Department in all age groups. It is due to the anovulatory cycles which are commonly seen in adolescent and peri-menopausal women. Abnormal uterine bleeding is caused by wide variety of organic or non-organic causes. Histopathological examination of endometrial sample remains the gold standard for diagnosis of endometrial pathology.
Aim: To study the clinicopathological spectrum of endometrium in abnormal uterine bleeding in peri-menopausal and post-menopausal age groups.
Material and Methods: The study included prospective analysis of 119 cases of endometrial samples in patients of abnormal uterine bleeding above 40 years of age. The specimens were routinely processed and H&E stained slides were studied. Patients were categorized into peri-menopausal (40-49 years) and post-menopausal (> 50 years) age group.
Results: A total of 119 specimens of endometrium were analyzed. Maximum number (73.94%) of cases were from peri-menopausal age group. The most common presenting complaint was menorrhagia (48.86%) followed by post-menopausal bleeding (26.05%). In peri-menopausal age group proliferative endometrium (35.22%) was the predominant histopathological pattern followed by endometrial hyperplasia (23.86%). Atrophic endometrium (25.80%) was the most frequent finding followed by endometrial hyperplasia (19.35%) in post-menopausal age group. Three cases of endometrial carcinoma were reported in post-menopausal age group only.
Conclusion: A thorough histopathological work up and clinical correlation is mandatory in cases of abnormal uterine bleeding above the age of 40 years to find out organic lesions. Careful screening can detect early cancer of endometrium which has excellent prognosis and it will help in further management.
Abnormal uterine bleeding; Peri-menopausal; Post-menopausal
Steroid induced skin atrophy is the most frequent and perhaps most important cutaneous side effect of topical glucocorticoid therapy. To date, it has not been described in vulvar skin. We describe a patient with significant vulvar skin atrophy following prolonged steroid application to treat vulvar dermatitis. The extensive atrophy in the perineum resulted in secondary ‘webbing’ and partial obstruction of genital hiatus and superimposed dyspareunia. Prolonged topical steroids may result in atrophic changes in vulvar skin. Therefore, further research in clinical correlates of steroid-induced atrophy in the vulvar region is warranted.
Mortality prediction models generally require clinical data or are derived from information coded at discharge, limiting adjustment for presenting severity of illness in observational studies using administrative data.
To develop and validate a mortality prediction model using administrative data available in the first 2 hospital days.
After dividing the dataset into derivation and validation sets, we created a hierarchical generalized linear mortality model that included patient demographics, comorbidities, medications, therapies, and diagnostic tests administered in the first 2 hospital days. We then applied the model to the validation set.
Patients aged ≥18 years admitted with pneumonia between July 2007 and June 2010 to 347 hospitals in Premier, Inc.’s Perspective database.
In hospital mortality.
The derivation cohort included 200,870 patients and the validation cohort had 50,037. Mortality was 7.2%. In the multivariable model, 3 demographic factors, 25 comorbidities, 41 medications, 7 diagnostic tests, and 9 treatments were associated with mortality. Factors that were most strongly associated with mortality included receipt of vasopressors, non-invasive ventilation, and bicarbonate. The model had a c-statistic of 0.85 in both cohorts. In the validation cohort, deciles of predicted risk ranged from 0.3% to 34.3% with observed risk over the same deciles from 0.1% to 33.7%.
A mortality model based on detailed administrative data available in the first 2 hospital days had good discrimination and calibration. The model compares favorably to clinically based prediction models and may be useful in observational studies when clinical data are not available.
The optimization of a purely negative depletion, enrichment process for circulating tumor cells, CTC's, in the peripheral blood of Head and Neck cancer patients is presented. The enrichment process uses a red cell lysis step followed by immunomagnetic labeling, and subsequent depletion, of CD45 positive cells. A number of relevant variables are quantified, or attempted to be quantified, which control the performance of the enrichment process. Six different immunomagnetic labeling combinations were evaluated as well as the significant difference in performance with respect to the blood source: buffy coats purchased from the Red Cross, fresh, peripheral blood from normal donors, and fresh peripheral blood from human cancer patients. After optimization, the process is able to reduce the number of normal blood cells in a cancer patient's blood from 4.05 × 109 to 8.04 × 103 cells/ml and still recover, on average, 2.32 CTC per ml of blood. For all of the cancer patient blood samples tested in which CTC were detected (20 out of 26 patients) the average recovery of CTCs was 21.7 per ml of blood, with a range of 282 to 0.53 CTC per ml of blood. Unlike a majority of other published studies, this study focused on quantifying as many factors as possible to facilitate both the optimization of the process as well as provide information for future performance comparisons. The authors are not aware any other reported study which has achieved the performance reported here (a 5.76 log10) in a purely negative enrichment mode of operation. Such a mode of operation of an enrichment process provides significant flexibility in that it has no bias with respect to what attributes define a CTC; thereby allowing the researcher or clinician to use any maker they choose to define whether the final, enrich product contains CTC's or other cell type relevant to the specific question (i.e. does the CTC have predominately epithelia or mesenchymal characteristics?).
Circulating tumor cells; Immunomagnetic cell separation; Immunocytochemistry; RT-PCR; Squamous cell carcinoma of the head and neck (HNSCC)
Socioeconomic status (SES) is an important determinant of health, but SES measures are frequently unavailable in commonly used datasets. Area-level SES measures are used as proxy measures of individual SES when the individual measures are lacking. Little is known about the agreement between individual-level versus area-level SES measures in mixed urban–rural settings.
We identified SES agreement by comparing information from telephone self-reported SES levels and SES calculated from area-level SES measures. We assessed the impact of this agreement on reported associations between SES and rates of childhood obesity, low birth weight <2500 g and smoking within the household in a mixed urban–rural setting.
750 households were surveyed with a response rate of 62%: 51% male, 89% Caucasian; mean child age 9.5 years. Individual-level self-reported income was more strongly associated with all three childhood health outcomes compared to area-level SES. We found significant disagreement rates of 22–31%. The weighted Cohen’s κ indices ranged from 0.15 to 0.22, suggesting poor agreement between individual-level and area-level measures.
In a mixed urban–rural setting comprised of both rural and urbanised areas, area-level SES proxy measures significantly disagree with individual SES measures, and have different patterns of association with health outcomes from individual-level SES measures. Area-level SES may be an unsuitable proxy for SES when individual rather than community characteristics are of primary concern.
Leptin, a hormone produced by body fat tissue, acts on hypothalamic receptors in the brain to regulate appetite and energy expenditure, and on neurons in the arcuate nucleus to signal that a individual has had enough to eat. Leptin enters the central nervous system at levels that depend on a individual’s body fat. Obese people, on average, show greater brain atrophy in old age, so it is valuable to know whether brain atrophy relates to leptin levels, which can be targeted by interventions. We therefore determined how plasma leptin levels, and BMI, relate to brain structure, and whether leptin levels might account for BMI’s effect on the brain. We measured regional brain volumes using tensor-based morphometry, in MRI scans of 517 elderly individuals with plasma leptin measured (mean: 13.3±0.6 ng/ml; mean age: 75.2±7.3 years; 321 men/196 women). We related plasma leptin levels to brain volumes at every location in the brain after adjusting for age, sex, and diagnosis and, later, also BMI. Plasma leptin levels were significantly higher (a) in women than men, and (b) in obese versus overweight, normal or underweight individuals. People with higher leptin levels showed deficits in frontal, parietal, temporal and occipital lobes, brainstem, and the cerebellum, irrespective of age, sex, or diagnosis. These associations persisted after controlling for BMI. Greater brain atrophy may occur in people with central leptin insufficiency, a marker of obesity. Therapeutic manipulation of leptin may be a promising direction for slowing brain decline.
Alzheimer’s disease; BMI; brain structure; leptin; MRI; obesity
There is considerable interest in defining new agents or targets for antithrombotic purposes. The 5-HT2A receptor is a G-protein coupled receptor (GPCR) expressed on many cell types, and a known therapeutic target for many disease states. This serotonin receptor is also known to regulate platelet function. Thus, in our FDA-approved drug repurposing efforts, we investigated the antiplatelet activity of cyproheptadine and pizotifen, two antidepressant 5-HT2A Receptor antagonists. Our results revealed that cyproheptadine and pizotifen reversed serotonin-enhanced ADP-induced platelet aggregation in vitro and ex vivo. And the inhibitory effects of these two agents were found to be similar to that of EMD 281014, a 5-HT2A Receptor antagonist under development. In separate experiments, our studies revealed that these 5-HT2A receptor antagonists have the capacity to reduce serotonin-enhanced ADP-induced elevation in intracellular calcium levels and tyrosine phosphorylation. Using flow cytometry, we also observed that cyproheptadine, pizotifen, and EMD 281014 inhibited serotonin-enhanced ADP-induced phosphatidylserine (PS) exposure, P-selectin expression, and glycoprotein IIb-IIIa activation. Furthermore, using a carotid artery thrombosis model, these agents prolonged the time for thrombotic occlusion in mice in vivo. Finally, the tail-bleeding time was investigated to assess the effect of cyproheptadine and pizotifen on hemostasis. Our findings indicated prolonged bleeding time in both cyproheptadine- and pizotifen-treated mice. Notably, the increases in occlusion and bleeding times associated with these two agents were comparable to that of EMD 281014, and to clopidogrel, a commonly used antiplatelet drug, again, in a fashion comparable to clopidogrel and EMD 281014. Collectively, our data indicate that the antidepressant 5-HT2A antagonists, cyproheptadine and pizotifen do exert antiplatelet and thromboprotective effects, but similar to clopidogrel and EMD 281014, their use may interfere with normal hemostasis.
A 55-year-old lady was admitted following a concern raised by family members who had noticed a change in behaviour in terms of declining mood, paranoia with expression of belief that she was being bugged, also reported smelling perfume and after shave lotion. She had a prior diagnosis of bipolar mood disorder and was on lithium but remained no-compliant with her prescribed medication in the 10 weeks before admission. Upon admission, necessary investigations were performed including CT scan and EEG. Her CT was normal but EEG was grossly abnormal. Neurology consultation was sought and a sleep deprived EEG and MRI was ordered. Meanwhile, carbamezipine was commenced in view of the change in diagnosis to that of temporal lobe epilepsy. The patient responded very well to this regime and improved in all spheres. She was finally discharged with follow-up appointments with both neurology and psychiatry clinics.
Productive herpesvirus infection requires a profound, time-controlled remodeling of the viral transcriptome and proteome. To gain insights into the genomic architecture and gene expression control in Kaposi's sarcoma-associated herpesvirus (KSHV), we performed a systematic genome-wide survey of viral transcriptional and translational activity throughout the lytic cycle. Using mRNA-sequencing and ribosome profiling, we found that transcripts encoding lytic genes are promptly bound by ribosomes upon lytic reactivation, suggesting their regulation is mainly transcriptional. Our approach also uncovered new genomic features such as ribosome occupancy of viral non-coding RNAs, numerous upstream and small open reading frames (ORFs), and unusual strategies to expand the virus coding repertoire that include alternative splicing, dynamic viral mRNA editing, and the use of alternative translation initiation codons. Furthermore, we provide a refined and expanded annotation of transcription start sites, polyadenylation sites, splice junctions, and initiation/termination codons of known and new viral features in the KSHV genomic space which we have termed KSHV 2.0. Our results represent a comprehensive genome-scale image of gene regulation during lytic KSHV infection that substantially expands our understanding of the genomic architecture and coding capacity of the virus.
Kaposi's sarcoma-associated herpesvirus (KSHV) is a cancer-causing agent in immunocompromised patients that establishes long-lasting infections in its hosts. Initially described in 1994 and extensively studied ever since, KSHV molecular biology is understood in broad outline, but many detailed questions are still to be resolved. After almost two decades, specific aspects pertaining to the organization of the KSHV genome as well as the fate of the viral transcripts during the productive stages of infection remain unexplored. Here we use a systematic genome-wide approach to investigate changes in gene and protein expression during the productive stage of infection known as the lytic cycle. We found that the viral genome has a large coding capacity, capable of generating at least 45% more products than initially anticipated by bioinformatic analyses alone, and that it uses multiple strategies to expand its coding capacity well beyond what is determined solely by the DNA sequence of its genome. We also provide an expanded and highly detailed annotation of known and new genomic features in KSHV. We have termed this new architectural and functional annotation KSHV 2.0. Our results indicate that viral genomes are more complex than anticipated, and that they are subject to tight mechanisms of regulation to ensure correct gene expression.
Telomeric 3′ overhangs can fold into a four-stranded DNA structure termed G-quadruplex (G4), a formation which inhibits telomerase. As telomerase activation is crucial for telomere maintenance in most cancer cells, several classes of G4 ligands have been designed to directly disrupt telomeric structure.
We exposed brain tumor cells to the G4 ligand 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4) and investigated proliferation, cell cycle dynamics, telomere length, telomerase activity and activated c-Myc levels.
Although all cell lines tested were sensitive to RHPS4, PFSK-1 central nervous system primitive neuroectodermal cells, DAOY medulloblastoma cells and U87 glioblastoma cells exhibited up to 30-fold increased sensitivity compared to KNS42 glioblastoma, C6 glioma and Res196 ependymoma cells. An increased proportion of S-phase cells were observed in medulloblastoma and high grade glioma cells whilst CNS PNET cells showed an increased proportion of G1-phase cells. RHPS4-induced phenotypes were concomitant with telomerase inhibition, manifested in a telomere length-independent manner and not associated with activated c-Myc levels. However, anti-proliferative effects were also observed in normal neural/endothelial cells in vitro and ex vivo.
This study warrants in vivo validation of RHPS4 and alternative G4 ligands as potential anti-cancer agents for brain tumors but highlights the consideration of dose-limiting tissue toxicities.
The genetic structure of the indigenous hunter-gatherer peoples of southern Africa, the oldest known lineage of modern human, is important for understanding human diversity. Studies based on mitochondrial1 and small sets of nuclear markers2 have shown that these hunter-gatherers, known as Khoisan, San, or Bushmen, are genetically divergent from other humans1,3. However, until now, fully sequenced human genomes have been limited to recently diverged populations4–8. Here we present the complete genome sequences of an indigenous hunter-gatherer from the Kalahari Desert and a Bantu from southern Africa, as well as protein-coding regions from an additional three hunter-gatherers from disparate regions of the Kalahari. We characterize the extent of whole-genome and exome diversity among the five men, reporting 1.3 million novel DNA differences genome-wide, including 13,146 novel amino acid variants. In terms of nucleotide substitutions, the Bushmen seem to be, on average, more different from each other than, for example, a European and an Asian. Observed genomic differences between the hunter-gatherers and others may help to pinpoint genetic adaptations to an agricultural lifestyle. Adding the described variants to current databases will facilitate inclusion of southern Africans in medical research efforts, particularly when family and medical histories can be correlated with genome-wide data.
Freezing of gait (FOG) is an elusive phenomenon that debilitates a large number of Parkinson’s disease (PD) patients regardless of stage of disease, medication status, or deep brain stimulation implantation. Sensory feedback cues, especially visual feedback cues, have been shown to alleviate FOG episodes or even prevent episodes from occurring. Here, we examine cortical information flow between occipital, parietal, and motor areas during the pre-movement stage of gait in a PD-with-FOG patient that had a strong positive behavioral response to visual cues, one PD-with-FOG patient without any behavioral response to visual cues, and age-matched healthy controls, before and after training with visual feedback. Results for this case study show differences in cortical information flow between the responding PD-with-FOG patient and the other two subject types, notably, an increased information flow in the beta range. Tentatively suggesting the formation of an alternative cortical sensory-motor pathway during training with visual feedback, these results are proposed as subject for further verification employing larger cohorts of patients.
FOG; visual feedback; occipital lobe; parkinsonian disorders; EEG-fMRI
We report the 3.98-Mbp first draft genome sequence of Sediminibacillus halophilus strain NSP9.3, a moderate halophile isolated from a seasonal salt marsh of the Great Rann of Kutch, India. Exploring the genome of this organism will facilitate the understanding of the mechanism(s) of osmotolerance and survival in differential osmolarity.
The 4.37-Mbp draft genome of a moderately halophilic Bacillus megaterium strain, MSP20.1, isolated from a saltern of the Little Rann of Kutch, India, is reported here. To understand the mechanism(s) of moderate halophilism and to isolate the gene(s) involved in osmotolerance and adaptation, the genome of MSP20.1 was sequenced.