query
stringlengths 11
289
| positive
stringlengths 1
4.52k
| negatives
listlengths 2
10
|
|---|---|---|
what is pms
|
Phelan-McDermid syndrome (PMS) is characterized by a variety of clinical symptoms with heterogeneous degrees of severity, including intellectual disability (ID), absent or delayed speech, and autism spectrum disorders (ASD). It results from a deletion of the distal part of chromosome 22q13 that in most cases includes the
|
[
"Mutations in SHANK3, which encodes a synaptic scaffolding protein, have been described in subjects with an autism spectrum disorder (ASD). To assess the quantitative contribution of SHANK3 to the pathogenesis of autism, we determined the frequency of DNA sequence and copy-number variants in this gene in 400 ASD-affected subjects ascertained in Canada. One de novo mutation and two gene deletions were discovered, indicating a contribution of 0.75% in this cohort. One additional SHANK3 deletion was characterized in two ASD-affected siblings from another collection, which brings the total number of published mutations in unrelated ASD-affected families to seven. The combined data provide support that haploinsufficiency of SHANK3 can cause a monogenic form of autism in sufficient frequency to warrant consideration in clinical diagnostic testing.",
"The 22q13.3 deletion syndrome (MIM 606232) is characterised by neonatal hypotonia, normal to accelerated growth, absent to severely delayed speech, global developmental delay, and minor dysmorphic facial features. We report the molecular characterisation of the deletion breakpoint in two unrelated chromosome 22q13.3 deletion cases. The deletions were characterised by FISH, checked for other abnormalities by array-CGH, and confirmed by Real-Time PCR, and finally the breakpoints were cloned, sequenced, and compared. Both cases show the cardinal features of the 22q13.3 deletion syndrome associated with a deletion involving the last 100 kb of chromosome 22q13.3. The cases show a breakpoint within the same 15 bp repeat unit, overlapping the results obtained by Wong and colleagues in 1997 and suggesting that a recurrent deletion breakpoint exists within the SHANK3 gene. The direct repeat involved in these 22q13 deletion cases is presumably able to form slipped (hairpin) structures, but it also has a strong potential for forming tetraplex structures. Three cases with a common breakpoint within SHANK3 share a number of common phenotypic features, such as mental retardation and developmental delay with severely delayed or absent expressive speech. The two cases presented here, having a deletion partially overlapping the commercial subtelomeric probe, highlight the difficulties in interpreting FISH results and suggest that many similar cases may be overlooked.",
"By array-CGH, we identified a cryptic deletion of about 3.4 Mb involving the chromosomal region 11q13.2q13.4 in a child with speech and developmental delay. Highly homologous segmental duplications related to the well-known olfactory receptor (OR)-containing clusters at 8p and 4p are located at the breakpoints of the imbalance and may be involved in its occurrence. Although these structural features are known to promote recurrent chromosomal rearrangements and previous studies had included the 11q13.2q13.4 deletion region among those considered potentially more unstable, neither deletions nor duplications of this region had been reported until now. Among the deleted genes, SHANK2 might play a role in the phenotype of the patient since it encodes a postsynaptic scaffolding protein similar to SHANK3, whose haploinsufficiency is a well-known cause of severe speech delay and autistic-like behavior, and recently deletions and mutations of SHANK2 have been described in patients with an autistic spectrum disorder or mental retardation.",
"Phelan-McDermid syndrome (22q13.3 deletion syndrome) is a contiguous gene disorder resulting from the deletion of the distal long arm of chromosome 22. SHANK3, a gene within the minimal critical region, is a candidate gene for the major neurological features of this syndrome. We report clinical and molecular data from a study of nine patients with overlapping interstitial deletions in 22q13 not involving SHANK3. All of these deletions overlap with the largest, but not with the smallest deletion associated with Phelan-McDermid syndrome. The deletion sizes and breakpoints varied considerably among our patients, with the largest deletion spanning 6.9 Mb and the smallest deletion spanning 2.7 Mb. Eight out of nine patients had a de novo deletion, while in one patient the origin of deletion was unknown. These patients shared clinical features common to Phelan-McDermid syndrome: developmental delay (11/12), speech delay (11/12), hypotonia (9/12), and feeding difficulties (7/12). Moreover, the majority of patients (8/12) exhibited macrocephaly. In the minimal deleted region, we identified two candidate genes, SULT4A1 and PARVB (associated with the PTEN pathway), which could be associated in our cohort with neurological features and macrocephaly/hypotonia, respectively. This study suggests that the haploinsufficiency of genes in the 22q13 region beside SHANK3 contributes to cognitive and speech development, and that these genes are involved in the phenotype associated with the larger Phelan-McDermid syndrome 22q13 deletions. Moreover, because the deletions in our patients do not involve the SHANK3 gene, we posit the existence of a new contiguous gene syndrome proximal to the smallest terminal deletions in the 22q13 region.",
"Multiple studies have confirmed the contribution of rare de novo copy number variations to the risk for autism spectrum disorders. But whereas de novo single nucleotide variants have been identified in affected individuals, their contribution to risk has yet to be clarified. Specifically, the frequency and distribution of these mutations have not been well characterized in matched unaffected controls, and such data are vital to the interpretation of de novo coding mutations observed in probands. Here we show, using whole-exome sequencing of 928 individuals, including 200 phenotypically discordant sibling pairs, that highly disruptive (nonsense and splice-site) de novo mutations in brain-expressed genes are associated with autism spectrum disorders and carry large effects. On the basis of mutation rates in unaffected individuals, we demonstrate that multiple independent de novo single nucleotide variants in the same gene among unrelated probands reliably identifies risk alleles, providing a clear path forward for gene discovery. Among a total of 279 identified de novo coding mutations, there is a single instance in probands, and none in siblings, in which two independent nonsense variants disrupt the same gene, SCN2A (sodium channel, voltage-gated, type II, α subunit), a result that is highly unlikely by chance.",
"SHANK genes code for scaffold proteins located at the post-synaptic density of glutamatergic synapses. In neurons, SHANK2 and SHANK3 have a positive effect on the induction and maturation of dendritic spines, whereas SHANK1 induces the enlargement of spine heads. Mutations in SHANK genes have been associated with autism spectrum disorders (ASD), but their prevalence and clinical relevance remain to be determined. Here, we performed a new screen and a meta-analysis of SHANK copy-number and coding-sequence variants in ASD. Copy-number variants were analyzed in 5,657 patients and 19,163 controls, coding-sequence variants were ascertained in 760 to 2,147 patients and 492 to 1,090 controls (depending on the gene), and, individuals carrying de novo or truncating SHANK mutations underwent an extensive clinical investigation. Copy-number variants and truncating mutations in SHANK genes were present in ∼1% of patients with ASD: mutations in SHANK1 were rare (0.04%) and present in males with normal IQ and autism; mutations in SHANK2 were present in 0.17% of patients with ASD and mild intellectual disability; mutations in SHANK3 were present in 0.69% of patients with ASD and up to 2.12% of the cases with moderate to profound intellectual disability. In summary, mutations of the SHANK genes were detected in the whole spectrum of autism with a gradient of severity in cognitive impairment. Given the rare frequency of SHANK1 and SHANK2 deleterious mutations, the clinical relevance of these genes remains to be ascertained. In contrast, the frequency and the penetrance of SHANK3 mutations in individuals with ASD and intellectual disability-more than 1 in 50-warrant its consideration for mutation screening in clinical practice.",
"Intact synaptic homeostasis is a fundamental prerequisite for a healthy brain. Thus, it is not surprising that altered synaptic morphology and function are involved in the molecular pathogenesis of so-called synaptopathies including autism, schizophrenia (SCZ) and Alzheimer's disease (AD). Intriguingly, various recent studies revealed a crucial role of postsynaptic ProSAP/Shank scaffold proteins in all of the aforementioned disorders. Considering these findings, we follow the hypothesis that ProSAP/Shank proteins are key regulators of synaptic development and plasticity with clear-cut isoform-specific roles. We thus propose a model where ProSAP/Shank proteins are in the center of a postsynaptic signaling pathway that is disrupted in several neuropsychiatric disorders.",
"Recent research has uncovered an important role for de novo variation in neurodevelopmental disorders. Using aggregated data from 9,246 families with autism spectrum disorder, intellectual disability, or developmental delay, we found that ∼1/3 of de novo variants are independently present as standing variation in the Exome Aggregation Consortium's cohort of 60,706 adults, and these de novo variants do not contribute to neurodevelopmental risk. We further used a loss-of-function (LoF)-intolerance metric, pLI, to identify a subset of LoF-intolerant genes containing the observed signal of associated de novo protein-truncating variants (PTVs) in neurodevelopmental disorders. LoF-intolerant genes also carry a modest excess of inherited PTVs, although the strongest de novo-affected genes contribute little to this excess, thus suggesting that the excess of inherited risk resides in lower-penetrant genes. These findings illustrate the importance of population-based reference cohorts for the interpretation of candidate pathogenic variants, even for analyses of complex diseases and de novo variation.",
"The 22q13.3 deletion syndrome, also known as Phelan-McDermid syndrome, is a contiguous gene disorder resulting from deletion of the distal long arm of chromosome 22. In addition to normal growth and a constellation of minor dysmorphic features, this syndrome is characterized by neurological deficits which include global developmental delay, moderate to severe intellectual impairment, absent or severely delayed speech, and neonatal hypotonia. In addition, more than 50% of patients show autism or autistic-like behavior, and therefore it can be classified as a syndromic form of autism spectrum disorders (ASD). The differential diagnosis includes Angelman syndrome, velocardiofacial syndrome, fragile X syndrome, and FG syndrome. Over 600 cases of 22q13.3 deletion syndrome have been documented. Most are terminal deletions of ∼100 kb to >9 Mb, resulting from simple deletions, ring chromosomes, and unbalanced translocations. Almost all of these deletions include the gene SHANK3 which encodes a scaffold protein in the postsynaptic densities of excitatory synapses, connecting membrane-bound receptors to the actin cytoskeleton. Two mouse knockout models and cell culture experiments show that SHANK3 is involved in the structure and function of synapses and support the hypothesis that the majority of 22q13.3 deletion syndrome neurological defects are due to haploinsufficiency of SHANK3, although other genes in the region may also play a role in the syndrome. The molecular connection to ASD suggests that potential future treatments may involve modulation of metabotropic glutamate receptors.",
"Chromosome 22q13 monosomy has been described as a contiguous gene syndrome. Localized in the critical region, SHANK3 is likely to play a key role in the expression of the clinical phenotype. SHANK3 mutations have also been reported in autistic patients without a syndromic phenotype. We report on a 20-year-old woman with mental retardation carrying a de novo translocation between chromosome Xq21.33 and 22q13.33, associated with a duplication on Xq21.33 and deletion on 22q13.33. As a child her development was characterized by disturbed social interaction, stereotypic hand movements and ritualistic behavior and she was considered at one time to have autistic features. All these traits match the 22q13 deletion syndrome (Phelan-McDermid syndrome, OMIM 606232), likely due to the deletion overlapping the last two exons of the SHANK3 gene. Our patient harbors the smallest and most distal SHANK3 deletion described to date, yet resulting in the full spectrum of the Phelan-McDermid syndrome. In addition, she has hypergonadotropic hypogonadism with low estrogen level, high FSH level, and irregular menstruation. Intriguingly, chromosome translocations affecting the chromosome band Xq21 can result in premature ovarian failure."
] |
Immunocytochemical study of canine lymphoma
|
Canine lymphoma is one of the most common canine neoplasms, but little is known regarding the effects of exposure to tobacco smoke on their biologic behavior. As cytology is the most frequent diagnostic method of canine lymphoma, the aims of this study were to perform an immunocytochemical study of canine lymphomas, including subtyping and cell proliferation analysis, and to establish their correlation with tobacco smoke exposure.
|
[
"Changing definitions and classifications of hematologic malignancies (HMs) complicate incidence comparisons. HAEMACARE classified HMs into groupings consistent with the latest World Health Organization classification and useful for epidemiologic and public health purposes. We present crude, age-specific and age-standardized incidence rates for European HMs according to these groupings, estimated from 66,371 lymphoid malignancies (LMs) and 21,796 myeloid malignancies (MMs) registered in 2000-2002 by 44 European cancer registries, grouped into 5 regions. Age-standardized incidence rates were 24.5 (per 100,000) for LMs and 7.55 for MMs. The commonest LMs were plasma cell neoplasms (4.62), small B-cell lymphocytic lymphoma/chronic lymphatic leukemia (3.79), diffuse B-cell lymphoma (3.13), and Hodgkin lymphoma (2.41). The commonest MMs were acute myeloid leukemia (2.96), other myeloproliferative neoplasms (1.76), and myelodysplastic syndrome (1.24). Unknown morphology LMs were commonest in Northern Europe (7.53); unknown morphology MMs were commonest in Southern Europe (0.73). Overall incidence was lowest in Eastern Europe and lower in women than in men. For most LMs, incidence was highest in Southern Europe; for MMs incidence was highest in the United Kingdom and Ireland. Differences in diagnostic and registration criteria are an important cause of incidence variation; however, different distribution of HM risk factors also contributes. The quality of population-based HM data needs further improvement.",
"The term \"lymphoma\" describes a heterogeneous group of disorders involving monoclonal proliferation of malignant lymphocytes. As a group, lymphomas are among the most common tumors of dogs. Yet our enumeration and understanding of the many subtypes of lymphoma have been relatively slow, perhaps in part because for many years lymphoma was treated as a singular entity rather than a group of distinct diseases. The recognition that the full spectrum of lymphoid malignancies seen in humans also occurs in dogs, and that these tumors retain not only morphologic similarities and biological behavior but also synonymous driver molecular abnormalities, sets an ideal stage for dual-purpose research that can accelerate progress for these diseases in both species. Specifically, dogs represent exceptional models for defining causality, understanding progression, and developing new treatments for lymphoma in comparatively brief windows of time. Unique advantages of canine models include (1) spontaneous disease occurring without an isogenic background or genetic engineering; (2) chronology of disease adapted to lifespan, (3) shared environment and societal status that allows dogs to be treated as \"patients,\" while at the same time being able to ethically explore translational innovations that are not possible in human subjects; and (4) organization of dogs into breeds with relatively homogeneous genetic backgrounds and distinct predisposition for lymphomas. Here, we will review recent studies describing intrinsic and extrinsic factors that contribute to the pathogenesis of canine and human lymphomas, as well as newly developed tools that will enhance the fidelity of these models to improve diagnosis and develop new treatments.",
"Marginal zone lymphomas (MZL) are a diverse group of indolent lymphoproliferative disorders that comprise three subtypes: nodal, splenic and mucosal associated marginal zone lymphomas (MALT). Histologic transformation (HT) to an aggressive lymphoma is a rare event that can occur in any subtype, and at lower frequency compared to other indolent non Hodgkin lymphomas (NHL) like follicular lymphoma. There are few data directly associated with risk and prognosis of transformation in MZL. However, recent advances in the understanding of molecular and genetic features of MALT have contributed to an evolving appreciation of HT in this disease. Optimal treatment of HT of MZL remains unknown. Much of the approach to managing transformed MZL is extrapolated from other indolent NHLs.",
"Non-Hodgkin lymphoma (NHL) comprises numerous biologically and clinically heterogeneous subtypes, with limited data examining the risk factors for these distinct disease entities. Many limitations exist when studying lymphoma epidemiology; therefore, until recently, little was known regarding the etiology of NHL subtypes. This review highlights the results of recent pooled analyses examining the risk factors for NHL subtypes. We outline the heterogeneity and commonality among the risk factors for NHL subtypes, with proposed subtype-specific as well as shared etiologic mechanisms. In addition, we describe how the study of lymphoma epidemiology may translate into prevention or therapeutic targeting as we continue to explore the complexities of lifestyle and genetic factors that impact lymphomagenesis.",
"While it has been well established that there are significant racial differences in lymphoid malignancies, registry-based studies have been limited by incomplete or missing data on stage, race, important clinical and laboratory prognostic factors, treatment, treatment response, and follow-up. To overcome some of these limitations, the authors conducted a retrospective cohort study of consecutive patients with a confirmed diagnosis of chronic lymphocytic leukemia (CLL) receiving care at MD Anderson Cancer Center and Duke University Medical Center. The authors identified 84 AA patients with untreated CLL who more commonly presented with poor-risk biological features such as unmutated IGHV gene, ZAP70 expression, and chromosome 17p or 11q deletion. . When compared to a group of non-black patients, the AA group had significantly shorter median event-free survival and overall survival. These results corroborate the findings of prior studies of CLL, but forward the field by providing additional clinical details to understand the nature of these racial disparities.",
"Burkitt lymphoma is characterized by deregulation of MYC, but the contribution of other genetic mutations to the disease is largely unknown. Here, we describe the first completely sequenced genome from a Burkitt lymphoma tumor and germline DNA from the same affected individual. We further sequenced the exomes of 59 Burkitt lymphoma tumors and compared them to sequenced exomes from 94 diffuse large B-cell lymphoma (DLBCL) tumors. We identified 70 genes that were recurrently mutated in Burkitt lymphomas, including ID3, GNA13, RET, PIK3R1 and the SWI/SNF genes ARID1A and SMARCA4. Our data implicate a number of genes in cancer for the first time, including CCT6B, SALL3, FTCD and PC. ID3 mutations occurred in 34% of Burkitt lymphomas and not in DLBCLs. We show experimentally that ID3 mutations promote cell cycle progression and proliferation. Our work thus elucidates commonly occurring gene-coding mutations in Burkitt lymphoma and implicates ID3 as a new tumor suppressor gene.",
"Vitamin D insufficiency is common globally and low levels are linked to higher cancer incidence. Although vitamin D insufficiency is related to inferior prognosis in some cancers, no data exist for chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). We evaluated the relationship of 25(OH)D serum levels with time-to-treatment (TTT) and overall survival (OS) in newly diagnosed CLL patients participating in a prospective cohort study (discovery cohort) and a separate cohort of previously untreated patients participating in an observational study (confirmation cohort). Of 390 CLL patients in the discovery cohort, 119 (30.5%) were 25(OH)D insufficient. After a median follow-up of 3 years, TTT (hazard ratio[HR] = 1.66; P = .005) and OS (HR = 2.39; P = .01) were shorter for 25(OH)D-insufficient patients. In the validation cohort, 61 of 153 patients (39.9%) were 25(OH)D insufficient. After a median follow-up of 9.9 years, TTT (HR = 1.59; P = .05) and OS (HR 1.63; P = .06) were again shorter for 25(OH)D-insufficient patients. On pooled multivariable analysis of patients in both cohorts adjusting for age, sex, Rai stage, CD38 status, ZAP-70 status, immunoglobulin heavy chain variable (IGHV) gene mutation status, CD49d status, and cytogenetic abnormalities assessed by interphase fluorescent in situ hybridization testing, 25(OH)D insufficiency remained an independent predictor of TTT (HR = 1.47; P = .008), although the association with OS was not significant (HR = 1.47; P = .07). Vitamin D insufficiency is associated with inferior TTT and OS in CLL patients. Whether normalizing vitamin D levels in deficient CLL patients would improve outcome merits clinical testing."
] |
Aujeszky's disease virus seroprevalence in wild boar in Switzerland
|
In parallel to the increase of wild boar abundance in the past decades, an increase of exposure to the Aujeszky's disease virus (ADV) has been reported in wild boar in several parts of Europe. Since high animal densities have been proposed to be one of the major factors influencing ADV seroprevalence in wild boar populations and wild boar abundance has increased in Switzerland, too, a re-evaluation of the ADV status was required in wild boar in Switzerland. We tested wild boar sera collected from 2008-2013 with a commercial ELISA for antibodies against ADV. To set our data in the European context, we reviewed scientific publications on ADV serosurveys in Europe for two time periods (1995-2007 and 2008-2014).
|
[
"Wild boars (Sus scrofa) are indigenous in many countries in the world. These free-living swine are known reservoirs for a number of viruses, bacteria and parasites that are transmissible to domestic animals and humans. Changes of human habitation to suburban areas, increased use of lands for agricultural purposes, increased hunting activities and consumption of wild boar meat have increased the chances of exposure of wild boars to domestic animals and humans. Wild boars can act as reservoirs for many important infectious diseases in domestic animals, such as classical swine fever, brucellosis and trichinellosis, and in humans, diseases such as hepatitis E, tuberculosis, leptospirosis and trichinellosis. For examples, wild boars are reservoirs for hepatitis E virus, and cluster cases of hepatitis E have been reported in Japan of humans who consumed wild boar meat. In Canada, an outbreak of trichinellosis was linked to the consumption of wild boar meat. The incidence of tuberculosis owing to Mycobacterium bovis has increased in wild boars, thus posing a potential concern for infections in livestock and humans. It has also been documented that six hunters contracted Brucella suis infections from wild swine in Florida. This article discusses the prevalence and risk of infectious agents in wild boars and their potential transmission to livestock and humans.",
"Prevalences of parasitic infections in pigs from different housing systems may vary, due to their contact with the environment, and this might have consequences for food safety. In this study, 40 organic, 9 free-range and 24 intensive farms were selected and a total of 845 serum samples were tested for antibodies specific for Toxoplasma and Trichinella using ELISA assays. The overall seroprevalence of Toxoplasma in the total number of 845 serum samples tested is 2.6%, ranging from 0.38% in intensively raised pigs to 5.62% in free-range pigs. Of the housing systems tested, 4% (intensive farms) to 33% (free-range farms) is infected with Toxoplasma gondii. The risk of detecting Toxoplasma antibodies in a free-range farm are statistically higher (almost 16 times higher) than in an intensive farm. We observed that the risk of detecting specific antibodies is twice as high as in free-range compared with organic farms. Seropositivity of Trichinella spiralis antibodies was 0.12-0.35% (depending on the cut-off value at the 99.5% or 97.5% level). There was a tendency that Trichinella seropositivity was higher in organic pig farming (0.24%), but this was not significant. This serological study in pigs from different farming systems shows that the seroprevalence of antibodies specific for T. gondii is higher and for Trichinella equivalent in pigs raised in systems where there is contact with the environment than in pigs raised in intensive, indoor farming systems. This indicates that the prevalence of parasitic infections is higher in outdoor farming systems than in indoor farming systems. The possible consequences for food safety are discussed.",
"Psychological stress is thought to contribute to reactivation of latent herpes simplex virus (HSV). Although several animal models have been developed in an effort to reproduce different pathogenic aspects of HSV keratitis or labialis, until now, no good animal model existed in which application of a psychological laboratory stressor results in reliable reactivation of the virus. Reported herein, disruption of the social hierarchy within colonies of mice increased aggression among cohorts, activated the hypothalamic-pituitary-adrenal axis, and caused reactivation of latent HSV type 1 in greater than 40% of latently infected animals. However, activation of the hypothalamic-pituitary-adrenal axis using restraint stress did not activate the latent virus. Thus, the use of social stress in mice provides a good model in which to investigate the neuroendocrine mechanisms that underlie behaviorally mediated reactivation of latent herpesviruses.",
"Prior to the recent discovery of the swine hepatitis E virus (swine HEV) in pigs from the midwestern United States, HEV was not considered endemic to this country. Since swine HEV is antigenically and genetically related to human strains of HEV, it was important to characterize this new virus further. The infectivity titer of a pool of swine HEV in pigs was determined in order to prepare a standardized reagent and to evaluate the dose response in pigs. Although the sequence of swine HEV varied extensively from those of most human strains of HEV, it was very closely related to the two strains of human HEV (US-1 and US-2) isolated in the United States. The U.S. strains which were recently recovered from two patients with clinical hepatitis E in the United States shared >/=97% amino acid identity with swine HEV in open reading frames 1 and 2. Phylogenetic analyses of different regions of the genome revealed that swine HEV and the U.S. strains grouped together and formed a distinct branch. These results suggested that swine HEV may infect humans. When we inoculated rhesus monkeys and a chimpanzee, experimental surrogates of humans, with swine HEV, the primates became infected. Furthermore, in a reciprocal experiment, specific-pathogen-free pigs were experimentally infected with the US-2 strain of human HEV that is genetically similar to swine HEV. These results provided experimental evidence for cross-species infection by the swine virus. Thus, humans appear to be at risk of infection with swine HEV or closely related viruses."
] |
Determinants of the outcomes after hip/knee arthroplasty in patients with osteoarthritis during the first postsurgical year
|
Objective To measure and identify the determinants of the outcomes after hip/knee arthroplasty (HA/KA) in patients with osteoarthritis during the first postsurgical year. Design In this prospective observational study, we evaluated the preoperative and postoperative (3, 6, and 12 months) outcomes of 626 patients who underwent HA (346 with median age 65 years, 59% female) or KA (280 with median age 66.5 years, 54% female) between 2008 and 2013. Generic and specific tools were used to measure health-related quality of life (HRQoL) and utility. Good outcome was defined as an improvement in WOMAC (Western Ontario and McMaster Universities Osteoarthritis Index) greater than or equal to the minimal important difference (MID). Regressions were performed to evaluate the relationship between preoperative and postoperative measures and evolution of WOMAC/good outcome. Results We observed an almost systematic improvement of all parameters for up to 12 months, but especially at the 3-month follow-up. The low number of comorbidities and the absence of postoperative complications were the common determinants of improvement of WOMAC total score after 12 months. Other parameters (background of the joint, preoperative function and length of hospital stay in KA group; place of discharge in HA group) affected the evolution of WOMAC scores. 87.09% of HA and 73.06% of KA patients experienced a good outcome. A small number of comorbidities, a worse preoperative function, a shortened hospital stay (KA only), and an absence of early postoperative complications (HA only) significantly predicted a good outcome. Conclusions Intermediate HRQoL following HA or KA improved quickly from preoperative levels for all instruments. More than 70% of patients achieved a good outcome defined as improved pain, stiffness and disability and the predictors are slightly close.
|
[
"Risk of revision following total knee arthroplasty (TKA) is higher in patients under 55 years, but little data are reported regarding non-revision outcomes. This study aims to identify predictors of dissatisfaction in these patients. We prospectively assessed 177 TKAs (157 consecutive patients, 99 women, mean age 50 years; 17 to 54) from 2008 to 2013. Age, gender, implant, indication, body mass index (BMI), social deprivation, range of movement, Kellgren-Lawrence (KL) grade of osteoarthritis (OA) and prior knee surgery were recorded. Pre- and post-operative Oxford Knee Score (OKS) as well as Short Form-12 physical (PCS) and mental component scores were obtained. Post-operative range of movement, complications and satisfaction were measured at one year. Overall, 44 patients with 44 TKAs (24.9%) under 55 years of age were unsure or dissatisfied with their knee. Significant predictors of dissatisfaction on univariate analysis included: KL grade 1/2 OA (59% dissatisfied), poor pre-operative OKS, complications, poor improvements in PCS and OKS and indication (primary OA 19% dissatisfied, previous meniscectomy 41%, multiply operated 42%, other surgery 29%, BMI > 40 kg/m2 31%, post-traumatic OA 45%, and inflammatory arthropathy 5%). Poor pre-operative OKS, poor improvement in OKS and post-operative stiffness independently predicted dissatisfaction on multivariate analysis. Patients receiving TKA younger than 55 years old should be informed about the increased risks of dissatisfaction. Offering TKA in KL 1/2 is questionable, with a dissatisfaction rate of 59%. Cite this article: Bone Joint J 2016;98-B:1625-34.",
"To document the disease burden of osteoarthritis and the benefits of total joint replacement by using the Short Form Health Survey (SF-36) general health status survey and evaluate other factors that could affect scores. Prospective study. All patients scheduled for primary total hip arthroplasty (THA) or total knee arthroplasty (TKA) (n = 622 preoperatively) in 2 years were surveyed using the SF-36, which assesses health-related quality of life (HRQOL) in patients' physical and social functioning and mental health. Follow-up surveys were administered 12 months after surgery to all patients and 3 and 24 months after surgery to a subset of patients. Preoperatively, patient scores were significantly lower than normative scores in the physical functioning, bodily pain, and social functioning domains. Preoperative scores were not different between THA and TKA patients. Women scored lower than men. Comorbid conditions were weakly associated with low SF-36 scores. Postoperatively, the largest incremental improvement in scores was seen at 3-month follow-up. Scores improved sooner and more substantially in THA vs TKA patients and in men vs women, paralleling improvement in clinical and subjective ratings of postoperative physical function and pain. The SF-36 has the sensitivity to document improvement in HRQOL after surgery and to reveal differences in THA vs TKA and in men vs women. However, routine use of outcome assessment instruments to monitor this patient population is costly and unjustified in our current healthcare environment.",
"Six hundred twenty-two primary total knee arthroplasties were studied prospectively in 512 patients. A group with no pain and one with severe pain at 5 years were statistically compared. The following were significant predictors of poor pain outcomes: age below 60 (17%) compared with above 60 (7%, P < .05). The first knee was most likely to be in the poor outcome group (13%) compared with the second knee (6%). In contrast, patients who underwent simultaneous bilateral arthroplasty faired better (2%, P < .01). Performing lateral release and sacrificing the posterior cruciate ligament also significantly predicted for poor pain outcomes but may have been influenced by selection bias. We conclude that avoiding surgery in patients younger than 60 and choosing a simultaneous approach to bilateral disease reduce the chance of poor pain outcomes.",
"The objective of this study is to characterize, based on clinical, radiographic, health-related, quality-of-life-related, and demographic variables, the profile of a large, homogeneous, cohort of patients undergoing knee or hip arthroplasty, in a public hospital. Current regulatory guidelines for structure-modifying agent are not clear regarding hard clinical endpoint. The \"need for surgery\" has been suggested as a potential relevant outcome, but, until now, it is poorly defined. By characterizing a large number of patients who undergo total hip or total knee replacement, this paper aims at providing a contribution to the better definition of the \"need for surgery\" in advanced OA of the lower limbs. Consecutive patients who underwent primary knee arthroplasty (KA) or hip arthroplasty (HA) between December 2008 and February 2013, in an academic hospital, and who were diagnosed with hip or knee osteoarthritis (OA) (ACR criteria). Data collected at baseline included demographic and clinical data; Kellgren-Lawrence radiological grading; Western Ontario and Mc Master Universities Arthritis Index (WOMAC); EuroQol five dimensions questionnaire and EuroQol visual analog scale; and 36-item Short Form Health Survey. 626 subjects were included, 346 with hip OA and 280 with knee OA. Significant differences between subjects in need of an HA or of a KA were seen in terms of age (66.5 years versus 65 for hip), duration of complaints (2188 days versus 1146.5 for hip), BMI (28.68 kg/m² versus 27.07), radiological status (severe OA were found in 79.85% in knee group and 68.73% in hip group), comorbidities (FCI higher in knee group), traumatic of surgical history (37 versus 6%), and health-related quality of life and function (patients with HA had a poorer clinical status regarding WOMAC and WOMAC subscale). Significant differences were observed between patients undergoing KA or HA. These differences might be useful to better understand the \"need for surgery\" status in these indications. This concept may help to define responders and failures to pharmacological treatment of OA.",
"We investigated beliefs of blacks with osteoarthritis (OA) regarding total knee replacement (TKR) surgery. These beliefs potentially related to the known racial disparity in the use of TKR. Ninety-four community-dwelling blacks aged 50 to 89 with knee OA in Harlem, NY, were assessed for arthritis knowledge, expectations, quality of life (QoL), and disability. Subjects have had OA for a median of 6 years and the disability was severe. Only 36% believed that TKR was likely to improve knee pain; 45% stated that TKR would not improve their current health. Mean QoL was 7.6 +/- 1.7 (max 10). Despite debilitating OA, African American patients perceive a high QoL, yet have low expectations from TKR and are therefore less likely to consider TKR as a treatment for OA.",
"To investigate and compare the impact of primary hip (THA) and knee (TKA) arthroplasty on quality of life in patients with osteoarthritis, to determine patients' satisfaction with total joint arthroplasty, and to detect the effect of patients' demographic and clinical characteristics on outcome. Three hundred seventy eight (378) patients with hip (174) and knee (204) osteoarthritis undergoing total joint arthroplasty (174 THA-204 TKA) were assessed pre- and post-operatively (6 weeks, 3, 6, and 12 months) using the Western Ontario and McMaster Osteoarthritis Index (WOMAC) and Centre for Epidemiological Studies Depression Scale (CES-D10). The patients' satisfaction with the results of total joint arthroplasty was also assessed. Differences were analyzed using general linear model for repeated measures. The one-year response rate was 97 % for THA and 90 % for TKA. WOMAC and CES-D10 scores improved significantly after one year for both THA and TKA (P < 0.0001). The improvement in WOMAC total score was significantly greater for TKA patients (P < 0.0001 at 12 months). WOMAC pain and stiffness improved earlier for THA (6 weeks), while TKA had equivalent improvements at 3 and 6 months respectively. Both THA/TKA displayed significant improvement of WOMAC function at 3 months but TKA had greater improvement. Age, body mass index, residence, education and social support were not significant predictors of quality of life after total joint arthroplasty. One year postoperatively 88 % of patients were satisfied. WOMAC and CES-D10 improved significantly one year postoperatively. Although pain and stiffness improved earlier in THA, functional improvement was inferior in THA compared to TKA.",
"Osteoarthritis is the most prevalent joint disease and a common cause of joint pain, functional loss, and disability. Conventional treatments demonstrate only modest clinical benefits without lesion reversal. Autologous mesenchymal stromal cell (MSC) treatments have shown feasibility, safety, and strong indications for clinical efficacy. We performed a randomized, active control trial to assess the feasibility and safety of treating osteoarthritis with allogeneic MSCs, and we obtain information regarding the efficacy of this treatment. We randomized 30 patients with chronic knee pain unresponsive to conservative treatments and showing radiological evidence of osteoarthritis into 2 groups of 15 patients. The test group was treated with allogeneic bone marrow MSCs by intra-articular injection of 40 × 10(6) cells. The control group received intra-articular hyaluronic acid (60 mg, single dose). Clinical outcomes were followed for 1 year and included evaluations of pain, disability, and quality of life. Articular cartilage quality was assessed by quantitative magnetic resonance imaging T2 mapping. Feasibility and safety were confirmed and indications of clinical efficacy were identified. The MSC-treated patients displayed significant improvement in algofunctional indices versus the active controls treated with hyaluronic acid. Quantification of cartilage quality by T2 relaxation measurements showed a significant decrease in poor cartilage areas, with cartilage quality improvements in MSC-treated patients. Allogeneic MSC therapy may be a valid alternative for the treatment of chronic knee osteoarthritis that is more logistically convenient than autologous MSC treatment. The intervention is simple, does not require surgery, provides pain relief, and significantly improves cartilage quality."
] |
Cross-Neutralisation of Tick-Borne Encephalitis Viruses Following Vaccination and Infection
|
The tick-borne encephalitis complex contains a number of flaviviruses that share close genetic homology, and are responsible for significant human morbidity and mortality with widespread geographical range. Although many members of this complex have been recognised for decades, licenced human vaccines with broad availability are only available for tick-borne encephalitis virus. While tick-borne encephalitis virus vaccines have been demonstrated to induce significant protective immunity, as determined by virus-neutralisation titres, vaccine breakthrough (clinical infection following complete vaccination), has been described. The aim of this study was to confirm the cross-neutralisation of tick-borne flaviviruses using mouse immune ascitic fluids, and to determine the magnitude of cross-neutralising antibody titres in sera from donors following tick-borne encephalitis vaccination, infection, and vaccine breakthrough. The results demonstrate that there is significant cross-neutralisation of representative members of the tick-borne encephalitis complex following vaccination and/or infection, and that the magnitude of immune responses varies based upon the exposure type. Donor sera successfully neutralised most of the viruses tested, with 85% of vaccinees neutralising Kyasanur forest disease virus and 73% of vaccinees neutralising Alkhumra virus. By contrast, only 63% of vaccinees neutralised Powassan virus, with none of these neutralisation titres exceeding 1:60. Taken together, the data suggest that tick-borne encephalitis virus vaccination may protect against most of the members of the tick-borne encephalitis complex including Kyasanur forest disease virus and Alkhumra virus, but that the neutralisation of Powassan virus following tick-borne encephalitis vaccination is minimal.
|
[
"During the recent chikungunya fever outbreak in French Polynesia in October 2014 to March 2015, we observed an abnormally high number of patients with neurological deficit. Clinical presentation and complementary exams were suggestive of Guillain-Barré syndrome (GBS) for nine patients. All nine had a recent dengue-like syndrome and tested positive for chikungunya virus (CHIKV) in serology or RT-PCR. GBS incidence was increased four- to nine-fold during this period, suggesting a link to CHIKV infection.",
"Genus flavivirus comprises many important human pathogens causing public health problems worldwide. Some flavivirus infections are characterized by a relatively high mortality rate and/or high sequelae rate in survivors. Because most flavivirus life cycles are maintained between arthropod vectors and amplifying/reservoir hosts in the absence of humans, eradication of flaviviruses might be extremely difficult. Flavivirus vaccine development is considered a reasonable method to prevent flavivirus infections. Some vaccines have been successfully developed, but others have not, regardless of much effort. This review article describes currently available flavivirus vaccines against yellow fever, Japanese encephalitis, and tick-borne encephalitis. In addition, the current status of dengue and West Nile virus vaccine development is reviewed and problems regarding their development are discussed.",
"Arthropod-borne viruses, or arboviruses, are viruses that are transmitted through the bites of mosquitoes, ticks, or sandflies. There are numerous arboviruses throughout the world capable of causing human disease spanning different viral families and genera. Recently, Jamestown Canyon, Powassan, chikungunya, and Zika viruses have emerged as increasingly important arboviruses that can cause human disease in North America. Unfortunately, there are currently no proven disease-modifying therapies for these arboviral diseases, so treatment is largely supportive. Given there are also no commercially available vaccines for these four arboviral infections, prevention is the key. To prevent mosquito or tick bites that might result in one of these arboviral diseases, people should wear long-sleeved shirts and pants while outside if feasible, apply insect repellant when going outdoors, using window screens or air conditioning to keep mosquitoes outside, and perform tick checks after being in wooded or brushy outdoor areas.",
"To investigate arbovirus transmission in North Dakota, we collected and screened mosquitoes for viral infection by Vero cell culture assay. Seven viruses were isolated from 13 mosquito species. Spatial and temporal distributions of the important vectors of West Nile virus (WNV), Cache Valley virus, Jamestown Canyon virus (JCV), and trivittatus virus are reported. Snowshoe hare virus, Potosi virus, and western equine encephalomyelitis virus were also isolated. The risks of Culex tarsalis and Aedes vexans transmitting WNV to humans were 61.4% and 34.0% in 2003-2006, respectively, but in 2003 when the largest epidemic was reported, risks for Ae. vexans and Cx. tarsalis in Cass County were 73.6% and 23.9%, respectively. Risk of humans acquiring an infectious bite was greatest from about the second week of July through most of August. West Nile virus sequences were of the WN02 genotype. Most JCV strains belonged to a single clade of genetically related strains. Cache Valley virus and JCV were prevalent during August and early September and during July and August, respectively."
] |
Perfluoroalkyl Acids in Drinking Water
|
Perfluoroalkyl acids (PFAAs), a group of synthetic organic chemicals with industrial and commercial uses, are of current concern because of increasing awareness of their presence in drinking water and their potential to cause adverse health effects. PFAAs are distinctive among persistent, bioaccumulative, and toxic (PBT) contaminants because they are water soluble and do not break down in the environment. This commentary discusses scientific and risk assessment issues that impact the development of drinking water guidelines for PFAAs, including choice of toxicological endpoints, uncertainty factors, and exposure assumptions used as their basis. In experimental animals, PFAAs cause toxicity to the liver, the immune, endocrine, and male reproductive systems, and the developing fetus and neonate. Low-dose effects include persistent delays in mammary gland development (perfluorooctanoic acid; PFOA) and suppression of immune response (perfluorooctane sulfonate; PFOS). In humans, even general population level exposures to some PFAAs are associated with health effects such as increased serum lipids and liver enzymes, decreased vaccine response, and decreased birth weight. Ongoing exposures to even relatively low drinking water concentrations of long-chain PFAAs substantially increase human body burdens, which remain elevated for many years after exposure ends. Notably, infants are a sensitive subpopulation for PFAA's developmental effects and receive higher exposures than adults from the same drinking water source. This information, as well as emerging data from future studies, should be considered in the development of health-protective and scientifically sound guidelines for PFAAs in drinking water.
|
[
"Contribution of diet and selected risk factors to the levels of four polyfluorinated compounds was evaluated. Data from National Health and Nutrition Examination Survey for the years 2003-2008 were used. Dietary factors accounted for 10.4% to 21.2% of the explained variation. Amount of milk consumed was found to be positively associated (p<0.01) with perfluorononanoic acid (PFNA) but negatively associated with perfluorooctanoate (PFOA) and perfluorooctane sulfonate (PFOS) (p<0.01). Amount of meat and fish consumed was positively associated (p<0.01) with PFNA and PFOS. Amount of non-alcoholic beverages consumed was positively associated (p<0.01) with PFNA and PFOA. Levels of PFOS increased (p<0.01) with increase in the amount of alcoholic beverages consumed. Total amount of alcohol consumed was positively associated (p<0.01) with PFNA. Levels of both PFOA and PFOS decreased with increase in total amount of caffeine consumed. Total amount of fat consumed was negatively associated with PFNA and positively associated with PFOS. Total calories consumed were negatively associated with perfluorohexane sulfonate (PFHxS) and PFOS but positively associated with PFNA. New to this study, positive correlations (p<0.01) between serum cholesterol and PFNA, PFOA, and PFOS were found. Serum albumin levels were negatively correlated with PFHxS but positively correlated with PFOA and PFOS. Males had statistically significantly higher levels of all four PFCs as compared to females and Mexican Americans had the lowest levels of all four PFCs than other race/ethnic groups. Levels of all four PFCs increased with increase in family income. Body mass index was negatively correlated with PFNA but positively associated with PFOA. There was a statistically significant decrease in the levels of PFOS over survey years 2003-2008.",
"Some cross-sectional epidemiological studies have reported positive associations of serum concentrations of non-high density lipoprotein cholesterol with serum perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA). However, the strength of the reported associations is inconsistent for exposure-response across three orders of magnitude of serum PFOS and/or PFOA concentrations. These positive associations are unexpected based on toxicological/mechanistic studies, suggesting that the associations may have a biological, rather than a causal, basis. This study tested the hypothesis that PFOS and PFOA distribute into serum lipoprotein fractions such that increases in serum lipoproteins would result in corresponding increases in serum concentrations of PFOS and PFOA. Based on observed binding of PFOS and PFOA to isolated β-lipoproteins in physiological saline (96% and 40% bound, respectively) in preliminary experiments using ultrafiltration and LC-MS/MS methods, binding to human donor plasma lipoprotein fractions was investigated by two density gradient methods. The majority of PFOS and PFOA recovered masses were found in lipoprotein-depleted plasma. Plasma density gradient fractionation data suggested that maximally 9% of PFOS distributes to lipoprotein-containing fractions, yet only 1% or less of PFOA is so distributed. These data do not support a strong role for plasma lipoprotein fractions in explaining the inconsistent dose-response associations reported in cross-sectional epidemiological studies.",
"Extensive data obtained in both experimental animals and humans demonstrate that steady-state plasma LDL-C concentrations are determined largely by the rate of LDL-C formation, Jt, and the level of LDL-R activity, Jm, located primarily in the liver. An increase in net cholesterol delivery to the liver suppresses Jm, slightly elevates Jt, and modestly raises the LDL-C level. Feeding lipids such as the 12:0, 14:0, and 16:0 saturated fatty acids further suppresses Jm, increases Jt, and markedly elevates the plasma LDL-C concentration. Feeding triacylglycerols containing the 18:1(c9) fatty acid restores hepatic receptor activity, decreases Jt, and modestly reduces the concentration of LDL-C in the plasma. The 18:2(c9, c12) compound has similar effects, although it is quantitatively less active than the monounsaturated fatty acid in restoring Jm. In contrast to these fatty acids that actively raise or lower hepatic receptor activity, a large group of compounds including the 4:0, 6:0, 8:0, 10:0, 18:0, and 18:1(t9) fatty acids have no demonstrable effect on any parameter of LDL-C metabolism. These fatty acids, therefore, can be added to animal and human diets with relative impunity. They will alter plasma LDL-C levels only to the extent that they replace the active saturated fatty acids (in which case they lower the LDL-C concentration) or unsaturated compounds (in which case they raise the plasma cholesterol level). All of these effects of cholesterol and the various fatty acids can be explained by the effects of these lipids in altering the size of the regulatory pool of cholesterol in the hepatocyte. However, many aspects of the cellular and molecular biology of these regulatory processes require additional investigation. In particular, new studies should focus on how the genetic background of an individual animal or human alters the quantitative response of its plasma LDL-C concentration to the dietary challenge of each of these types of lipids.",
"The peroxisome proliferator-activated receptors (PPAR) belong to the nuclear hormone receptor superfamily and there are three primary subtypes, PPARalpha, beta, and gamma. These receptors regulate important physiological processes that impact lipid homeostasis, inflammation, adipogenesis, reproduction, wound healing, and carcinogenesis. These nuclear receptors have important roles in reproduction and development and their expression may influence the responses of an embryo exposed to PPAR agonists. PPARs are relevant to the study of the biological effects of the perfluorinated alkyl acids as these compounds, including perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), activate PPARalpha. Exposure of the rodent to PFOA or PFOS during gestation results in neonatal deaths, developmental delay and growth deficits. Studies in PPARalpha knockout mice demonstrate that the developmental effects of PFOA, but not PFOS, depend on expression of PPARalpha. This review provides an overview of PPARalpha, beta, and gamma protein and mRNA expression during mouse, rat, and human development. The review presents the results from many published studies and the information is organized by organ system and collated to show patterns of expression at comparable developmental stages for human, mouse, and rat. The features of the PPAR nuclear receptor family are introduced and what is known or inferred about their roles in development is discussed relative to insights from genetically modified mice and studies in the adult.",
"Perfluorooctanoic acid (PFOA, 'C8') and perfluoroctane sulphonate (PFOS) are environmentally stable compounds with industrial and consumer uses and long half-lives in humans. Concern has been raised over chronic exposure effects to human health, especially in relation to cholesterol metabolism. Here, we explore the association between exposure to PFOA and PFOS and the in vivo expression of genes involved in cholesterol metabolism. We studied 290 individuals exposed to background levels of PFOS and elevated concentrations of PFOA through drinking water. Using adjusted linear regression models, we found inverse associations between serum PFOA levels and the whole blood expression level of genes involved in cholesterol transport (NR1H2, NPC1 and ABCG1; p=0.002, 0.026 and 0.014 respectively). A positive association was seen between PFOS and a transcript involved in cholesterol mobilisation (NCEH1; p=0.018), and a negative relationship with a transcript involved in cholesterol transport (NR1H3; p=0.044). When sexes were analysed separately, reductions in the levels of mRNAs involved in cholesterol transport were seen with PFOA in men (NPC1, ABCG1, and PPARA; p=0.025, 0.024 and 0.012 respectively) and in women (NR1H2 expression; p=0.019), whereas an increase in the levels of a cholesterol mobilisation transcript (NCEH1; p=0.036) was noted in women alone. PFOS was positively associated with expression of genes involved in both cholesterol mobilisation and transport in women (NCEH1 and PPARA; p=0.003 and 0.039 respectively), but no effects were evident in men. This is the first report of associations between the in vivo expression of genes involved in cholesterol metabolism and exposure to PFOA or PFOS, suggested that exposure to these compounds may promote a hypercholesterolaemic environment, with wider implications for human disease.",
"Perfluorooctanoic acid (PFOA) is a man-made surfactant with a number of industrial applications. It has a long half-life environmentally and biologically. Past studies suggest a direct relationship between plasma cholesterol and PFOA serum concentrations in humans and an inverse one in rodents fed standard rodent chow, making it difficult to examine mechanisms responsible for the potential PFOA-induced hypercholesterolemia and altered sterol metabolism. To examine dietary modification of PFOA-induced effects, C57BL/6 and BALB/c mice were fed PFOA in a fat- and cholesterol-containing diet. When fed these high fat diets, PFOA ingestion resulted in marked hypercholesterolemia in male and female C57BL/6 mice and less robust hypercholesterolemia in male BALB/c mice. The PFOA-induced hypercholesterolemia appeared to be the result of increased liver masses and altered expression of genes associated with hepatic sterol output, specifically bile acid production. mRNA levels of genes associated with sterol input were reduced only in C57BL/6 females, the mice with the greatest increase in plasma cholesterol levels. Strain-specific PFOA-induced changes in cholesterol concentrations in mammary tissues and ovaries paralleled changes in plasma cholesterol levels. mRNA levels of sterol-related genes were reduced in ovaries of C57BL/6 but not in BALB/c mice and not in mammary tissues. Our data suggest that PFOA ingestion leads to hypercholesterolemia in mice fed fat and cholesterol and effects are dependent upon the genetic background and gender of the mice with C57BL/6 female mice being most responsive to PFOA."
] |
Meta-Analysis of the Prognostic Value of Tetraspanin CD82 in Malignant Neoplasms
|
Tetraspanin CD82, also known as KAI1, was revealed as an attractive prognostic tumor biomarker in recent studies. However, some results of these studies remained debatable and inconclusive. Therefore, we conducted a meta-analysis to clarify the precise predictive value of CD82 in various neoplasms. Qualified studies were identified up to April 27, 2017, by searching PubMed, EMBASE, and the Web of Science. In total, 29 eligible studies were ultimately enrolled in this meta-analysis. Pooled hazard ratios (HRs) with 95% CIs of overall survival and disease/recurrence/progression-free survival were calculated to evaluate the correct prognostic role of CD82. Statistical analysis demonstrated that high expression of CD82 was significantly associated with enhanced overall survival (HR =0.56, 95% CI: 0.47-0.67) and disease/recurrence/progression-free survival (HR =0.42, 95% CI: 0.30-0.59) in cancer patients. Furthermore, we also conducted the subgroup analysis and the results revealed that CD82 was associated with favorable outcomes in cancer patients. Taken together, CD82 could be a promising biomarker for predicting the prognosis of patients with malignant neoplasms, and the biological functions of CD82 are of great research value of the subject.
|
[
"KAI1 (CD82) belongs to the transmembrane 4 superfamily in which members have inhibitory effects on tumor cell motility and metastasis. During reverse transcription-PCR analysis, we found a splice variant of KAI1 (spliced-KAI1) in which exon 7 was deleted. This exon encodes the 28 amino acids that span from the distal part of the second extracellular loop to the proximal part of the fourth transmembrane region. Expression of spliced-KAI1 was observed in metastatic tissues of gastric cancer patients with poor prognosis after operation. Genomic DNA analysis revealed that this variant was derived from the alternative splicing of exon 7. Immunoprecipitation showed that the interaction of spliced-KAI1 with integrin alpha(3)beta(1) was weaker than that of wild-type KAI1. Wild-type KAI1, but not spliced-KAI1, colocalized with E-cadherin, an adherens junction protein. Also, mouse colon adenocarcinoma cells stably expressing spliced-KAI1 (CT-26/spliced-KAI1) showed increased in vivo tumorigenicity, as well as increased in vitro invasive potential and cell-extracellular matrix adhesion compared with wild-type KAI1-expressing cells. In metastatic lung and liver tissues from mice inoculated with CT-26/spliced-KAI1 cells, the expression of wild-type KAI1 was nearly absent and spliced-KAI1 was dominant, and weak interaction of KAI1 with integrin alpha(3)beta(1) was observed. These results indicate that there is a functional difference between wild-type KAI1 and spliced-KAI1 in respect to cell motility, adhesion, tumor growth and metastasis, and expression of spliced-KAI1 may be a marker for poor prognostic factors in gastric and other cancers.",
"The recent identification of metastasis suppressor genes, uniquely responsible for negatively controlling cancer metastasis, are providing inroads into the molecular machinery involved in metastasis. While the normal function of a few of these genes is known; the molecular events associated with their loss that promotes tumor metastasis is largely not understood. KAI1/CD82, whose loss is associated with a wide variety of metastatic cancers, belongs to the tetraspanin family. Despite intense scrutiny, many aspects of how CD82 specifically functions as a metastasis suppressor and its role in normal biology remain to be determined. This review will focus on the molecular events associated with CD82 loss, the potential impact on signaling pathways that regulate cellular processes associated with metastasis, and its relationship with other metastasis suppressor genes.",
"The prevention and treatment of prostate cancer metastasis continue to provide a significant clinical challenge. Identification of the rate limiting steps of metastasis and their underlying molecular mechanisms may lead to new therapeutic targets and also allow more accurate risk stratification for clinical metastases. We review the literature supporting growth of disseminated tumor cells at the secondary site as a key rate limiting step in metastasis. We also reviewed the definition, identification and characterization of metastasis suppressor genes, and discuss their evolving role in regulating this step. We performed MEDLINE searches and manual bibliographic reviews on the specific steps of metastasis, including growth at the secondary site. In addition, we performed a comprehensive literature review to identify genes fitting the classic definition of a metastasis suppressor gene. The literature was also searched to assess the status of each gene in clinical cancer and evaluate functional support for the potential involvement of each gene in regulating growth at the secondary site. Clinical studies in prostate cancer and other cancer types suggest that dissemination to the secondary site is often an early clinical event. However, not all patients with tumor cells at the secondary site have overt metastatic lesions even in the absence of therapy, suggesting that growth at the secondary site may be highly inefficient. Complimentary approaches have allowed researchers to document and quantify the inefficiency of cancer cell growth at the secondary site. Regarding the mechanism of growth control, many studies support a role for the interaction of a cancer cell and the microenvironment at the secondary site influencing whether growth into metastasis may occur. The 7 genes that suppress metastasis without affecting primary tumor growth that have been identified are KAI1, CD44, mitogen activated protein kinase (MAPK) kinase 4, nm23-H1, nm23-H2, KiSS1 and BrMS1. Three of these genes (KAI1, CD44 and MAPK kinase 4) act as metastasis suppressor genes of prostate cancer, while the remainder have yet to be tested in this cancer type. Loss of expression has been demonstrated for most of these genes during the clinical progression of prostate cancer to metastasis. MAPK kinase 4 and KiSS1 appear to suppress metastasis by inhibiting cancer cell growth at the secondary site. Interestingly many metastasis suppressor genes have common roles in growth control, adhesion and cytoskeletal reorganization, suggesting a common mechanism of metastasis suppression. Proposed candidate pathways include signaling through Src kinase and Rac GTPase. The findings discussed support growth at the secondary site as a clinical target for metastasis treatment and prevention. Metastasis suppressor genes may offer valuable mechanistic insight for guiding specific therapeutic strategies, which may include drug induced reactivation of metastasis suppressor genes and their signaling pathways. Clinical assessment of metastasis suppressor gene product status in disseminated cancer cells may improve the accuracy of predicting the prognosis in patients with clinically localized disease.",
"The KAI1/CD82 gene, the product of which is a member of the transmembrane-4 superfamily, is a suppressor of metastasis; as a result, it is inversely associated with tumor progression and is a favorable prognostic factor in some tumors. This study was performed to determine the prognostic value of KAI1/CD82 protein levels in nonsmall cell lung carcinoma (NSCLC). In addition, levels of KAIl/CD82 expression in metastatic lesions were determined and compared with those in primary NSCLC lesions. KAI1/CD82 expression in 200 NSCLC patients who underwent potentially curative surgery was immunohistochemically detected with C33, an anti-KAI1/CD82 monoclonal antibody. According to the degree of KAI1/CD82 positive cancer cells within the tumor tissue, each sample was classified as KAI1/CD82 positive, KAI1/CD82 reduced, or KAI1/CD82 negative. Sixty-five samples (32.5%) were KAI1/CD82 positive, 31 (15.5%) were reduced, and 104 (52%) were negative. There was no significant association between KAI1/CD82 expression and clinicopathologic factors, but patients who were positive for KAI1/CD82 expression had significantly favorable prognoses for overall survival (P = 0.0026) and disease free survival (DFS; P = 0.0007) compared with the other groups. In particular, among patients with adenocarcinoma, similar results were even more significant. In multivariate analysis, immunohistochemical KAI1/CD82 expression in patients with NSCLC was an independent prognostic factor for overall survival and DFS; in those with adenocarcinoma, it was an even more valuable factor. In some patients with NSCLC, especially those with adenocarcinoma, KAI1/CD82 expression levels in metastatic lesions were diminished compared with levels of expression in the primary lung lesions. The immunohistochemically determined level of KAI1/CD82 expression in NSCLC cells within tumor tissue appears to be a favorable prognostic factor for overall survival as well as DFS. The results of this study suggest that decreased KAI1/CD82 expression may be associated with tumor progression and enhanced metastatic potential in some patients with this disease.",
"The present study aimed to explore the effect and mechanism of the Kangai 1 (KAI1) gene in regulating the migration and invasion of gastric carcinoma cells, and the prognostic significance of this gene in gastric cancer patients. Immunohistochemistry and in situ hybridization were used to investigate the role of KAI1 in the progression and prognosis of gastric cancer. The pEGFP-N1-KAI1 plasmid was transfected into human gastric carcinoma SGC7901 cells using liposomes. The effect of transfection with the KAI1 gene was measured using a reverse transcription-semi-quantitative polymerase chain reaction (RT-sqPCR) assay. The Transwell chamber assay was used to study the metastatic and invasive ability of SGC7901 cells. Gastric cancer metastasis-associated genes, including hypoxia-inducible factor (HIF)-1α, matrix metalloproteinase (MMP)-2, MMP-9, basic fibroblast growth factor (bFGF), and urease plasminogen activator (uPA) were measured by RT-sqPCR prior to and following transfection with the KAI1 gene. The expression of KAI1 protein and mRNA was associated with the differentiation degree of gastric cancer, presence of lymph node metastasis, tumor-node-metastasis stage, depth of invasion and the survival time of patients. The migratory and invasive abilities of SGC7901 cells were significantly decreased subsequent to transfection with the KAI1 gene, and the expression of bFGF and uPA was downregulated. It was concluded that the tumor suppressor gene KAI1 inhibits the migration and invasion of gastric carcinoma cells, possibly by suppressing the expression of uPA. Patients that expressed KAI1 may demonstrate an improved prognosis."
] |
E5 regulates epithelial proliferation and differentiation in human papillomavirus18 cells
|
Deregulation of proliferation and differentiation-dependent signalling pathways is a hallmark of human papillomavirus (HPV) infection. Although the manipulation of these pathways by E6 and E7 has been extensively studied, controversies surround the role of the E5 oncoprotein during a productive virus life cycle. By integrating primary keratinocytes harbouring wild type or E5 knockout HPV18 genomes with pharmacological and gain/loss of function models, this study aimed to provide molecular information about the role of E5 in epithelial proliferation and differentiation. We show that E5 contributes to cell cycle progression and unscheduled host DNA synthesis in differentiating keratinocytes. E5 function correlates with increased EGFR activation in differentiating cells and blockade of this pathway impairs differentiation-dependent cell cycle progression of HPV18 containing cells. Our findings provide a functional requirement of enhanced EGFR signalling for suprabasal cellular DNA synthesis during the virus life cycle. They also reveal an unrecognised contribution of E5 towards the impaired keratinocyte differentiation observed during a productive HPV infection. E5 suppresses a signalling axis consisting of the keratinocyte growth factor receptor (KGFR) pathway. Inhibition of this pathway compensates for the loss of E5 in knockout cells and re-instates the delay in differentiation. The negative regulation of KGFR involves suppression by the EGFR pathway. Thus our data reveal an unappreciated role for E5-mediated EGFR signalling in orchestrating the balance between proliferation and differentiation in suprabasal cells.
|
[
"E5 proteins are amongst the least understood of the Human Papillomavirus (HPV) encoded gene products. They are small, membrane-integrated proteins known to modulate a number of critical host pathways associated with pathogenesis including growth factor receptor signaling and immune evasion. Their role in the virus life cycle is less clear, indicating a role in the productive stages of the life cycle. However, a mechanism for this is currently lacking. Here we describe the identification of a novel binding partner of E5, YIPF4 using yeast two-hybrid analysis. YIPF4 is also a poorly characterized membrane spanning protein. Mutagenesis studies implicated the transmembrane regions of each protein as important for their interaction. Binding to YIPF4 was found for all E5 proteins tested suggesting that this interaction may mediate a conserved E5 function. In normal human keratinocytes YIPF4 expression was down-regulated upon differentiation and this reduction was partially rescued in cells harbouring HPV. Despite the conserved nature of the interaction with E5, siRNA mediated depletion of YIPF4 failed to impede two well-characterized functions of E5, namely EGFR trafficking or HLA class I presentation. Continued studies of YIPF4 are warranted to determine its role in the PV life cycle.",
"E5 oncoprotein activity from high risk human papillomaviruses (HPVs) is associated with growth factor receptor signaling, but the function of this protein is not well understood. In this study, we investigated the role of HPV-16 E5 on the cell cycle progression during EGF-stimulation. Wild-type and NIH 3T3 cells over-expressing human EGF-receptor were transfected with HPV-16 E5 gene and the cell cycle progression was characterized. This analysis showed that the E5-expressing cells increased DNA synthesis (S-phase) by around 40%. Cell cycle protein analysis of E5-expressing cells showed a reduction in the half-life of p27(Kip1) protein as compared to control cells (18.4 vs. 12.7 h), an effect that was enhanced in EGF-stimulated cells (12.8 vs. 3.6 h). Blockage of EGF-receptor activity abrogated E5 signals as well as p27(Kip1) down-regulation. These results suggest that E5 and the EGF-receptor cooperate to enhance cell cycle entry and progression through regulating p27(Kip1) expression at protein level.",
"Activation of the productive phase of the human papillomavirus (HPV) life cycle in differentiated keratinocytes is coincident with high-level expression of E1E4 protein. To determine the role of E1E4 in the HPV replication cycle, we constructed HPV18 mutant genomes in which expression of the full-length E1E4 protein was abrogated. Undifferentiated keratinocytes containing mutant genomes showed enhanced proliferation when compared to cells containing wildtype genomes, but there were no differences in maintenance of viral episomes. Following differentiation, cells with mutant genomes exhibited reduced levels of viral DNA amplification and late gene expression, compared to wildtype genome-containing cells. This indicates that HPV18 E1E4 plays an important role in regulating HPV late functions, and it may also function in the early phase of the replication cycle. Our finding that full-length HPV18 E1E4 protein plays a significant role in promoting viral genome amplification concurs with a similar report with HPV31, but is in contrast to an HPV11 study where viral DNA amplification was not dependent on full-length E1E4 expression, and to HPV16 where only C-terminal truncations in E1E4 abrogated vegetative genome replication. This suggests that type-specific differences exist between various E1E4 proteins.",
"A family of five-pass transmembrane proteins (FinGERs) were identified from the protein sequence database. The family includes yeast Yip1p, Yip4p, Yip5p, and Yif1p, and also their plant, insects, nematode, and mammalian homologues, suggesting their conserved function in a broad range of species. Eight family members were found in human. Multiple sequence alignment revealed three regions conserved among all family members. All of the human family members were expressed widely in various tissues. The human proteins were localized in and around the Golgi apparatus and may also be in the ER to some extent. The Golgi apparatus was fragmented by overexpression of the five of the family members. Some of the members were found to interact by yeast two-hybrid analysis, suggesting the formation of a complex. These results suggest that FinGERs function in maintenance of the Golgi structure and/or transport between the ER and the Golgi apparatus."
] |
Immunogenicity of H5N1 and H7N9 avian influenza vaccines in nave ferrets
|
Conventional inactivated avian influenza vaccines have performed poorly in past vaccine trials, leading to the hypothesis that they are less immunogenic than seasonal influenza vaccines. We tested this hypothesis by comparing the immunogenicity of the H5N1 and H7N9 vaccines (avian influenza vaccines) to a seasonal trivalent inactivated influenza vaccine in naïve ferrets, administered with or without the adjuvants MF59 or AS03. Vaccine immunogenicity was assessed by measuring neutralizing antibody titers against hemagglutinin and neuraminidase and by hemagglutinin -specific IgG levels. Two doses of unadjuvanted vaccines induced low or no HA-specific IgG responses and hemagglutination-inhibiting titers. Adjuvanted vaccines induced comparable IgG-titers, but poorer neutralizing antibody titers for the H5 vaccine. All adjuvanted vaccines elicited detectable anti- neuraminidase -antibodies with the exception of the H5N1 vaccine, likely due to the low amounts of neuraminidase in the vaccine. Overall, the H5N1 vaccine had poorer capacity to induce neutralizing antibodies, but not HA-specific IgG, compared to H7N9 or trivalent inactivated influenza vaccine.
|
[
"Data are needed from large clinical trials of paediatric, adult, and elderly people to find the appropriate antigen dose and vaccination schedule for the 2009 pandemic influenza A H1N1. We therefore report preliminary safety and immunogenicity results after one injection of a licensed monovalent pandemic H1N1 vaccine in the USA. We randomly assigned healthy children (aged 6-35 months and 3-9 years) and adults (18-64 years and >or=65 years) to vaccine containing per dose 7.5 microg (children and adults), 15 microg (children and adults), or 30 microg (adults only) haemagglutinin in two placebo-controlled, observer-masked, multicentre phase 2 studies done in the USA. Participants were allocated with an interactive voice-response system or computer-generated randomisation lists with opaque scratchable patches. Primary outcome was haemagglutination inhibition antibody response 21 days after the first of two planned vaccinations (interim analysis of studies in progress). Analyses were by full-analysis set. The trials are registered with ClinicalTrials.gov as NCT00953524 and NCT00952419. 410 of 423 children and 724 of 750 adults given an active vaccine, and 50 of 51 children and 95 of 99 adults given placebo were assessed for immunogenicity on day 21. After active vaccination, 45 of 101 (45%; 95% CI 35-55) to 47 of 94 (50%; 40-61) infants aged 6-35 months, 75 of 109 (69%; 59-77) to 80 of 106 (75%; 66-83) 3-9-year-old children, 134 of 141 (95%; 90-98) to 144 of 144 (100%; 98-100) of 18-64-year-old adults, and 93 of 100 (93%; 86-96) to 93 of 98 (95%; 89-98) elderly adults were seroprotected (proportion with titres >or=1:40). No vaccine-related serious adverse events occurred. Injection-site and systemic reactions were reported by up to about 50% of every age and vaccine group, with no noticeable differences between vaccine and placebo groups. One dose of vaccine was highly immunogenic in adults, suggesting that it afforded sufficient protection against this pandemic influenza A H1N1 virus. Two doses of vaccine will probably be needed in children younger than 9 years. Safety and reactogenicity of the vaccine were acceptable and similar to those of seasonal vaccine. Office of the Assistant Secretary for Preparedness and Response, and Biomedical Advanced Research and Development Authority.",
"A miniaturized neuraminidase inhibition (NI) assay using HA-mismatched H6 reassortant viruses was performed to examine the neuraminidase (NA)-specific antibody response in ferrets immunized with live-attenuated influenza vaccine (LAIV) strains. The strains tested possessed different NAs derived from seasonal H1N1 and H3N2, 2009 pandemic H1N1, and the highly pathogenic influenza H5N1 virus. The anti-NA antibodies from the 2009 pandemic strain (A/California/7/2009) immunized ferrets cross-reacted with the NA of H5N1 but not with the NA of seasonal H1N1 viruses. The plaque size reduction assay confirmed the cross-reactivity between the NAs of A/California/7/2009 and the H5N1 virus. Sequence and structural analyses of these N1 NA proteins showed that the NA of the 2009 pandemic H1N1 strain shared at least 22 more amino acids in the head domain with the NAs of the avian H5N1 strains than with the NAs of seasonal human H1N1 viruses. Our data demonstrated LAIV-induced NA antibody responses in ferrets and cross-reactive NA antibodies induced by 2009 pandemic H1N1 and H5N1 LAIV viruses.",
"Humans infected by the highly pathogenic H5N1 avian influenza viruses (HPAIV) present unusually high concentrations in serum of proinflammatory cytokines and chemokines, which are believed to contribute to the high pathogenicity of these viruses. The hemagglutinins (HAs) of avian influenza viruses preferentially bind to sialic acids attached through α2,3 linkages (SAα2,3) to the terminal galactose of carbohydrates on the host cell surface, while the HAs from human strains bind to α2,6-linked SA (SAα2,6). To evaluate the role of the viral receptor specificity in promoting innate immune responses in humans, we generated recombinant influenza viruses, one bearing the HA and neuraminidase (NA) genes from the A/Vietnam/1203/2004 H5N1 HPAIV in an influenza A/Puerto Rico/8/1934 (A/PR/8/34) backbone with specificity for SAα2,3 and the other a mutant virus (with Q226L and G228S in the HA) with preferential receptor specificity for SAα2,6. Viruses with preferential affinity for SAα2,3 induced higher levels of proinflammatory cytokines and interferon (IFN)-inducible genes in primary human dendritic cells (DCs) than viruses with SAα2,6 binding specificity, and these differences were independent of viral replication, as shown by infections with UV-inactivated viruses. Moreover, human primary macrophages and respiratory epithelial cells showed higher expression of proinflammatory genes after infection with the virus with SAα2,3 affinity than after infection with the virus with SAα2,6 affinity. These data indicate that binding to SAα2,3 by H5N1 HPAIV may be sensed by human cells differently than binding to SAα2,6, inducing an exacerbated innate proinflammatory response in infected individuals.",
"In preparing for the threat of a pandemic of avian H5N1 influenza virus, we need to consider the significant delay (4 to 6 months) necessary to produce a strain-matched vaccine. As some degree of cross-reactivity between seasonal influenza vaccines and H5N1 virus has been reported, this was further explored in the ferret model to determine the targets of protective immunity. Ferrets were vaccinated with two intramuscular inoculations of trivalent inactivated split influenza vaccine or subcomponent vaccines, with and without adjuvant, and later challenged with a lethal dose of A/Vietnam/1203/2004 (H5N1) influenza virus. We confirmed that vaccination with seasonal influenza vaccine afforded partial protection against lethal H5N1 challenge and showed that use of either AlPO(4) or Iscomatrix adjuvant with the vaccine resulted in complete protection against disease and death. The protection was due exclusively to the H1N1 vaccine component, and although the hemagglutinin contributed to protection, the dominant protective response was targeted toward the neuraminidase (NA) and correlated with sialic acid cleavage-inhibiting antibody titers. Purified heterologous NA formulated with Iscomatrix adjuvant was also protective. These results suggest that adjuvanted seasonal trivalent vaccine could be used as an interim measure to decrease morbidity and mortality from H5N1 prior to the availability of a specific vaccine. The data also highlight that an inducer of cross-protective immunity is the NA, a protein whose levels are not normally monitored in vaccines and whose capacity to induce immunity in recipients is not normally assessed."
] |
Effects of 2,2',4,4'-tetrabromodiphenyl ether on liver metabolism in mice and human hepatocellular carcinoma cells
|
Polybrominated diphenyl ethers (PBDEs) were used as flame-retardant additives in a wide range of polymers. The generations born when environmental concentrations of PBDEs reached their maximum account in the United States for one-fifth of the total population. We hypothesized that exposure to PBDEs during sensitive developmental windows might result in long-lasting changes in liver metabolism. The present study was based on experiments with CD-1 mice and human hepatocellular carcinoma cells (human hepatoma cell line, HepG2). Pregnant mice were exposed to 0.2 mg/kg 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) from gestation day 8 until postnatal day 21. The metabolic health-related outcomes were analyzed on postnatal day 21 and postnatal week 20 in male offspring. Several groups of metabolic genes, including ribosomal and mitochondrial genes, were significantly upregulated in the liver at both points. Genes regulated via mechanistic target of rapamycin (mTOR) pathway, the gatekeeper of metabolic homeostasis, were whether up- or downregulated at both measurement points. On postnatal day 21, but not week 20, both mTOR complexes in the liver were activated, as measured by phosphorylation of their targets. mTOR complexes were also activated by BDE-47 in HepG2 cells
|
[
"The amount of milk removed from rats during 1 hour's suckling after a separation period of 8 hours was estimated by weighing their pups. In the 1st experiment litters of 9 pups were suckled by all rats. At test sucklings held daily on days 6-15 of lactation, pups were reallocated to give 3, 6, 9, 12 or 15 pups per dam. The largest amount of milk was removed at suckling by groups of 12 pups. In a 2nd experiment, rats were constantly sucked by litters of either 3, 6, 9, 12 or 15 pups from birth to slaughter at 15 days post partum. Rats with 12 and 15 pups yielded the most milk, nearly twice as much as those suckling only 6 or 9 pups. Litters of 3 pups consumed less than 1/3 of the amount consumed by litters of 6 or 9 pups.",
"Three brominated flame retardants (BFRs), tetrabromobisphenol A (TBBPA), hexabromocyclododecane (HBCD) and polybrominated diphenyl ethers (PBDEs), were measured in 103 human milk samples collected from Beijing in 2011. The donors' personal information, such as dietary habit and socioeconomic and lifestyle factors, was obtained by questionnaires. Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) analysis indicated that the levels of TBBPA ranged from <LOD to 12.46 ng g(-1) lipid weight (lw), with a median value of 0.10 ng g(-1) lw. Three HBCD diastereoisomers (α-, β- and γ-HBCD) were also measured using UPLC-MS/MS. The levels of ΣHBCDs ranged from <LOD to 78.28 ng g(-1) lw, with a median value of 2.40 ng g(-1) lw. α-HBCD was generally the most abundant of the three isomers. Eight PBDE congeners, BDE-28, 47, 99, 100, 153, 154, 183 and 209, were measured using gas chromatography coupled with a mass spectrometry (GC/MS). The concentrations of ΣPBDEs ranged from 0.22 to 135.41 ng g(-1) lw, with a median value of 3.24 ng g(-1) lw. BDE-209 dominated the PBDE profile in the majority of the human milk samples. The mean estimated daily intakes (EDIs) of TBBPA, ΣHBCDs and ΣPBDEs by breast-fed infants were 2.34, 24.89 and 71.27 ng kg(-1) bw day(-1), respectively. No significant correlation was found between the BFR levels in milk and the mother's diet, place of residence, smoking habit, nursing duration or computer use habit. In contrast, the mother's age, body mass index (BMI), education level and number of computers in the home were related to the levels of some types of BFRs. More research is needed to further investigate the major source(s) of exposure, the effect of each potential factor and the possible toxicological impact of high daily BFR intake on infants.",
"BDE-47 is the most prevalent congener of polybrominated diphenyl ethers, which are widely used flame retardants, and is known for endocrine and behavioral disrupting properties in animals. Transient effect on spontaneous motor activity in rats following perinatal exposure to BDE-47 at low doses, relevant to human exposure, was reported in our previous study. The objective of this study was to screen for the long-term effects on gene expression in the brain of rats perinatally exposed to BDE-47. Wistar dams were exposed to BDE-47 (0.002 and 0.2 mg kg(-1) body weight) from gestation day 15 to postnatal day (PND) 20. Total RNA was extracted from the whole brain at PND10 and the brain frontal lobes at PND41 and hybridized to whole-genome RNA expression microarrays. The genes, differentially expressed 1.5-fold, were analyzed with the DAVID bioinformatics resources for cluster and gene-term enrichment. At PND41, clusters of genes involved in nerve impulse transmission, nervous system development and functioning, and core biosynthetic process were altered, including several downregulated genes of cation channels. Representation of LINE1 RNA was decreased significantly. Altered expression of genes involved in neurodevelopment occured at least 3 weeks after the last exposure and the behavioral manifestation of low dose BDE-47 toxicity.",
"Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that exists in two separate complexes, mTORC1 and mTORC2, that function to control cell size and growth in response to growth factors, nutrients, and cellular energy levels. Low molecular weight GTP-binding proteins of the Rheb and Rag families are key regulators of the mTORC1 complex, but regulation of mTORC2 is poorly understood. Here, we report that Rac1, a member of the Rho family of GTPases, is a critical regulator of both mTORC1 and mTORC2 in response to growth-factor stimulation. Deletion of Rac1 in primary cells using an inducible-Cre/Lox approach inhibits basal and growth-factor activation of both mTORC1 and mTORC2. Rac1 appears to bind directly to mTOR and to mediate mTORC1 and mTORC2 localization at specific membranes. Binding of Rac1 to mTOR does not depend on the GTP-bound state of Rac1, but on the integrity of its C-terminal domain. This function of Rac1 provides a means to regulate mTORC1 and mTORC2 simultaneously.",
"Outbred CD-1 mice received subcutaneous injections on neonatal days 1-5 with DES (0.0001-1000 microg/kg per day), a model xenoestrogen. At 17 days of age, uterine wet weight increase in response to estrogen was altered in neonatally DES-treated mice compared to controls. The response varied depending on the neonatal DES dose; a low dose (0.01 microg/kg) caused an enhanced uterine response but higher neonatal doses dampened the response. Western blots and immunolocalization of estrogen receptor alpha (ERalpha) showed high ER levels at DES 0.01 microg/kg, but decreased levels at higher doses compared to controls. Genes responding through ER-mediated pathways (c-fos, proliferating cell nuclear antigen (PCNA), and lactoferrin (LF)) mirrored altered wet weight responses, i.e., enhancement at low doses and dampening at higher doses. A similar dose-response curve was seen in 4 months old ovariectomized DES-treated mice suggesting the altered response was long-term. These data suggest xenoestrogen exposure during critical developmental windows alters hormone programming so that the uterus responds abnormally to estrogen later in life, and that the response differs following high versus low doses of neonatal exposure.",
"An endocrine-disrupting chemical (EDC) is an exogenous chemical, or mixture of chemicals, that can interfere with any aspect of hormone action. The potential for deleterious effects of EDC must be considered relative to the regulation of hormone synthesis, secretion, and actions and the variability in regulation of these events across the life cycle. The developmental age at which EDC exposures occur is a critical consideration in understanding their effects. Because endocrine systems exhibit tissue-, cell-, and receptor-specific actions during the life cycle, EDC can produce complex, mosaic effects. This complexity causes difficulty when a static approach to toxicity through endocrine mechanisms driven by rigid guidelines is used to identify EDC and manage risk to human and wildlife populations. We propose that principles taken from fundamental endocrinology be employed to identify EDC and manage their risk to exposed populations. We emphasize the importance of developmental stage and, in particular, the realization that exposure to a presumptive \"safe\" dose of chemical may impact a life stage when there is normally no endogenous hormone exposure, thereby underscoring the potential for very low-dose EDC exposures to have potent and irreversible effects. Finally, with regard to the current program designed to detect putative EDC, namely, the Endocrine Disruptor Screening Program, we offer recommendations for strengthening this program through the incorporation of basic endocrine principles to promote further understanding of complex EDC effects, especially due to developmental exposures.",
"Numerous studies indirectly implicate Rac GTPases in cancer. To investigate if Rac3 contributes to normal or malignant cell function, we generated rac3 null mutants through gene targeting. These mice were viable, fertile, and lacked an obvious external phenotype. This shows Rac3 function is dispensable for embryonic development. Bcr/Abl is a deregulated tyrosine kinase that causes chronic myelogenous leukemia and Ph-positive acute lymphoblastic leukemia in humans. Vav1, a hematopoiesis-specific exchange factor for Rac, was constitutively tyrosine phosphorylated in primary lymphomas from Bcr/Abl P190 transgenic mice, suggesting inappropriate Rac activation. rac3 is expressed in these malignant hematopoietic cells. Using lysates from BCR/ABL transgenic mice that express or lack rac3, we detected the presence of activated Rac3 but not Rac1 or Rac2 in the malignant precursor B-lineage lymphoblasts. In addition, in female P190 BCR/ABL transgenic mice, lack of rac3 was associated with a longer average survival. These data are the first to directly show a stimulatory role for Rac in leukemia in vivo. Moreover, our data suggest that interference with Rac3 activity, for example, by using geranyl-geranyltransferase inhibitors, may provide a positive clinical benefit for patients with Ph-positive acute lymphoblastic leukemia."
] |
are traditional healers accepted in south africa?
|
Traditional healers are acceptable and highly accessible health practitioners throughout sub-Saharan Africa. Patients in South Africa often seek concurrent traditional and allopathic treatment leading to medical pluralism.
|
[
"To measure the direct cost burdens (health care expenditure as a percent of total household expenditure) for households in rural South Africa, and examine the expenditure and use patterns driving those burdens, in a setting with free public primary health care and hospital exemptions for the poor. Data on illness events, treatment patterns and health expenditure in the previous month were assessed from a cross-sectional survey of 280 households conducted in the Agincourt Health and Demographic Surveillance site, South Africa. On average, a household experiencing illness incurred a direct cost burden of 4.5% of total household expenditure. A visit to a public clinic generated a mean burden of 1.3%. Complex sequences of treatments led 20% of households to incur a burden over 10%, with transport costs generating 42% of this burden. An outpatient public hospital visit generated a burden of 8.2%, as only 58% of those eligible obtained an exemption; inpatient stays incurred a burden of 45%. Consultations with private providers incurred a mean burden of 9.5%. About 38% of individuals who reported illness did not take any treatment action, 55% of whom identified financial and perceived supply-side barriers as reasons. The low overall mean cost burden of 4.5% suggests that free primary care and hospital exemptions provided financial protection. However, transport costs, the difficulty of obtaining hospital exemptions, use of private providers, and complex treatment patterns meant state-provided protection had limitations. The significant non-use of care shows the need for other measures such as more outreach services and more exemptions in rural areas. The findings also imply that fee removal anywhere must be accompanied by wider measures to ensure improved access.",
"This paper describes current support for mental health care user participation in policy development and implementation in South Africa and suggests strategies for improving participation. The World Health Organization (WHO) Mental Health Policy Checklist and WHO Mental Health Legislation Checklist were completed. Between August 2006 and August 2009 96 semi-structured interviews with national, regional and district stakeholders were conducted. Most respondents felt that inclusion of user perspectives in policy processes would improve policy development. In practice, mental health care user consultation in policy development and implementation has been limited during the 16 years of democracy in South Africa. Strategies to create a supportive environment for user participation include social action directed at reducing stigma, advocating for acceptance of users' rights to participate in decision making, crafting a supportive regulatory framework to promote participation, and equipping providers and policy makers to support inclusion. User capacity for participation could be strengthened through early and effective access to treatment and support, development of a national user lobby, skills training and practical exposure to the policy and service development environment.",
"While South Africa has a new policy framework supporting the integration of mental health care into primary health care, this is not sufficient to ensure transformation of the health care system towards integrated primary mental health care. Health systems strengthening is needed, incorporating, inter alia, capacity building and resource inputs, as well as good governance for ensuring that the relevant policy imperatives are implemented. To identify systemic factors within institutional and policy contexts that are likely to facilitate or impede the implementation of integrated mental health care in South Africa. Semi-structured qualitative interviews were conducted with 17 key stakeholders in the Department of Health and Department of Social Development at national level, at provincial level in the North West Province, and at district level in the Dr Kenneth Kaunda district. Participants were purposively identified based on their positions and job responsibilities. Interview questions were guided by a hybrid of Siddiqi et al.'s governance framework principles and Mikkelsen-Lopez et al.'s health system governance approach. Data were analysed using framework analysis in NVivo. Facilitative factors included the recent mental health care policy framework and national action plan that embraces integrated care using a task sharing model and provides policy imperatives for the establishment of district mental health teams to facilitate the development and implementation of district mental health care plans; the roll out of the integrated chronic disease service delivery platform that can be leveraged to increase access and resources as well as decrease stigma; and the presence of NGOs that can assist with service delivery. Challenges included the low prioritisation and stigmatisation of mental illness; weak managerial and planning capacity to develop and implement mental health care plans at provincial and district level; poor pre-service training of generalists in mental health care; weak orientation to integrated care; high staff turnover; weak intersectoral coordination; infrastructural constraints; and no dedicated mental health budget. This study identifies strategies to support and improve integrated mental health care in primary health care services."
] |
The anti-LINGO-1 antibody has immunomodulatory effects
|
To evaluate whether the anti-LINGO-1 antibody has immunomodulatory effects.
|
[
"Meningeal inflammation, including the presence of semi-organized tertiary lymphoid tissue, has been associated with cortical pathology at autopsy in secondary progressive multiple sclerosis (SPMS). Accessible and robust biochemical markers of cortical inflammation for use in SPMS clinical trials are needed. Increased levels of chemokines in the cerebrospinal fluid (CSF) can report on inflammatory processes occurring in the cerebral cortex of MS patients. A multiplexed chemokine array that included BAFF, a high sensitivity CXCL13 assay and composite chemokine scores were developed to explore differences in lymphoid (CXCL12, CXCL13, CCL19 and CCL21) and inflammatory (CCL2, CXCL9, CXCL10 and CXCL11) chemokines in a small pilot study. Paired CSF and serum samples were obtained from healthy controls (n=12), relapsing-remitting MS (RRMS) (n=21) and SPMS (N=12). A subset of the RRMS patients (n = 9) was assessed upon disease exacerbation and 1 month later following iv methylprednisone. SPMS patients were sampled twice to ascertain stability. Both lymphoid and inflammatory chemokines were elevated in RRMS and SPMS with the highest levels found in the active RRMS group. Inflammatory and lymphoid chemokine signatures were defined and generally correlated with each other. This small exploratory clinical study shows the feasibility of measuring complex and potentially more robust chemokine signatures in the CSF of MS patients during clinical trials. No differences were found between stable RRMS and SPMS. Future trials with larger patient cohorts with this chemokine array are needed to further characterize the differences, or the lack thereof, between stable RRMS and SPMS.",
"Gene-expression analysis is increasingly important in biological research, with real-time reverse transcription PCR (RT-PCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. Given the increased sensitivity, reproducibility and large dynamic range of this methodology, the requirements for a proper internal control gene for normalization have become increasingly stringent. Although housekeeping gene expression has been reported to vary considerably, no systematic survey has properly determined the errors related to the common practice of using only one control gene, nor presented an adequate way of working around this problem. We outline a robust and innovative strategy to identify the most stably expressed control genes in a given set of tissues, and to determine the minimum number of genes required to calculate a reliable normalization factor. We have evaluated ten housekeeping genes from different abundance and functional classes in various human tissues, and demonstrated that the conventional use of a single gene for normalization leads to relatively large errors in a significant proportion of samples tested. The geometric mean of multiple carefully selected housekeeping genes was validated as an accurate normalization factor by analyzing publicly available microarray data. The normalization strategy presented here is a prerequisite for accurate RT-PCR expression profiling, which, among other things, opens up the possibility of studying the biological relevance of small expression differences.",
"Accessory cell surface molecules, such as T cell antigen CD2 and its ligand lymphocyte function-associated antigen 3 (LFA-3; CD58), are critical costimulatory pathways for optimal T cell activation in response to antigens. Interaction of CD2 with cell surface LFA-3 not only increases T cell/accessory cell adhesion, but also induces signal transduction events involved in the regulation of T cell responses. In this report, we show that specific interactions of LFA-3 with CD2 can result in T cell unresponsiveness to antigenic or mitogenic stimuli in vitro. By deletion of certain regions of the extracellular domain of LFA-3, we localized the CD2 binding site to the first domain of LFA-3. We then demonstrated that a soluble, purified first domain-LFA-3/IgG1 fusion protein (LFA3TIP) interacts with CD2 and binds to the same CD2 epitope as purified multimeric or cell surface-expressed LFA-3. LFA3TIP inhibits tetanus toxoid, hepatitis B surface antigen, anti-CD3 mAb, Con A, and phytohemagglutinin P-induced T cell proliferation, as well as xenogeneic and allogeneic mixed lymphocyte reactions (MLR). Unlike anti-LFA-3 or anti-CD2 monoclonal antibodies (mAbs) which inhibit T cell responses by blocking LFA-3/CD2 binding, LFA3TIP is capable of rendering T cells unresponsive to antigenic stimuli in situations where T cell activation is independent of CD2/LFA-3 interactions. Furthermore, LFA3TIP, but not blocking anti-CD2 mAbs, is capable of inducing T cell unresponsiveness to secondary stimulation in allogeneic MLR. This inhibition of T cell responses by LFA3TIP occurs through a different mechanism from that of mAbs to LFA-3 or CD2.",
"Quantitative studies are commonly realised in the biomedical research to compare RNA expression in different experimental or clinical conditions. These quantifications are performed through their comparison to the expression of the housekeeping gene transcripts like glyceraldehyde-3-phosphate dehydrogenase (G3PDH), albumin, actins, tubulins, cyclophilin, hypoxantine phsophoribosyltransferase (HRPT), L32. 28S, and 18S rRNAs are also used as internal standards. In this paper, it is recalled that the commonly used internal standards can quantitatively vary in response to various factors. Possible variations are illustrated using three experimental examples. Preferred types of internal standards are then proposed for each of these samples and thereafter the general procedure concerning the choice of an internal standard and the way to manage its uses are discussed."
] |
is dexmedetomidine a sedative?
|
Using dexmedetomidine (Dex) as a sedative agent may benefit the clinical outcomes of post-surgery patients. We reviewed randomized controlled trials (RCTs) to assess whether use of a Dex could improve the outcomes in post-surgery critically ill adults.
|
[
"Despite the increasing use of anesthesiologist-administered sedation for monitored anesthesia care (MAC) or general anesthesia in patients undergoing ERCP, limited prospective data exist on the effectiveness, safety, and cost of this approach. To prospectively assess sedation-related adverse events (SRAEs), patient- and procedure-related risk factors associated with SRAEs, and endoscopist and patient satisfaction with anesthesiologist-administered sedation. Single-center, prospective cohort study. Tertiary-care referral center. A total of 528 consecutive patients undergoing ERCP. Anesthesiologist-administered MAC or general anesthesia. SRAEs, endoscopist and patient satisfaction. There were 120 intraprocedure SRAEs during 109 of the 528 ERCPs (21% of cases). Intraprocedure SRAEs included hypotension (38 events), arrhythmia (20 events), O(2) desaturation to less than 85% (66 events), unplanned intubation (16 events), and procedure termination (1 event). Thirty postprocedure SRAEs occurred in a total of 22 patients (4% of cases), including hypotension (5 events), endotracheal intubation (2 events), and arrhythmia (12 events). Patient-related variables associated with adverse intraprocedure events were American Society of Anesthesiologists class (P = .004) and body mass index (kg/m(2)) (P = .02). On a 10-point scale, mean endoscopist satisfaction with sedation was 9.2 (standard deviation 1.8) and patient satisfaction with sedation was 9.9 (standard deviation 0.7). The approach to sedation was not randomized. Higher American Society of Anesthesiologists class and body mass index are associated with an increased rate of cardiac and respiratory events during ERCP. Cardiac and respiratory events are generally minor, and MAC can be considered a safe option for most ERCP patients. Despite the frequency of minor sedation-related events, procedure interruption or premature termination was rare in the setting of anesthesiologist-administered sedation.",
"The use of dexmedetomidine may have benefits on the clinical outcomes of cardiac surgery. We conducted a meta-analysis comparing the postoperative complications in patients undergoing cardiac surgery with dexmedetomidine versus other perioperative medications to determine the influence of perioperative dexmedetomidine on cardiac surgery patients. Randomized or quasi-randomized controlled trials comparing outcomes in patients who underwent cardiac surgery with dexmedetomidine, another medication, or a placebo were retrieved from EMBASE, PubMed, the Cochrane Library, and Science Citation Index. A total of 1702 patients in 14 studies met the selection criteria among 1,535 studies that fit the research strategy. Compared to other medications, dexmedetomidine has combined risk ratios of 0.28 (95% confidence interval [CI] 0.15, 0.55, P = 0.0002) for ventricular tachycardia, 0.35 (95% CI 0.20, 0.62, P = 0.0004) for postoperative delirium, 0.76 (95% CI 0.55, 1.06, P = 0.11) for atrial fibrillation, 1.08 (95% CI 0.74, 1.57, P = 0.69) for hypotension, and 2.23 (95% CI 1.36, 3.67, P = 0.001) for bradycardia. In addition, dexmedetomidine may reduce the length of intensive care unit (ICU) and hospital stay. This meta-analysis revealed that the perioperative use of dexmedetomidine in patients undergoing cardiac surgery can reduce the risk of postoperative ventricular tachycardia and delirium, but may increase the risk of bradycardia. The estimates showed a decreased risk of atrial fibrillation, shorter length of ICU stay and hospitalization, and increased risk of hypotension with dexmedetomidine.",
"To compare the efficacy, safety, and cost of continuous infusions of lorazepam, midazolam, and propofol in a critically ill trauma/surgery patient population. A prospective, randomized, nonblinded, single center. A 16-bed intensive care unit. A total of 30 ventilated patients who were 18-70 yrs of age and required pharmacologic sedation. Patients with renal and/or liver failure, a history of alcohol abuse, a head injury, or in a coma were excluded. Patients were randomized by block design to receive lorazepam, midazolam, or propofol. Initial boluses and infusion rates were as follows: lorazepam 0.05 mg/kg, then 0.007 mg/kg/hr; midazolam 0.05 mg/kg, then 0.003 mg/kg/hr; and propofol 0.25 mg/kg, then 0.06 mg/kg/hr. Sedation was assessed and agents titrated every 5-10 mins to achieve > or =2 and <5 on the modified Ramsay scale. Once adequate response was achieved, agents were titrated to maintain the desired level of sedation. Maintenance doses of lorazepam 0.02+/-0.01 mg/kg/hr, midazolam 0.04+/-0.03 mg/kg/hr, and propofol 2.0+/-1.5 mg/kg/hr achieved the desired level of sedation 68%, 79%, and 62% of the time, respectively. Oversedation occurred most often with lorazepam, compared with midazolam and propofol, at 14%, 6%, and 7% of the assessment times, respectively. Undersedation occurred most frequently with propofol compared with lorazepam and midazolam, at 31%, 18%, and 16% of the assessment times, respectively. The mean number of dosage changes per day was 7.8+/-4.3 for lorazepam, 4.4+/-2.9 for midazolam, and 5.6+/-6.0 for propofol (p = .91). Sedation costs per patient day (mean +/- SD) were $48+/-$76 (lorazepam), $182+/-$98 (midazolam), and $273+/-$200 (propofol) (p = .005). The potential savings, if all study patients had received lorazepam, is $14,208 compared with $8,808 if all received midazolam. The data suggest that lorazepam appears to be a cost-effective choice for sedation; however, oversedation may be problematic. Midazolam is the most titratable drug in our population, avoiding excessive oversedation or undersedation. Trauma patients may respond inadequately to propofol even at higher doses. Lorazepam may be the sedative of choice in critically ill trauma/surgery patients.",
"Agitated delirium is common in patients undergoing mechanical ventilation, and is often treated with haloperidol despite concerns about safety and efficacy. Use of conventional sedatives to control agitation can preclude extubation. Dexmedetomidine, a novel sedative and anxiolytic agent, may have particular utility in these patients. We sought to compare the efficacy of haloperidol and dexmedetomidine in facilitating extubation. We conducted a randomised, open-label, parallel-groups pilot trial in the medical and surgical intensive care unit of a university hospital. Twenty patients undergoing mechanical ventilation in whom extubation was not possible solely because of agitated delirium were randomised to receive an infusion of either haloperidol 0.5 to 2 mg/hour or dexmedetomidine 0.2 to 0.7 microg/kg/hr, with or without loading doses of 2.5 mg haloperidol or 1 mug/kg dexmedetomidine, according to clinician preference. Dexmedetomidine significantly shortened median time to extubation from 42.5 (IQR 23.2 to 117.8) to 19.9 (IQR 7.3 to 24) hours (P = 0.016). Dexmedetomidine significantly decreased ICU length of stay, from 6.5 (IQR 4 to 9) to 1.5 (IQR 1 to 3) days (P = 0.004) after study drug commencement. Of patients who required ongoing propofol sedation, the proportion of time propofol was required was halved in those who received dexmedetomidine (79.5% (95% CI 61.8 to 97.2%) vs. 41.2% (95% CI 0 to 88.1%) of the time intubated; P = 0.05). No patients were reintubated; three receiving haloperidol could not be successfully extubated and underwent tracheostomy. One patient prematurely discontinued haloperidol due to QTc interval prolongation. In this preliminary pilot study, we found dexmedetomidine a promising agent for the treatment of ICU-associated delirious agitation, and we suggest this warrants further testing in a definitive double-blind multi-centre trial. Clinicaltrials.gov NCT00505804.",
"Delirium after cardiac surgery is associated with serious long-term negative outcomes and high costs. The aim of this study is to evaluate neurobehavioral, hemodynamic, and sedative characteristics of dexmedetomidine, compared with the current postoperative sedative protocol (remifentanil) in patients undergoing open heart surgery with cardiopulmonary bypass (CPB). One hundred and forty two eligible patients who underwent cardiac surgery on CPB between April 2012 and March 2013 were randomly divided into two groups. Patients received either dexmedetomidine (range, 0.2 to 0.8 μg/kg/hr; n=67) or remifentanil (range, 1,000 to 2,500 μg/hr, n=75). The primary end point was the prevalence of delirium estimated daily via the confusion assessment method for intensive care. When the delirium incidence was compared with the dexmedetomidine group (6 of 67 patients, 8.96%) and the remifentanil group (17 of 75 patients, 22.67%) it was found to be significantly less in the dexmedetomidine group (p<0.05). There were no statistically significant differences between two groups in the extubation time, ICU stay, total hospital stay, and other postoperative complications including hemodynamic side effects. This preliminary study suggests that dexmedetomidine as a postoperative sedative agent is as sociated with significantly lower rates of delirium after cardiac surgery.",
"The aim of the present study is to compare the safety, efficacy and cost effectiveness of anesthetic regimens by compound, using etomidate and propofol in elderly patients undergoing gastroscopy. A total of 200 volunteers (65-79 years of age) scheduled for gastroscopy under anesthesia were randomly divided into the following groups: P, propofol (1.5-2.0 mg/kg); E, etomidate (0.15-0.2 mg/kg); P+E, propofol (0.75-1 mg/kg) followed by etomidate (0.075-0.1 mg/kg); and E+P, etomidate (0.075-0.01 mg/kg) followed by propofol (0.75-1 mg/kg). Vital signs and bispectral index were monitored at different time points. Complications, induction and examination time, anesthesia duration, and recovery and discharge time were recorded. At the end of the procedure, the satisfaction of patients, endoscopists and the anesthetist were evaluated. The recovery (6.1±1.2 h) and discharge times (24.8±2.8 h) in group E were significantly longer compared with groups P, P+E and E+P (P<0.05). The occurrence of injection pain in group P+E was significantly higher compared with the other three groups (P<0.05). In addition, the incidence of myoclonus and post-operative nausea and vomiting were significantly higher in group P+E compared with the other three groups (P<0.05). There was no statistical difference among the four groups with regards to the patients' immediate, post-procedure satisfaction (P>0.05). Furthermore, there was no difference in the satisfaction of anesthesia, as evaluated by the anesthetist and endoscopist, among the four groups (P>0.05). The present study demonstrates that anesthesia for gastroscopy in elderly patients can be safely and effectively accomplished using a drug regimen that combines propofol with etomidate. The combined use of propofol and etomidate has unique characteristics which improve hemodynamic stability, cause minimal respiratory depression and less side effects, provide rapid return to full activity and result in high levels of satisfaction."
] |
Prostate cancer and histone H3K18 acetylation
|
Histones undergo extensive post-translational modifications and this epigenetic regulation plays an important role in modulating transcriptional programs capable of driving cancer progression. Acetylation of histone H3K18, associated with gene activation, is enhanced by P300 and opposed by the deacetylase Sirtuin2 (SIRT2). As these enzymes represent an important target for cancer therapy, we sought to determine whether the underlying genes are altered during prostate cancer (PCa) progression.
|
[
"Histone acetyltransferases (HATs) GCN5 and PCAF (GCN5/PCAF) and CBP and p300 (CBP/p300) are transcription co-activators. However, how these two distinct families of HATs regulate gene activation remains unclear. Here, we show deletion of GCN5/PCAF in cells specifically and dramatically reduces acetylation on histone H3K9 (H3K9ac) while deletion of CBP/p300 specifically and dramatically reduces acetylations on H3K18 and H3K27 (H3K18/27ac). A ligand for nuclear receptor (NR) PPARδ induces sequential enrichment of H3K18/27ac, RNA polymerase II (Pol II) and H3K9ac on PPARδ target gene Angptl4 promoter, which correlates with a robust Angptl4 expression. Inhibiting transcription elongation blocks ligand-induced H3K9ac, but not H3K18/27ac, on the Angptl4 promoter. Finally, we show GCN5/PCAF and GCN5/PCAF-mediated H3K9ac correlate with, but are surprisingly dispensable for, NR target gene activation. In contrast, CBP/p300 and their HAT activities are essential for ligand-induced Pol II recruitment on, and activation of, NR target genes. These results highlight the substrate and site specificities of HATs in cells, demonstrate the distinct roles of GCN5/PCAF- and CBP/p300-mediated histone acetylations in gene activation, and suggest an important role of CBP/p300-mediated H3K18/27ac in NR-dependent transcription.",
"Histone deacetylases (HDACs) are subdivided into three classes--HDAC I, HDAC II, and Sir2. Sirt proteins are mammalian members of the Sir2 family of NAD+ (nicotinamide adenine dinucleotide)-dependent protein deacetylases. The balance between acetylation and deacetylation of histone and non-histone proteins, regulated by protein acetyltransferases and deacetylases, affects the expression of genes involved in a variety of cellular processes. In addition, HDAC1 is acetylated and regulated by p300, a transcriptional co-activator with protein acetyltransferase activity, suggesting that protein acetyltransferases and deacetylases they control the activities of each other. Although the regulation of HDAC1 by p300 is well characterized, the relationship between Sir2 homologs and p300 is not understood. Here, we report that p300 interacts with Sirt2, a member of the Sir2 family, and triggers the acetylation and subsequent down-regulation of the deacetylation activity of Sirt2, and that the acetylation of Sirt2 by p300 relieves the inhibitory effect of Sirt2 on the transcriptional activity of p53. These observations demonstrate that p300 can inactivate Sirt2 by acetylation and that p300 may regulate the activity of p53 indirectly through Sirt2 in addition to its direct modification of p53.",
"PTIP, a protein with tandem BRCT domains, has been implicated in DNA damage response. However, its normal cellular functions remain unclear. Here we show that while ectopically expressed PTIP is capable of interacting with DNA damage response proteins including 53BP1, endogenous PTIP, and a novel protein PA1 are both components of a Set1-like histone methyltransferase (HMT) complex that also contains ASH2L, RBBP5, WDR5, hDPY-30, NCOA6, SET domain-containing HMTs MLL3 and MLL4, and substoichiometric amount of JmjC domain-containing putative histone demethylase UTX. PTIP complex carries robust HMT activity and specifically methylates lysine 4 (K4) on histone H3. Furthermore, PA1 binds PTIP directly and requires PTIP for interaction with the rest of the complex. Moreover, we show that hDPY-30 binds ASH2L directly. The evolutionarily conserved hDPY-30, ASH2L, RBBP5, and WDR5 likely constitute a subcomplex that is shared by all human Set1-like HMT complexes. In contrast, PTIP, PA1, and UTX specifically associate with the PTIP complex. Thus, in cells without DNA damage agent treatment, the endogenous PTIP associates with a Set1-like HMT complex of unique subunit composition. As histone H3 K4 methylation associates with active genes, our study suggests a potential role of PTIP in the regulation of gene expression.",
"Epigenetic alterations are common in prostate cancer, yet how these modifications contribute to carcinogenesis is poorly understood. We investigated whether specific histone modifications are prognostic for prostate cancer relapse, and whether the expression of epigenetic genes is altered in prostate tumorigenesis. Global levels of histone H3 lysine-18 acetylation (H3K18Ac) and histone H3 lysine-4 dimethylation (H3K4diMe) were assessed immunohistochemically in a prostate cancer cohort of 279 cases. Epigenetic gene expression was investigated in silico by analysis of microarray data from 23 primary prostate cancers (8 with biochemical recurrence and 15 without) and 7 metastatic lesions. H3K18Ac and H3K4diMe are independent predictors of relapse-free survival, with high global levels associated with a 1.71-fold (P < 0.0001) and 1.80-fold (P = 0.006) increased risk of tumor recurrence, respectively. High levels of both histone modifications were associated with a 3-fold increased risk of relapse (P < 0.0001). Epigenetic gene expression profiling identified a candidate gene signature (DNMT3A, MBD4, MLL2, MLL3, NSD1, and SRCAP), which significantly discriminated nonmalignant from prostate tumor tissue (P = 0.0063) in an independent cohort. This study has established the importance of histone modifications in predicting prostate cancer relapse and has identified an epigenetic gene signature associated with prostate tumorigenesis. Our findings suggest that targeting the epigenetic enzymes specifically involved in a particular solid tumor may be a more effective approach. Moreover, testing for aberrant expression of epigenetic genes such as those identified in this study may be beneficial in predicting individual patient response to epigenetic therapies.",
"Nuclear receptors regulate gene expression by direct activation of target genes and inhibition of AP-1. Here we report that, unexpectedly, activation by nuclear receptors requires the actions of CREB-binding protein (CBP) and that inhibition of AP-1 activity is the apparent result of competition for limiting amounts of CBP/p300 in cells. Utilizing distinct domains, CBP directly interacts with the ligand-binding domain of multiple nuclear receptors and with the p160 nuclear receptor coactivators, which upon cloning have proven to be variants of the SRC-1 protein. Because CBP represents a common factor, required in addition to distinct coactivators for function of nuclear receptors, CREB, and AP-1, we suggest that CBP/p300 serves as an integrator of multiple signal transduction pathways within the nucleus.",
"Nuclear receptors (NRs) are ligand-regulated, DNA-binding transcription factors that function in the chromatin environment of the nucleus to alter the expression of subsets of hormone-responsive genes. It is clear that chromatin, rather than being a passive player, has a profound effect on both transcriptional repression and activation mediated by NRs. NRs act in conjunction with at least three general classes of cofactors to regulate transcription in the context of chromatin: (a) chromatin remodelers; (b) corepressors; and (c) coactivators, many of which have distinct enzymatic activities that remodel nucleosomes or covalently modify histones (e.g. acetylases, deacetylases, methyltransferases, and kinases). In this paper, we will present a brief overview of these enzymes, their activities, and how they assist NRs in the repression or activation of transcription in the context of chromatin.",
"Transcriptional activators, several different coactivators, and general transcription factors are necessary to access specific loci in the dense chromatin structure to allow precise initiation of RNA polymerase II (Pol II) transcription. Histone acetyltransferase (HAT) complexes were implicated in loosening the chromatin around promoters and thus in gene activation. Here we demonstrate that the 2 MDa GCN5 HAT-containing metazoan TFTC/STAGA complexes contain a histone H2A and H2B deubiquitinase activity. We have identified three additional subunits of TFTC/STAGA (ATXN7L3, USP22, and ENY2) that form the deubiquitination module. Importantly, we found that this module is an enhancer of position effect variegation in Drosophila. Furthermore, we demonstrate that ATXN7L3, USP22, and ENY2 are required as cofactors for the full transcriptional activity by nuclear receptors. Thus, the deubiquitinase activity of the TFTC/STAGA HAT complex is necessary to counteract heterochromatin silencing and acts as a positive cofactor for activation by nuclear receptors in vivo."
] |
What is the role of p53 in aging and vascular dysfunction?
|
Chronological aging is linked to cellular senescence, and there is accumulating evidence for a pathological role of cellular senescence in age-related disorders such as obesity, diabetes, and heart failure. The protein p53 has a central role in cellular senescence, and p53 expression in cardiomyocytes, vascular endothelial cells, adipocytes, and immune cells leads to the development of heart failure and diabetes. It is widely accepted that formation of capillary networks is critical for morphogenesis of organs and maintenance of homeostasis. Capillary rarefaction and hypoxia promote pathological changes in the myocardium of the failing heart, causing systolic dysfunction. Capillary rarefaction and hypoxia also cause dysfunction of brown adipose tissue (BAT), leading to systemic metabolic disorders with promotion of diabetes. Vascular endothelial cell senescence develops in heart failure and diabetes and is responsible for progression of these age-related disorders. In a murine model of left ventricular pressure overload, increased expression of p53 in vascular endothelial cells and bone marrow cells promotes inflammatory cell infiltration into the heart, contributing to cardiac remodeling and systolic dysfunction. Metabolic stress up-regulates p53 expression in endothelial cells, while reducing the phosphorylation of endothelial nitric oxide synthase (eNOS) and glucose transporter (GLUT)1 expression in these cells. These changes lead to suppression of mitochondrial biogenesis and glucose uptake in the skeletal muscle and promote the development of systemic metabolic dysfunction. Suppression of vascular aging and vascular dysfunction is critically important for maintenance of organ homeostasis and is essential for prevention or treatment of heart failure, obesity, and diabetes.
|
[
"Adipose tissue can undergo rapid expansion during times of excess caloric intake. Like a rapidly expanding tumor mass, obese adipose tissue becomes hypoxic due to the inability of the vasculature to keep pace with tissue growth. Consequently, during the early stages of obesity, hypoxic conditions cause an increase in the level of hypoxia-inducible factor 1alpha (HIF1alpha) expression. Using a transgenic model of overexpression of a constitutively active form of HIF1alpha, we determined that HIF1alpha fails to induce the expected proangiogenic response. In contrast, we observed that HIF1alpha initiates adipose tissue fibrosis, with an associated increase in local inflammation. \"Trichrome- and picrosirius red-positive streaks,\" enriched in fibrillar collagens, are a hallmark of adipose tissue suffering from the early stages of hypoxia-induced fibrosis. Lysyl oxidase (LOX) is a transcriptional target of HIF1alpha and acts by cross-linking collagen I and III to form the fibrillar collagen fibers. Inhibition of LOX activity by beta-aminoproprionitrile treatment results in a significant improvement in several metabolic parameters and further reduces local adipose tissue inflammation. Collectively, our observations are consistent with a model in which adipose tissue hypoxia serves as an early upstream initiator for adipose tissue dysfunction by inducing a local state of fibrosis.",
"Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen in vitro and an angiogenic inducer in a variety of in vivo models. Hypoxia has been shown to be a major inducer of VEGF gene transcription. The tyrosine kinases Flt-1 (VEGFR-1) and Flk-1/KDR (VEGFR-2) are high-affinity VEGF receptors. The role of VEGF in developmental angiogenesis is emphasized by the finding that loss of a single VEGF allele results in defective vascularization and early embryonic lethality. VEGF is critical also for reproductive and bone angiogenesis. Substantial evidence also implicates VEGF as a mediator of pathological angiogenesis. In situ hybridization studies demonstrate expression of VEGF mRNA in the majority of human tumors. Anti-VEGF monoclonal antibodies and other VEGF inhibitors block the growth of several tumor cell lines in nude mice. Clinical trials with various VEGF inhibitors in a variety of malignancies are ongoing. Very recently, an anti-VEGF monoclonal antibody (bevacizumab; Avastin) has been approved by the Food and Drug Administration as a first-line treatment for metastatic colorectal cancer in combination with chemotherapy. Furthermore, VEGF is implicated in intraocular neovascularization associated with diabetic retinopathy and age-related macular degeneration.",
"We describe the generation and characterization of the first inducible 'fatless' model system, the FAT-ATTAC mouse (fat apoptosis through targeted activation of caspase 8). This transgenic mouse develops identically to wild-type littermates. Apoptosis of adipocytes can be induced at any developmental stage by administration of a FK1012 analog leading to the dimerization of a membrane-bound, adipocyte-specific caspase 8-FKBP fusion protein. Within 2 weeks of dimerizer administration, FAT-ATTAC mice show near-knockout levels of circulating adipokines and markedly reduced levels of adipose tissue. FAT-ATTAC mice are glucose intolerant, have diminished basal and endotoxin-stimulated systemic inflammation, are less responsive to glucose-stimulated insulin secretion and show increased food intake independent of the effects of leptin. Most importantly, we show that functional adipocytes can be recovered upon cessation of treatment, allowing the study of adipogenesis in vivo, as well as a detailed examination of the importance of the adipocyte in the regulation of multiple physiological functions and pathological states.",
"Wound infection risk is inversely related to subcutaneous tissue oxygenation, which is reduced in obese patients and may be reduced even more during laparoscopic procedures. We evaluated subcutaneous tissue oxygenation (PsqO(2)) in 20 patients with a body mass index (BMI) > or=40 kg/m(2) (obese group) and 15 patients with BMI <30 kg/m(2) (non-obese group) undergoing laparoscopic surgery with standardized anaesthesia technique and fluid administration. Arterial oxygen tension was maintained near 150 mmHg. PsqO(2) was measured from a surrogate wound on the upper arm. A mean FIO(2) of 51% (13%) was required in obese patients to reach an arterial oxygen tension of 150 mmHg; however, a mean FIO(2) of only 40% (7%) was required to reach the same oxygen tension in non-obese patients (P=0.007). PsqO(2) was significantly less in obese patients: 41 (10) vs 57 (15) mmHg (P<0.001). Obese patients having laparoscopic surgery require a significantly greater FIO(2) to reach an arterial oxygen tension of about 150 mmHg than non-obese patients; they also have significantly lower subcutaneous oxygen tensions. Both factors probably contribute to an increased infection risk in obese patients.",
"Hypoxia-inducible factor 1 (HIF-1) activates the transcription of genes that are involved in crucial aspects of cancer biology, including angiogenesis, cell survival, glucose metabolism and invasion. Intratumoral hypoxia and genetic alterations can lead to HIF-1alpha overexpression, which has been associated with increased patient mortality in several cancer types. In preclinical studies, inhibition of HIF-1 activity has marked effects on tumour growth. Efforts are underway to identify inhibitors of HIF-1 and to test their efficacy as anticancer therapeutics.",
"Chronic inflammation and reduced adiponectin are widely observed in the white adipose tissue in obesity. However, the cause of the changes remains to be identified. In this study, we provide experimental evidence that hypoxia occurs in adipose tissue in obese mice and that adipose hypoxia may contribute to the endocrine alterations. The adipose hypoxia was demonstrated by a reduction in the interstitial partial oxygen pressure (Po(2)), an increase in the hypoxia probe signal, and an elevation in expression of the hypoxia response genes in ob/ob mice. The adipose hypoxia was confirmed in dietary obese mice by expression of hypoxia response genes. In the adipose tissue, hypoxia was associated with an increased expression of inflammatory genes and decreased expression of adiponectin. In dietary obese mice, reduction in body weight by calorie restriction was associated with an improvement of oxygenation and a reduction in inflammation. In cell culture, inflammatory cytokines were induced by hypoxia in primary adipocytes and primary macrophages of lean mice. The transcription factor NF-kappaB and the TNF-alpha gene promoter were activated by hypoxia in 3T3-L1 adipocytes and NIH3T3 fibroblasts. In addition, adiponectin expression was reduced by hypoxia, and the reduction was observed in the gene promoter in adipocytes. These data suggest a potential role of hypoxia in the induction of chronic inflammation and inhibition of adiponectin in the adipose tissue in obesity.",
"Lysyl oxidase is an extracellular enzyme that controls the maturation of collagen and elastin. Lysyl oxidase and collagen III often show similar expression patterns in fibrotic tissues. Therefore, we investigated the influence of lysyl oxidase overexpression on the promoter activity of human COL3A1 gene. Our results showed that when COS-7 cells overexpressed the mature form of lysyl oxidase, the activity of the human COL3A1 promoter was increased up to an average of 12 times when tested by luciferase reporter assay. The effect was specific, because other promoters were not affected. Moreover, lysyl oxidase effect was abolished by beta-aminopropionitrile, a specific inhibitor of its catalytic activity. Electrophoretic mobility shift assay showed a binding activity in the region from -101 to -77 that was significantly increased by lysyl oxidase overexpression. The binding was specifically competed by the cold probe, and the mutagenesis of this region abolished both the binding activity in gel retardation and lysyl oxidase stimulation of COL3A1 promoter in transfection experiments. We identified the binding activity as Ku antigen in its two components: Ku80 and Ku70. This study suggests a new coordinated mechanism by which lysyl oxidase might control the development of fibrosis."
] |
Effect of recombinant Slit2 on peripheral immune cell infiltration after surgical brain injury in a rat model
|
Peripheral immune cell infiltration to the brain tissue at the perisurgical site can promote neuroinflammation after surgical brain injury (SBI). Slit2, an extracellular matrix protein, has been reported to reduce leukocyte migration. This study evaluated the effect of recombinant Slit2 and the role of its receptor roundabout1 (Robo1) and its downstream mediator Slit-Robo GTPase activating protein 1 (srGAP1)-Cdc42 on peripheral immune cell infiltration after SBI in a rat model.
|
[
"The rho family of small G proteins, including rho, rac and cdc42, are involved in many cellular processes, including cell transformation by ras and the organization of the actin cytoskeleton. Additionally, rac has a role in the regulation of phagocyte NADPH oxidase. Guanine nucleotide dissociation inhibitors (GDIs) of the rhoGDI family bind to these G proteins and regulate their activity by preventing nucleotide dissociation and by controlling their interaction with membranes. We report the structure of rhoGDI, determined by a combination of X-ray crystallography and NMR spectroscopy. NMR spectroscopy and selective proteolysis show that the N-terminal 50-60 residues of rhoGDI are flexible and unstructured in solution. The 2.5 A crystal structure of the folded core of rhoGDI, comprising residues 59-204, shows it to have an immunoglobulin-like fold, with an unprecedented insertion of two short beta strands and a 310 helix. There is an unusual pocket between the beta sheets of the immunoglobulin fold which may bind the C-terminal isoprenyl group of rac. NMR spectroscopy shows that the N-terminal arm is necessary for binding rac, although it remains largely flexible even in the complex. The rhoGDI structure is notable for the existence of both a structured and a highly flexible domain, both of which appear to be required for the interaction with rac. The immunoglobulin-like fold of the structured domain is unusual for a cytoplasmic protein. The presence of equivalent cleavage sites in rhoGDI and the closely related D4/Ly-GDI (rhoGDI-2) suggest that proteolytic cleavage between the flexible and structured regions of rhoGDI may have a role in the regulation of the activity of members of this family. There is no detectable similarity between the structure of rhoGDI and the recently reported structure of rabGDI, which performs the same function as rhoGDI for the rab family of small G proteins.",
"Rho GTPases are molecular switches that regulate many essential cellular processes, including actin dynamics, gene transcription, cell-cycle progression and cell adhesion. About 30 potential effector proteins have been identified that interact with members of the Rho family, but it is still unclear which of these are responsible for the diverse biological effects of Rho GTPases. This review will discuss how Rho GTPases physically interact with, and regulate the activity of, multiple effector proteins and how specific effector proteins contribute to cellular responses. To date most progress has been made in the cytoskeleton field, and several biochemical links have now been established between GTPases and the assembly of filamentous actin. The main focus of this review will be Rho, Rac and Cdc42, the three best characterized mammalian Rho GTPases, though the genetic analysis of Rho GTPases in lower eukaryotes is making increasingly important contributions to this field.",
"The small GTPases Cdc42 and Rac regulate a variety of biological processes, including actin polymerization, cell proliferation, and JNK/mitogen-activated protein kinase activation, conceivably via distinct effectors. Whereas the effector for mitogen-activated protein kinase activation appears to be p65PAK, the identity of effector(s) for actin polymerization remains unclear. We have found a putative effector for Drosophila Cdc42, Genghis Khan (Gek), which binds to Dcdc42 in a GTP-dependent and effector domain-dependent manner. Gek contains a predicted serine/threonine kinase catalytic domain that is 63% identical to human myotonic dystrophy protein kinase and has protein kinase activities. It also possesses a large coiled-coil domain, a putative phorbol ester binding domain, a pleckstrin homology domain, and a Cdc42 binding consensus sequence that is required for its binding to Dcdc42. To study the in vivo function of gek, we generated mutations in the Drosophila gek locus. Egg chambers homozygous for gek mutations exhibit abnormal accumulation of F-actin and are defective in producing fertilized eggs. These phenotypes can be rescued by a wild-type gek transgene. Our results suggest that this multidomain protein kinase is an effector for the regulation of actin polymerization by Cdc42.",
"The angiogenic sprout has been compared to the growing axon, and indeed, many proteins direct pathfinding by both structures. The Roundabout (Robo) proteins are guidance receptors with well-established functions in the nervous system; however, their role in the mammalian vasculature remains ill defined. Here we show that an endothelial-specific Robo, Robo4, maintains vascular integrity. Activation of Robo4 by Slit2 inhibits vascular endothelial growth factor (VEGF)-165-induced migration, tube formation and permeability in vitro and VEGF-165-stimulated vascular leak in vivo by blocking Src family kinase activation. In mouse models of retinal and choroidal vascular disease, Slit2 inhibited angiogenesis and vascular leak, whereas deletion of Robo4 enhanced these pathologic processes. Our results define a previously unknown function for Robo receptors in stabilizing the vasculature and suggest that activating Robo4 may have broad therapeutic application in diseases characterized by excessive angiogenesis and/or vascular leak.",
"We have identified a human Rho protein, RhoE, which has unusual structural and biochemical properties that suggest a novel mechanism of regulation. Within a region that is highly conserved among small GTPases, RhoE contains amino acid differences specifically at three positions that confer oncogenicity to Ras (12, 59, and 61). As predicted by these substitutions, which impair GTP hydrolysis in Ras, RhoE binds GTP but lacks intrinsic GTPase activity and is resistant to Rho-specific GTPase-activating proteins. Replacing all three positions in RhoE with conventional amino acids completely restores GTPase activity. In vivo, RhoE is found exclusively in the GTP-bound form, suggesting that unlike previously characterized small GTPases, RhoE may be normally maintained in an activated state. Thus, amino acid changes in Ras that are selected during tumorigenesis have evolved naturally in this Rho protein and have similar consequences for catalytic function. All previously described Rho family proteins are modified by geranylgeranylation, a lipid attachment required for proper membrane localization. In contrast, the carboxy-terminal sequence of RhoE predicts that, like Ras proteins, RhoE is normally farnesylated. Indeed, we have found that RhoE in farnesylated in vivo and that this modification is required for association with the plasma membrane and with an unidentified cellular structure that may play a role in adhesion. Thus, two unusual structural features of this novel Rho protein suggest a striking evolutionary divergence from the Rho family of GTPases.",
"The Rho family of GTPases plays key roles in the regulation of cell motility and morphogenesis. They also regulate protein kinase cascades, gene expression, and cell cycle progression. This multiplicity of roles requires that the Rho GTPases interact with a wide variety of downstream effector proteins. An understanding of their functions at a molecular level therefore requires the identification of the entire set of such effectors. Towards this end, we performed a two-hybrid screen using the TC10 GTPase as bait and identified a family of putative effector proteins related to MSE55, a murine stromal and epithelial cell protein of 55 kDa. We have named this family the Borg (binder of Rho GTPases) proteins. Complete open reading frames have been obtained for Borg1 through Borg3. We renamed MSE55 as Borg5. Borg1, Borg2, Borg4, and Borg5 bind both TC10 and Cdc42 in a GTP-dependent manner. Surprisingly, Borg3 bound only to Cdc42. An intact CRIB (Cdc42, Rac interactive binding) domain was required for binding. No interaction of the Borgs with Rac1 or RhoA was detectable. Three-hemagglutinin epitope (HA(3))-tagged Borg3 protein was mostly cytosolic when expressed ectopically in NIH 3T3 cells, with some accumulation in membrane ruffles. The phenotype induced by Borg3 was reminiscent of that caused by an inhibition of Rho function and was reversed by overexpression of Rho. Surprisingly, it was independent of the ability to bind Cdc42. Borg3 also inhibited Jun kinase activity by a mechanism that was independent of Cdc42 binding. HA(3)-Borg3 expression caused substantial delays in the spreading of cells on fibronectin surfaces after replating, and the spread cells lacked stress fibers. We propose that the Borg proteins function as negative regulators of Rho GTPase signaling.",
"The MLK (mixed lineage) ser/thr kinases are most closely related to the MAP kinase kinase kinase family. In addition to a kinase domain, MLK1, MLK2 and MLK3 each contain an SH3 domain, a leucine zipper domain and a potential Rac/Cdc42 GTPase-binding (CRIB) motif. The C-terminal regions of the proteins are essentially unrelated. Using yeast two-hybrid analysis and in vitro dot-blots, we show that MLK2 and MLK3 interact with the activated (GTP-bound) forms of Rac and Cdc42, with a slight preference for Rac. Transfection of MLK2 into COS cells leads to strong and constitutive activation of the JNK (c-Jun N-terminal kinase) MAP kinase cascade, but also to activation of ERK (extracellular signal-regulated kinase) and p38. When expressed in fibroblasts, MLK2 co-localizes with active, dually phosphorylated JNK1/2 to punctate structures along microtubules. In an attempt to identify proteins that affect the activity and localization of MLK2, we have screened a yeast two-hybrid cDNA library. MLK2 and MLK3 interact with members of the KIF3 family of kinesin superfamily motor proteins and with KAP3A, the putative targeting component of KIF3 motor complexes, suggesting a potential link between stress activation and motor protein function."
] |
Dynamic nuclear deformation during cell migration through constrictions
|
The ability of cells to migrate through tissues and interstitial spaces is an essential factor during development and tissue homeostasis, immune cell mobility, and in various human diseases. Deformation of the nucleus and its associated lamina during 3-D migration is gathering increasing interest in the context of cancer metastasis, with the underlying hypothesis that a softer nucleus, resulting from reduced levels of lamin A/C, may aid tumour spreading. However, current methods to study the migration of cells in confining three dimensional (3-D) environments are limited by their imprecise control over the confinement, physiological relevance, and/or compatibility with high resolution imaging techniques. We describe the design of a polydimethylsiloxane (PDMS) microfluidic device composed of channels with precisely-defined constrictions mimicking physiological environments that enable high resolution imaging of live and fixed cells. The device promotes easy cell loading and rapid, yet long-lasting (>24 hours) chemotactic gradient formation without the need for continuous perfusion. Using this device, we obtained detailed, quantitative measurements of dynamic nuclear deformation as cells migrate through tight spaces, revealing distinct phases of nuclear translocation through the constriction, buckling of the nuclear lamina, and severe intranuclear strain. Furthermore, we found that lamin A/C-deficient cells exhibited increased and more plastic nuclear deformations compared to wild-type cells but only minimal changes in nuclear volume, implying that low lamin A/C levels facilitate migration through constrictions by increasing nuclear deformability rather than compressibility. The integration of our migration devices with high resolution time-lapse imaging provides a powerful new approach to study intracellular mechanics and dynamics in a variety of physiologically-relevant applications, ranging from cancer cell invasion to immune cell recruitment.
|
[
"The mechanical properties of cells play an essential role in numerous physiological processes. Organized networks of semiflexible actin filaments determine cell stiffness and transmit force during mechanotransduction, cytokinesis, cell motility and other cellular shape changes. Although numerous actin-binding proteins have been identified that organize networks, the mechanical properties of actin networks with physiological architectures and concentrations have been difficult to measure quantitatively. Studies of mechanical properties in vitro have found that crosslinked networks of actin filaments formed in solution exhibit stress stiffening arising from the entropic elasticity of individual filaments or crosslinkers resisting extension. Here we report reversible stress-softening behaviour in actin networks reconstituted in vitro that suggests a critical role for filaments resisting compression. Using a modified atomic force microscope to probe dendritic actin networks (like those formed in the lamellipodia of motile cells), we observe stress stiffening followed by a regime of reversible stress softening at higher loads. This softening behaviour can be explained by elastic buckling of individual filaments under compression that avoids catastrophic fracture of the network. The observation of both stress stiffening and softening suggests a complex interplay between entropic and enthalpic elasticity in determining the mechanical properties of actin networks.",
"Mutations in LMNA, the gene that encodes nuclear lamins A and C, cause up to eight different diseases collectively referred to as \"laminopathies.\" These diseases affect striated muscle, adipose tissue, peripheral nerve, and bone, or cause features of premature aging. We investigated the consequences of LMNA mutations on nuclear architecture in skin fibroblasts from 13 patients with different laminopathies. Western-blotting showed that none of the mutations examined led to a decrease in cellular levels of lamin A or C. Regardless of the disease, we observed honeycomb nuclear structures and nuclear envelope blebs in cells examined by immunofluorescence microscopy. Concentrated foci of lamin A/C in the nucleoplasm were also observed. Only mutations in the head and tail domains of lamins A and C significantly altered the nuclear architecture of patient fibroblasts. These results confirm that mutations in lamins A and C may lead to a weakening of a structural support network in the nuclear envelope in fibroblasts and that nuclear architecture changes depend upon the location of the mutation in different domains of lamin A/C.",
"There has been a great deal of interest in the mechanism of lamellipodial protrusion (Pollard, T., and G. Borisy. 2003. Cell. 112:453-465). However, one of this mechanism's endpoints, the force of protrusion, has never been directly measured. We place an atomic force microscopy cantilever in the path of a migrating keratocyte. The deflection of the cantilever, which occurs over a period of approximately 10 s, provides a direct measure of the force exerted by the lamellipodial leading edge. Stall forces are consistent with approximately 100 polymerizing actin filaments per micrometer of the leading edge, each working as an elastic Brownian ratchet and generating a force of several piconewtons. However, the force-velocity curves obtained from this measurement, in which velocity drops sharply under very small loads, is not sensitive to low loading forces, and finally stalls rapidly at large loads, are not consistent with current theoretical models for the actin polymerization force. Rather, the curves indicate that the protrusive force generation is a complex multiphase process involving actin and adhesion dynamics.",
"Cell migration through solid tissue often involves large contortions of the nucleus, but biological significance is largely unclear. The nucleoskeletal protein lamin-A varies both within and between cell types and was shown here to contribute to cell sorting and survival in migration through constraining micropores. Lamin-A proved rate-limiting in 3D migration of diverse human cells that ranged from glioma and adenocarcinoma lines to primary mesenchymal stem cells (MSCs). Stoichiometry of A- to B-type lamins established an activation barrier, with high lamin-A:B producing extruded nuclear shapes after migration. Because the juxtaposed A and B polymer assemblies respectively conferred viscous and elastic stiffness to the nucleus, subpopulations with different A:B levels sorted in 3D migration. However, net migration was also biphasic in lamin-A, as wild-type lamin-A levels protected against stress-induced death, whereas deep knockdown caused broad defects in stress resistance. In vivo xenografts proved consistent with A:B-based cell sorting, and intermediate A:B-enhanced tumor growth. Lamins thus impede 3D migration but also promote survival against migration-induced stresses.",
"Despite decades of research, cancer metastasis remains an incompletely understood process that is as complex as it is devastating. In recent years, there has been an increasing push to investigate the biomechanical aspects of tumorigenesis, complementing the research on genetic and biochemical changes. In contrast to the high genetic variability encountered in cancer cells, almost all metastatic cells are subject to the same physical constraints as they leave the primary tumor, invade surrounding tissues, transit through the circulatory system, and finally infiltrate new tissues. Advances in live cell imaging and other biophysical techniques, including measurements of subcellular mechanics, have yielded stunning new insights into the physics of cancer cells. While much of this research has been focused on the mechanics of the cytoskeleton and the cellular microenvironment, it is now emerging that the mechanical properties of the cell nucleus and its connection to the cytoskeleton may play a major role in cancer metastasis, as deformation of the large and stiff nucleus presents a substantial obstacle during the passage through the dense interstitial space and narrow capillaries. Here, we present an overview of the molecular components that govern the mechanical properties of the nucleus, and we discuss how changes in nuclear structure and composition observed in many cancers can modulate nuclear mechanics and promote metastatic processes. Improved insights into this interplay between nuclear mechanics and metastatic progression may have powerful implications in cancer diagnostics and therapy and may reveal novel therapeutic targets for pharmacological inhibition of cancer cell invasion.",
"While the molecular and biophysical mechanisms underlying cell protrusion on two-dimensional substrates are well understood, our knowledge of the actin structures driving protrusion in three-dimensional environments is poor, despite relevance to inflammation, development and cancer. Here we report that, during chemotactic migration through microchannels with 5 μm × 5 μm cross-sections, HL60 neutrophil-like cells assemble an actin-rich slab filling the whole channel cross-section at their front. This leading edge comprises two distinct F-actin networks: an adherent network that polymerizes perpendicular to cell-wall interfaces and a 'free' network that grows from the free membrane at the cell front. Each network is polymerized by a distinct nucleator and, due to their geometrical arrangement, the networks interact mechanically. On the basis of our experimental data, we propose that, during interstitial migration, medial growth of the adherent network compresses the free network preventing its retrograde movement and enabling new polymerization to be converted into forward protrusion.",
"SCAR--also known as WAVE--is a key regulator of actin dynamics. Activation of SCAR enhances the nucleation of new actin filaments through the Arp2/3 complex, causing a localized increase in the rate of actin polymerization . In vivo, SCAR is held in a large regulatory complex, which includes PIR121 and Nap1 proteins, whose precise role is unclear. It was initially thought to hold SCAR inactive until needed , but recent data suggest that it is essential for SCAR function . Here, we show that disruption of the gene that encodes Nap1 (napA) causes loss of SCAR function. Cells lacking Nap1 are small and rounded, with diminished actin polymerization and small pseudopods. Furthermore, several aspects of the napA phenotype are more severe than those evoked by the absence of SCAR alone. In particular, napA mutants have defects in cell-substrate adhesion and multicellular development. Despite these defects, napA(-) cells move and chemotax surprisingly effectively. Our results show that the members of the complex have unexpectedly diverse biological roles."
] |
Effects of apolipoprotein M on sepsis-induced acute lung injury in a mouse model
|
It had been demonstrated that apolipoprotein M (apoM) is an important carrier of sphingosine-1-phosphate (S1P) in blood, and the S1P has critical roles in the pathogenesis of sepsis-induced acute lung injury (ALI). In the present study, we investigated whether apoM has beneficial effects in a mouse model after lipopolysaccharide (LPS)-induced ALI. Forty-eight mice were divided into two groups: male C57BL/6 wild-type (apoM
|
[
"The novel immunomodulator FTY720 is effective in experimental models of transplantation and autoimmunity, and is currently undergoing Phase III clinical trials for prevention of kidney graft rejection. In contrast to conventional immunosuppressants, FTY720 does not impair T- and B-cell activation, proliferation and effector function, but interferes with cell traffic between lymphoid organs and blood. The molecular basis for the mode of action of the drug has only recently been established. FTY720, after phosphorylation, acts as a high-affinity agonist at the G protein-coupled sphingosine 1-phosphate receptor-1 (S1P(1)) on thymocytes and lymphocytes, thereby inducing aberrant internalization of the receptor. This renders the cells unresponsive to the serum lipid sphingosine 1-phosphate (S1P), depriving them from an obligatory signal to egress from lymphoid organs. As a consequence, lymphocytes are unable to recirculate to peripheral inflammatory tissues and graft sites but remain functional in the lymphoid compartment. In addition to the effects on lymphocyte recirculation, the drug acts on endothelial cells and preserves vascular integrity by enhancing adherens junction assembly and endothelial barrier function. The available data establish S1P(1) as a key target for FTY720, and further point to therapeutically relevant effects of the drug on lymphocytes and vascular endothelium.",
"Maintenance of vascular integrity is critical for homeostasis, and temporally and spatially regulated vascular leak is a central feature of inflammation. Sphingosine-1-phosphate (S1P) can regulate endothelial barrier function, but the sources of the S1P that provide this activity in vivo and its importance in modulating different inflammatory responses are unknown. We report here that mutant mice engineered to selectively lack S1P in plasma displayed increased vascular leak and impaired survival after anaphylaxis, administration of platelet-activating factor (PAF) or histamine, and exposure to related inflammatory challenges. Increased leak was associated with increased interendothelial cell gaps in venules and was reversed by transfusion with wild-type erythrocytes (which restored plasma S1P levels) and by acute treatment with an agonist for the S1P receptor 1 (S1pr1). S1pr1 agonist did not protect wild-type mice from PAF-induced leak, consistent with plasma S1P levels being sufficient for S1pr1 activation in wild-type mice. However, an agonist for another endothelial cell Gi-coupled receptor, Par2, did protect wild-type mice from PAF-induced vascular leak, and systemic treatment with pertussis toxin prevented rescue by Par2 agonist and sensitized wild-type mice to leak-inducing stimuli in a manner that resembled the loss of plasma S1P. Our results suggest that the blood communicates with blood vessels via plasma S1P to maintain vascular integrity and regulate vascular leak. This pathway prevents lethal responses to leak-inducing mediators in mouse models.",
"Sphingosine 1-phosphate (S1P, 1) regulates vascular barrier and lymphoid development, as well as lymphocyte egress from lymphoid organs, by activating high-affinity S1P1 receptors. We used reversible chemical probes (i) to gain mechanistic insights into S1P systems organization not accessible through genetic manipulations and (ii) to investigate their potential for therapeutic modulation. Vascular (but not airway) administration of the preferred R enantiomer of an in vivo-active chiral S1P1 receptor antagonist induced loss of capillary integrity in mouse skin and lung. In contrast, the antagonist did not affect the number of constitutive blood lymphocytes. Instead, alteration of lymphocyte trafficking and phenotype required supraphysiological elevation of S1P1 tone and was reversed by the antagonist. In vivo two-photon imaging of lymph nodes confirmed requirements for obligate agonism, and the data were consistent with the presence of a stromal barrier mechanism for gating lymphocyte egress. Thus, chemical modulation reveals differences in S1P-S1P1 'set points' among tissues and highlights both mechanistic advantages (lymphocyte sequestration) and risks (pulmonary edema) of therapeutic intervention.",
"Lipoproteins modulate vascular cell function in inflammation. In this study, we analyzed whether plasma concentrations of lipoproteins and apolipoproteins in human sepsis are related to patient survival and the activation of blood monocytes and platelets. Observational study. Medical and surgical intensive care units (ICU) of a university hospital. 151 consecutive patients after sepsis criteria had been met for the first time. None. Plasma lipoproteins, apolipoproteins, platelet CD62P-expression, monocyte HLA-DR-expression, SAPS II-scores (Simplified Acute Physiology Score) and 30-day-mortality were recorded. Total cholesterol, high-density-lipoprotein (HDL) and low-density-lipoprotein (LDL) cholesterol, apolipoprotein (apo)-AI and apo-B were all found to be significantly lower in non-survivors than in survivors. In contrast to other (apo)lipoproteins, apo-AI and HDL cholesterol further decreased in non-survivors during the ICU stay. Logistic regression analysis revealed apo-AI to be an independent predictor of 30-day-mortality. A significant inverse correlation was found for apo-AI/HDL-cholesterol and platelet activation. Later in the course of the disease, HLA-DR expression on monocytes correlated positively to apo-AI and apo-CI concentrations and inversely to the apo-E concentration. Low apo-AI is independently related to 30-day mortality in human sepsis and the decrease in apo-AI/HDL cholesterol correlates to increased platelet activation. Moreover, changes in apolipoproteins supposed to modulate lipopolysaccharide effects, such as apo-CI and apo-E, correlate to monocyte activation.",
"Acute lung injury is a critical illness syndrome consisting of acute hypoxemic respiratory failure with bilateral pulmonary infiltrates that are not attributed to left atrial hypertension. Despite recent advances in our understanding of the mechanism and treatment of acute lung injury, its incidence and outcomes in the United States have been unclear. We conducted a prospective, population-based, cohort study in 21 hospitals in and around King County, Washington, from April 1999 through July 2000, using a validated screening protocol to identify patients who met the consensus criteria for acute lung injury. A total of 1113 King County residents undergoing mechanical ventilation met the criteria for acute lung injury and were 15 years of age or older. On the basis of this figure, the crude incidence of acute lung injury was 78.9 per 100,000 person-years and the age-adjusted incidence was 86.2 per 100,000 person-years. The in-hospital mortality rate was 38.5 percent. The incidence of acute lung injury increased with age from 16 per 100,000 person-years for those 15 through 19 years of age to 306 per 100,000 person-years for those 75 through 84 years of age. Mortality increased with age from 24 percent for patients 15 through 19 years of age to 60 percent for patients 85 years of age or older (P<0.001). We estimate that each year in the United States there are 190,600 cases of acute lung injury, which are associated with 74,500 deaths and 3.6 million hospital days. Acute lung injury has a substantial impact on public health, with an incidence in the United States that is considerably higher than previous reports have suggested.",
"Spleen T-lymphocytes, especially CD4(+) T-cells, have been demonstrated to be involved in broad immunomodulation and host-defense activity in vivo. Apolipoprotein M gene (apoM) may have an important role in the regulation of immunoprocess and inflammation, which could be hypothesized to the apoM containing sphingosine-1-phosphate (S1P). In the present study we demonstrate that the splenic CD4(+) T-lymphocytes were obviously decreased in the apoM gene deficient (apoM(-/-)) mice compared to the wild type (apoM(+/+)). Moreover, these mice were treated with lipopolysaccharide (LPS) and it was found that even more pronounced decreasing CD4(+) T-lymphocytes occurred in the spleen compared to the apoM(+/+) mice. The similar phenomena were found in the ratio of CD4(+)/CD8(+) T-lymphocytes. After administration of LPS, the hepatic mRNA levels of tumor necrosis factor-α (TNF-α) and monocyte chemotactic protein-1 (MCP-1) were markedly increased; however, there were no statistical differences observed between apoM(+/+) mice and apoM(-/-) mice. The present study demonstrated that apoM might facilitate the maintenance of CD4(+) T-lymphocytes or could modify the T-lymphocytes subgroups in murine spleen, which may further explore the importance of apoM in the regulation of the host immunomodulation, although the detailed mechanism needs continuing investigation.",
"Several Cre reporter strains of mice have been described, in which a lacZ gene is turned on in cells expressing Cre recombinase, as well as their daughter cells, following Cre-mediated excision of a loxP-flanked transcriptional \"stop\" sequence. These mice are useful for cell lineage tracing experiments as well as for monitoring the expression of Cre transgenes. The green fluorescent protein (GFP) and variants such as EYFP and ECFP offer an advantage over lacZ as a reporter, in that they can be easily visualized without recourse to the vital substrates required to visualize beta-gal in living tissue. In view of the general utility of targeting the ubiquitously expressed ROSA26 locus, we constructed a generic ROSA26 targeting vector. We then generated two reporter lines of mice by inserting EYFP or ECFP cDNAs into the ROSA26 locus, preceded by a loxP-flanked stop sequence. These strains were tested by crossing them with transgenic strains expressing Cre in a ubiquitous (beta-actin-Cre) or a cell-specific (Isl1-Cre and En1-Cre) pattern. The resulting EYFP or ECFP expression patterns indicated that the reporter strains function as faithful monitors of Cre activity. In contrast to existing lacZ reporter lines, where lacZ expression cannot easily be detected in living tissue, the EYFP and ECFP reporter strains are useful for monitoring the expression of Cre and tracing the lineage of these cells and their descendants in cultured embryos or organs. The non-overlapping emission spectra of EYFP and ECFP make them ideal for double labeling studies in living tissues."
] |
MicroRNAs are functionally regulated in the nuclei of the avian song control circuit
|
Adult neurogenesis and the incorporation of adult-born neurons into functional circuits requires precise spatiotemporal coordination across molecular networks regulating a wide array of processes, including cell proliferation, apoptosis, neurotrophin signaling, and electrical activity. MicroRNAs (miRs) - short, non-coding RNA sequences that alter gene expression by post-transcriptional inhibition or degradation of mRNA sequences - may be involved in the global coordination of such diverse biological processes. To test the hypothesis that miRs related to adult neurogenesis and related cellular processes are functionally regulated in the nuclei of the avian song control circuit, we used microarray analyses to quantify changes in expression of miRs and predicted target mRNAs in the telencephalic nuclei HVC, the robust nucleus of arcopallium (RA), and the basal ganglia homologue Area X in breeding and nonbreeding Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelli).
|
[
"The primary goal of this case study was to describe the speech, prosody, and voice characteristics of a mother and daughter with a breakpoint in a balanced 7;13 chromosomal translocation that disrupted the transcription gene, FOXP2 (cf. J. B. Tomblin et al., 2005). As with affected members of the widely cited KE family, whose communicative disorders have been associated with a point mutation in the FOXP2 gene, both mother and daughter had cognitive, language, and speech challenges. A 2nd goal of the study was to illustrate in detail, the types of speech, prosody, and voice metrics that can contribute to phenotype sharpening in speech-genetics research. A speech, prosody, and voice assessment protocol was administered twice within a 4-month period. Analyses were aided by comparing profiles from the present speakers (the TB family) with those from 2 groups of adult speakers: 7 speakers with acquired (with one exception) spastic or spastic-flaccid dysarthria and 14 speakers with acquired apraxia of speech. The descriptive and inferential statistical findings for 13 speech, prosody, and voice variable supported the conclusion that both mother and daughter had spastic dysarthria, an apraxia of speech, and residual developmental distortion errors. These findings are consistent with, but also extend, the reported communicative disorders in affected members of the KE family. A companion article (K. J. Ballard, L. D. Shriberg, J. R. Duffy, & J. B. Tomblin, 2006) reports information from the orofacial and speech motor control measures administered to the same family; reports on neuropsychological and neuroimaging findings are in preparation.",
"We previously discovered that mutations of the human FOXP2 gene cause a monogenic communication disorder, primarily characterized by difficulties in learning to make coordinated sequences of articulatory gestures that underlie speech. Affected people have deficits in expressive and receptive linguistic processing and display structural and/or functional abnormalities in cortical and subcortical brain regions. FOXP2 provides a unique window into neural processes involved in speech and language. In particular, its role as a transcription factor gene offers powerful functional genomic routes for dissecting critical neurogenetic mechanisms. Here, we employ chromatin immunoprecipitation coupled with promoter microarrays (ChIP-chip) to successfully identify genomic sites that are directly bound by FOXP2 protein in native chromatin of human neuron-like cells. We focus on a subset of downstream targets identified by this approach, showing that altered FOXP2 levels yield significant changes in expression in our cell-based models and that FOXP2 binds in a specific manner to consensus sites within the relevant promoters. Moreover, we demonstrate significant quantitative differences in target expression in embryonic brains of mutant mice, mediated by specific in vivo Foxp2-chromatin interactions. This work represents the first identification and in vivo verification of neural targets regulated by FOXP2. Our data indicate that FOXP2 has dual functionality, acting to either repress or activate gene expression at occupied promoters. The identified targets suggest roles in modulating synaptic plasticity, neurodevelopment, neurotransmission, and axon guidance and represent novel entry points into in vivo pathways that may be disturbed in speech and language disorders.",
"The human capacity to acquire complex language seems to be without parallel in the natural world. The origins of this remarkable trait have long resisted adequate explanation, but advances in fields that range from molecular genetics to cognitive neuroscience offer new promise. Here we synthesize recent developments in linguistics, psychology and neuroimaging with progress in comparative genomics, gene-expression profiling and studies of developmental disorders. We argue that language should be viewed not as a wholesale innovation, but as a complex reconfiguration of ancestral systems that have been adapted in evolutionarily novel ways.",
"A program of stringently-regulated gene expression is thought to be a fundamental component of the circadian clock. Although recent work has implicated a role for E-box-dependent transcription in circadian rhythmicity, the contribution of other enhancer elements has yet to be assessed. Here, we report that cells of the suprachiasmatic nuclei (SCN) exhibit a prominent circadian oscillation in cAMP response element (CRE)-mediated gene expression. Maximal reporter gene expression occurred from late-subjective night to mid-subjective day. Cycling of CRE-dependent transcription was not observed in other brain regions, including the supraoptic nucleus and piriform cortex. Levels of the phospho-active form of the transcription factor CREB (P-CREB) varied as a function of circadian time. Peak P-CREB levels occurred during the mid- to late-subjective night. Furthermore, photic stimulation during the subjective night, but not during the subjective day, triggered a marked increase in CRE-mediated gene expression in the SCN. Reporter gene experiments showed that activation of the p44/42 mitogen-activated protein kinase signaling cascade is required for Ca2+-dependent stimulation of CRE-mediated transcription in the SCN. These findings reveal the CREB/CRE transcriptional pathway to be circadian-regulated within the SCN, and raise the possibility that this pathway provides signaling information essential for normal clock function.",
"The aim of this study was to determine whether miR-210 can affect the apoptosis and proliferation of human U251 glioma cells from down-regulating SIN3A. The expression of miRNA-210 was detected by quantitative real-time PCR in normal brain tissue and glioma samples. The apoptosis and proliferation ability of U251 cells were analyzed by MTT and flow cytometry assay after anti-miR-210 transfection. For the regulation mechanism analysis of miR-210, TargetScan, PicTar, and microRNA were selected to predict some potential target genes of miR-210. The predicted gene was identified to be the direct and specific target gene of miR-210 by luciferase activities assay and Western blot. RNA interference technology was used to confirm that the apoptosis and proliferation effects of miR-210 were directly induced by SIN3A. The expression of miR-210 increased significantly in glioma in comparison with normal brain tissue. The silence of miR-210 expression could inhibit the proliferation of U251 cells and induce the apoptosis. Mechanism analysis revealed that SIN3A was a specific and direct target gene of miR-210. The siRNA-SIN3A could down-regulate the expression of SIN3A protein, which was up-regulated in U251 cells by anti-miR-210 transfection, and our experiments found that silence of SIN3A could inhibit the apoptosis and sharply increase the proliferation of U251 cells. The regulation effects of anti-miR-210 on apoptosis and proliferation can be reversed respectively by the expression silence of SIN3A. Aberrantly expressed miR-210 regulates human U251 glioma cells apoptosis and proliferation partly through directly down-regulating SIN3A protein expression. This might offer a new potential therapeutic stratagem for glioma.",
"New neurons are constantly added to the high vocal center (HVC) of adult male canaries, Serinus canaria. Singing and testosterone (T) are known to promote this addition, but it is not known whether either variable can act on its own and what is their effect when acting together. We studied this question by castrating adult male canaries in late summer and quantifying their song in early fall. Intact birds served as controls. A 5 d systemic treatment of two daily injections of the cell birth marker 3H-thymidine started 10 d after surgery. Twenty days after the first 3H-thymidine injection and for a period of 1 month, we quantified the singing of all birds, which were then killed. Amount of singing, syllable diversity, and song stability were similar in intacts and castrates. When castrates and intacts that sang comparable amounts were compared, the number of 3H-labeled HVC neurons was 2.6 times higher in intacts than in castrates. In castrates with plasma T levels that were undetectable, the mean amount of singing was positively related to the number of new neurons. We suggest that singing and gonadal factors promote, separately, the recruitment of new neurons and that when they exert this effect together they do so in an additive manner.",
"The problem of identifying differentially expressed genes in designed microarray experiments is considered. Lonnstedt and Speed (2002) derived an expression for the posterior odds of differential expression in a replicated two-color experiment using a simple hierarchical parametric model. The purpose of this paper is to develop the hierarchical model of Lonnstedt and Speed (2002) into a practical approach for general microarray experiments with arbitrary numbers of treatments and RNA samples. The model is reset in the context of general linear models with arbitrary coefficients and contrasts of interest. The approach applies equally well to both single channel and two color microarray experiments. Consistent, closed form estimators are derived for the hyperparameters in the model. The estimators proposed have robust behavior even for small numbers of arrays and allow for incomplete data arising from spot filtering or spot quality weights. The posterior odds statistic is reformulated in terms of a moderated t-statistic in which posterior residual standard deviations are used in place of ordinary standard deviations. The empirical Bayes approach is equivalent to shrinkage of the estimated sample variances towards a pooled estimate, resulting in far more stable inference when the number of arrays is small. The use of moderated t-statistics has the advantage over the posterior odds that the number of hyperparameters which need to estimated is reduced; in particular, knowledge of the non-null prior for the fold changes are not required. The moderated t-statistic is shown to follow a t-distribution with augmented degrees of freedom. The moderated t inferential approach extends to accommodate tests of composite null hypotheses through the use of moderated F-statistics. The performance of the methods is demonstrated in a simulation study. Results are presented for two publicly available data sets."
] |
Curcumin suppresses corneal neovascularization formation
|
To investigate whether curcumin suppressed corneal neovascularization (CNV) formation
|
[
"After oral administration of 400 mg curcumin to rats, about 60% of the dose was absorbed. No curcumin was detectable in urine. The urinary excretion of conjugated glucuronides and sulfates significantly increased. No curcumin was present in heart blood. Only traces (less than 5 microgram/ml) in portal blood and negligible quantities in liver and kidney (< 20 micrograms/tissue) were observed from 15 min upto 24 h after administration of curcumin. At the end of 24 h the concentration of curcumin remaining in the lower part of the gut namely caecum and large intestine amounted to 38% of the quantity administered.",
"Curcumin, a component of turmeric (Curcuma longa), has been reported to suppress beta-catenin response transcription (CRT), which is aberrantly activated in colorectal cancer. However, the effects of its natural analogs (demethoxycurcumin [DMC] and bisdemethoxycurcumin [BDMC]) and metabolite (tetrahydrocurcumin [THC]) on the Wnt/beta-catenin pathway have not been investigated. Here, we show that DMC and BDMC suppressed CRT that was activated by Wnt3a conditioned-medium (Wnt3a-CM) without altering the level of intracellular beta-catenin, and inhibited the growth of various colon cancer cells, with comparable potency to curcumin. Additionally, DMC and BDMC down-regulated p300, which is a positive regulator of the Wnt/beta-catenin pathway. Notably, THC also inhibited CRT and cell proliferation, but to a much lesser degree than curcumin, DMC, or BDMC, indicating that the conjugated bonds in the central seven-carbon chain of curcuminoids are essential for the inhibition of Wnt/beta-catenin pathway and the anti-proliferative activity of curcuminoids. Thus, our findings suggest that curcumin derivatives inhibit the Wnt/beta-catenin pathway by decreasing the amount of the transcriptional coactivator p300.",
"Age-related macular degeneration (AMD) is a complex disease that has potential involvement of inflammatory and oxidative stress-related pathways in its pathogenesis. In search of effective therapeutic agents, we tested curcumin, a naturally occurring compound with known anti-inflammatory and antioxidative properties, in a rat model of light-induced retinal degeneration (LIRD) and in retina-derived cell lines. We hypothesized that any compound effective against LIRD, which involves significant oxidative stress and inflammation, would be a candidate for further characterization for its potential application in AMD. We observed significant retinal neuroprotection in rats fed diets supplemented with curcumin (0.2% in diet) for 2 weeks. The mechanism of retinal protection from LIRD by curcumin involves inhibition of NF-kappaB activation and down-regulation of cellular inflammatory genes. When tested on retina-derived cell lines (661W and ARPE-19), pretreatment of curcumin protected these cells from H(2)O(2)-induced cell death by up-regulating cellular protective enzymes, such as HO-1, thioredoxin. Since, curcumin with its pleiotropic activities can modulate the expression and activation of many cellular regulatory proteins such as NF-kappaB, AKT, NRF2, and growth factors, which in turn inhibit cellular inflammatory responses and protect cells; we speculate that curcumin would be an effective nutraceutical compound for preventive and augmentative therapy of AMD.",
"Wnt genes comprise a large family of secreted polypeptides that are expressed in spatially and tissue-restricted patterns during vertebrate embryonic development. Mutational analysis in mice has shown the importance of Wnts in controlling diverse developmental processes such as patterning of the body axis, central nervous system and limbs, and the regulation of inductive events during organogenesis. Although many components of the Wnt signalling pathway have been identified, little is known about how Wnts and their cognate Frizzled receptors signal to downstream effector molecules. Here we present evidence that a new member of the low-density lipoprotein (LDL)-receptor-related protein family, LRP6 (ref. 3), is critical for Wnt signalling in mice. Embryos homozygous for an insertion mutation in the LRP6 gene exhibit developmental defects that are a striking composite of those caused by mutations in individual Wnt genes. Furthermore, we show a genetic enhancement of a Wnt mutant phenotype in mice lacking one functional copy of LRP6. Together, our results support a broad role for LRP6 in the transduction of several Wnt signals in mammals.",
"Curcumin, an extract from the plant Curcuma longa with well-known antioxidant and anti-inflammatory activities, was tested as protective agent against excitotoxicity in rat retinal cultures. A 24 h-treatment with curcumin reduced N-methyl-D: -aspartate (NMDA)-mediated excitotoxic cell damage, estimated as decrease of cell viability and increase in apoptosis. The protection was associated with decrease of NMDA receptor-mediated Ca(2+) rise and reduction in the level of phosphorylated NR1 subunit of the NMDA receptor. These results enlighten a new pharmacological action of the plant extract, possibly mediated by a modulation of NMDA receptor activity.",
"The adenomatous polyposis coli gene (APC) is a tumor suppressor gene that is inactivated in most colorectal cancers. Mutations of APC cause aberrant accumulation of beta-catenin, which then binds T cell factor-4 (Tcf-4), causing increased transcriptional activation of unknown genes. Here, the c-MYC oncogene is identified as a target gene in this signaling pathway. Expression of c-MYC was shown to be repressed by wild-type APC and activated by beta-catenin, and these effects were mediated through Tcf-4 binding sites in the c-MYC promoter. These results provide a molecular framework for understanding the previously enigmatic overexpression of c-MYC in colorectal cancers.",
"Turmeric (Curcuma longa) is extensively used as a household remedy for various diseases. For the last few decades, work has been done to establish the biological activities and pharmacological actions of curcumin, the principle constituent of turmeric. Curcumin has proven to be beneficial in the prevention and treatment of a number of inflammatory diseases due to its anti-inflammatory activity. Arachidonic acid-derived lipid mediators that are intimately involved in inflammation are biosynthesized by pathways dependent on cyclooxygenase (COX) and lipoxygenase (LOX) enzymes. The role of LOX and COX isoforms, particularly COX-2, in the inflammation has been well established. At cellular and molecular levels, curcumin has been shown to regulate a number of signaling pathways, including the eicosanoid pathway involving COX and LOX. A number of studies have been conducted that support curcumin-mediated regulation of COX and LOX pathways, which is an important mechanism by which curcumin prevents a number of disease processes, including the cancer. The specific regulation of 5-LOX and COX-2 by curcumin is not fully established; however, existing evidence indicates that curcumin regulates LOX and COX-2 predominately at the transcriptional level and, to a certain extent, the posttranslational level. Thus, the curcumin-selective transcriptional regulatory action of COX-2, and dual COX/LOX inhibitory potential of this naturally occurring agent provides distinctive advantages over synthetic COX/LOX inhibitors, such as nonsteroidal anti-inflammatory drugs. In this review, we discuss evidence that supports the regulation of COX and LOX enzymes by curcumin as the key mechanism for its beneficial effects in preventing various inflammatory diseases."
] |
Plasmodium falciparum drug resistance in Mozambique.
|
One of the fundamental steps toward malaria control is the use of antimalarial drugs. The success of antimalarial treatment can be affected by the presence of drug-resistant populations of Plasmodium falciparum. To assess resistance, we used molecular methods to examine 351 P. falciparum isolates collected from 4 sentinel sites in Mozambique for K13, pfmdr1, pfcrt, and pfdhps polymorphisms and for plasmepsin2 (pfpm2) and pfmdr1 copy numbers. We found multiple copies of pfpm2 in 1.1% of isolates. All isolates carried K13 wild-type alleles (3D7-like), except 4 novel polymorphisms (Leu619Leu, Phe656Ile, Val666Val, Gly690Gly). Prevalence of isolates with pfcrt mutant (K76T) allele was low (2.3%). Prevalence of isolates with pfdhps mutant alleles (A437G and K540E) was >80%, indicating persistence of sulfadoxine/pyrimethamine resistance; however, markers of artemisinin were absent, and markers of piperaquine resistance were low. Piperaquine resistance isolates may spread in Mozambique as dihydroartemisinin/piperaquine drug pressure increases.
|
[
"Artemisinin resistance in Plasmodium falciparum threatens to reduce the efficacy of artemisinin combination therapies (ACTs), thus compromising global efforts to eliminate malaria. Recent treatment failures with dihydroartemisinin-piperaquine, the current first-line ACT in Cambodia, suggest that piperaquine resistance may be emerging in this country. We explored the relation between artemisinin resistance and dihydroartemisinin-piperaquine failures, and sought to confirm the presence of piperaquine-resistant P falciparum infections in Cambodia. In this prospective cohort study, we enrolled patients aged 2-65 years with uncomplicated P falciparum malaria in three Cambodian provinces: Pursat, Preah Vihear, and Ratanakiri. Participants were given standard 3-day courses of dihydroartemisinin-piperaquine. Peripheral blood parasite densities were measured until parasites cleared and then weekly to 63 days. The primary outcome was recrudescent P falciparum parasitaemia within 63 days. We measured piperaquine plasma concentrations at baseline, 7 days, and day of recrudescence. We assessed phenotypic and genotypic markers of drug resistance in parasite isolates. The study is registered with ClinicalTrials.gov, number NCT01736319. Between Sept 4, 2012, and Dec 31, 2013, we enrolled 241 participants. In Pursat, where artemisinin resistance is entrenched, 37 (46%) of 81 patients had parasite recrudescence. In Preah Vihear, where artemisinin resistance is emerging, ten (16%) of 63 patients had recrudescence and in Ratanakiri, where artemisinin resistance is rare, one (2%) of 60 patients did. Patients with recrudescent P falciparum infections were more likely to have detectable piperaquine plasma concentrations at baseline compared with non-recrudescent patients, but did not differ significantly in age, initial parasite density, or piperaquine plasma concentrations at 7 days. Recrudescent parasites had a higher prevalence of kelch13 mutations, higher piperaquine 50% inhibitory concentration (IC50) values, and lower mefloquine IC50 values; none had multiple pfmdr1 copies, a genetic marker of mefloquine resistance. Dihydroartemisinin-piperaquine failures are caused by both artemisinin and piperaquine resistance, and commonly occur in places where dihydroartemisinin-piperaquine has been used in the private sector. In Cambodia, artesunate plus mefloquine may be a viable option to treat dihydroartemisinin-piperaquine failures, and a more effective first-line ACT in areas where dihydroartemisinin-piperaquine failures are common. The use of single low-dose primaquine to eliminate circulating gametocytes is needed in areas where artemisinin and ACT resistance is prevalent. National Institute of Allergy and Infectious Diseases.",
"Artemisinin-based combination therapies are the recommended first-line treatments of falciparum malaria in all countries with endemic disease. There are recent concerns that the efficacy of such therapies has declined on the Thai-Cambodian border, historically a site of emerging antimalarial-drug resistance. In two open-label, randomized trials, we compared the efficacies of two treatments for uncomplicated falciparum malaria in Pailin, western Cambodia, and Wang Pha, northwestern Thailand: oral artesunate given at a dose of 2 mg per kilogram of body weight per day, for 7 days, and artesunate given at a dose of 4 mg per kilogram per day, for 3 days, followed by mefloquine at two doses totaling 25 mg per kilogram. We assessed in vitro and in vivo Plasmodium falciparum susceptibility, artesunate pharmacokinetics, and molecular markers of resistance. We studied 40 patients in each of the two locations. The overall median parasite clearance times were 84 hours (interquartile range, 60 to 96) in Pailin and 48 hours (interquartile range, 36 to 66) in Wang Pha (P<0.001). Recrudescence confirmed by means of polymerase-chain-reaction assay occurred in 6 of 20 patients (30%) receiving artesunate monotherapy and 1 of 20 (5%) receiving artesunate-mefloquine therapy in Pailin, as compared with 2 of 20 (10%) and 1 of 20 (5%), respectively, in Wang Pha (P=0.31). These markedly different parasitologic responses were not explained by differences in age, artesunate or dihydroartemisinin pharmacokinetics, results of isotopic in vitro sensitivity tests, or putative molecular correlates of P. falciparum drug resistance (mutations or amplifications of the gene encoding a multidrug resistance protein [PfMDR1] or mutations in the gene encoding sarco-endoplasmic reticulum calcium ATPase6 [PfSERCA]). Adverse events were mild and did not differ significantly between the two treatment groups. P. falciparum has reduced in vivo susceptibility to artesunate in western Cambodia as compared with northwestern Thailand. Resistance is characterized by slow parasite clearance in vivo without corresponding reductions on conventional in vitro susceptibility testing. Containment measures are urgently needed. (ClinicalTrials.gov number, NCT00493363, and Current Controlled Trials number, ISRCTN64835265.)",
"Western Cambodia is recognized as the epicenter of Plasmodium falciparum multidrug resistance. Recent reports of the efficacy of dihydroartemisinin (DHA)-piperaquine (PP), the latest of the artemisinin-based combination therapies (ACTs) recommended by the WHO, have prompted further investigations. The clinical efficacy of dihydroartemisinin-piperaquine in uncomplicated falciparum malaria was assessed in western and eastern Cambodia over 42 days. Day 7 plasma piperaquine concentrations were measured and day 0 isolates tested for in vitro susceptibilities to piperaquine and mefloquine, polymorphisms in the K13 gene, and the copy number of the Pfmdr-1 gene. A total of 425 patients were recruited in 2011 to 2013. The proportion of patients with recrudescent infections was significantly higher in western (15.4%) than in eastern (2.5%) Cambodia (P <10(-3)). Day 7 plasma PP concentrations and median 50% inhibitory concentrations (IC50) of PP were independent of treatment outcomes, in contrast to median mefloquine IC50, which were found to be lower for isolates from patients with recrudescent infections (18.7 versus 39.7 nM; P = 0.005). The most significant risk factor associated with DHA-PP treatment failure was infection by parasites carrying the K13 mutant allele (odds ratio [OR], 17.5; 95% confidence interval [CI], 1 to 308; P = 0.04). Our data show evidence of P. falciparum resistance to PP in western Cambodia, an area of widespread artemisinin resistance. New therapeutic strategies, such as the use of triple ACTs, are urgently needed and must be tested. (This study has been registered at the Australian New Zealand Clinical Trials Registry under registration no. ACTRN12614000344695.).",
"Studies of 652 adults and children with acute uncomplicated falciparum malaria were done to determine the optimum treatment of multidrug-resistant Plasmodium falciparum malaria on the Thai-Burmese border. Single-dose artesunate (4 mg/kg) plus mefloquine (25 mg of base/kg) gave more rapid symptomatic and parasitologic responses than high-dose mefloquine alone but did not improve cure rates. Three days of artesunate (total dose, 10 mg/kg) plus mefloquine was 98% effective compared with a 28-day failure rate of 31% with high-dose mefloquine alone (relative risk [RR], 0.06; 95% confidence interval [CI], 0.02-0.2; P < .0001). By day 63, the reinfection adjusted failure rates were 2% and 44%, respectively (P < .0001). Artesunate also prevented high-grade failures. Both drugs were well tolerated. No adverse effects were attributable to artesunate. Vomiting was reduced significantly by giving mefloquine on day 2 of treatment (RR, 0.40; 95% CI, 0.20-0.79; P = .009. Artesunate (10 mg/kg over 3 days) plus mefloquine (25 mg/kg) is currently the most effective treatment for falciparum malaria in this area of increasing mefloquine resistance."
] |
Risk of second cancers associated with receipt of cisplatin-based chemotherapy in randomized controlled trials
|
Case reports, retrospective analyses, and observational studies have linked the use of cisplatin to increased risk of second cancers, especially life-threatening secondary leukemia. We therefore performed a systematic review and meta-analysis to evaluate the risk of second cancers associated with receipt of cisplatin-based chemotherapy in randomized controlled trials (RCTs).
|
[
"Induction chemotherapy (IC) before radiotherapy lowers distant failure (DF) rates in locally advanced squamous cell carcinoma of the head and neck (SCCHN). The goal of this phase III trial was to determine whether IC before chemoradiotherapy (CRT) further improves survival compared with CRT alone in patients with N2 or N3 disease. Treatment-naive patients with nonmetastatic N2 or N3 SCCHN were randomly assigned to CRT alone (CRT arm; docetaxel, fluorouracil, and hydroxyurea plus radiotherapy 0.15 Gy twice per day every other week) versus two 21-day cycles of IC (docetaxel 75 mg/m(2) on day 1, cisplatin 75 mg/m(2) on day 1, and fluorouracil 750 mg/m(2) on days 1 to 5) followed by the same CRT regimen (IC + CRT arm). The primary end point was overall survival (OS). Secondary end points included DF-free survival, failure pattern, and recurrence-free survival (RFS). A total of 285 patients were randomly assigned. The most common grade 3 to 4 toxicities during IC were febrile neutropenia (11%) and mucositis (9%); during CRT (both arms combined), they were mucositis (49%), dermatitis (21%), and leukopenia (18%). Serious adverse events were more common in the IC arm (47% v 28%; P = .002). With a minimum follow-up of 30 months, there were no statistically significant differences in OS (hazard ratio, 0.91; 95% CI, 0.59 to 1.41), RFS, or DF-free survival. IC did not translate into improved OS compared with CRT alone. However, the study was underpowered because it did not meet the planned accrual target, and OS was higher than predicted in both arms. IC cannot be recommended routinely in patients with N2 or N3 locally advanced SCCHN.",
"Despite the use of resection and postoperative radiotherapy, high-risk squamous-cell carcinoma of the head and neck frequently recurs in the original tumor bed. We tested the hypothesis that concurrent postoperative administration of cisplatin and radiotherapy would improve the rate of local and regional control. Between September 9, 1995, and April 28, 2000, 459 patients were enrolled. After undergoing total resection of all visible and palpable disease, 231 patients were randomly assigned to receive radiotherapy alone (60 to 66 Gy in 30 to 33 fractions over a period of 6 to 6.6 weeks) and 228 patients to receive the identical treatment plus concurrent cisplatin (100 mg per square meter of body-surface area intravenously on days 1, 22, and 43). After a median follow-up of 45.9 months, the rate of local and regional control was significantly higher in the combined-therapy group than in the group given radiotherapy alone (hazard ratio for local or regional recurrence, 0.61; 95 percent confidence interval, 0.41 to 0.91; P=0.01). The estimated two-year rate of local and regional control was 82 percent in the combined-therapy group, as compared with 72 percent in the radiotherapy group. Disease-free survival was significantly longer in the combined-therapy group than in the radiotherapy group (hazard ratio for disease or death, 0.78; 95 percent confidence interval, 0.61 to 0.99; P=0.04), but overall survival was not (hazard ratio for death, 0.84; 95 percent confidence interval, 0.65 to 1.09; P=0.19). The incidence of acute adverse effects of grade 3 or greater was 34 percent in the radiotherapy group and 77 percent in the combined-therapy group (P<0.001). Four patients who received combined therapy died as a direct result of the treatment. Among high-risk patients with resected head and neck cancer, concurrent postoperative chemotherapy and radiotherapy significantly improve the rates of local and regional control and disease-free survival. However, the combined treatment is associated with a substantial increase in adverse effects.",
"The aims of this study were to substantiate the previously reported activity of ifosfamide in patients with advanced, persistent, or recurrent carcinosarcoma (mixed mesodermal sarcoma) of the uterus, and to determine whether the addition of cisplatin results in an improved response or survival. Secondarily, we sought to determine the toxicity of ifosfamide-cisplatin in this patient population. Patients were randomized to receive ifosfamide (1.5 g/m(2)/day) times 5 days every 3 weeks for eight courses with mesna uroprotection, with or without cisplatin (20 mg/m(2)/day) times 5 days. No patient had received previous chemotherapy. Of 224 patients entered on this study, 30 were ineligible for a variety of reasons, leaving 194 evaluable patients. Early in the study, the dose of the combination regimen was reduced by 20% (1 day) because of toxicity. The investigational arms were balanced for age, grade, and Gynecologic Oncology Group performance status. Percentages of adverse effects reported in 191 patients receiving chemotherapy included (ifosfamide/cisplatin-ifosfamide) grade 3 or 4 granulocytopenia (36/60), grade 3 or 4 anemia (8/17), grade 3 or 4 central nervous system toxicity (19/14), and grade 3 or 4 peripheral neuropathy (1/12). Treatment may have contributed to the deaths of 6 patients treated with full doses of ifosfamide and cisplatin for 5 days. The proportion of patients responding to ifosfamide alone versus ifosfamide-cisplatin therapy was (0.36 versus 0.54) overall, 0.47 versus 0.61 for pelvic, 0.21 versus 0.54 for lung, and 0.33 versus 0.40 for \"other\" metastatic sites of measurable disease. The relative odds ratio of response adjusted for measurable sites of disease was 1.82 (P = 0.03, one-tailed test; 95% lower confidence limit, 1.06). Progression-free survival (PFS) and survival data suggest that the combination offers a slight prolongation of PFS (relative risk, 0.73; 95% upper confidence limit, 0.94; P = 0.02, one-tailed test), but no significant survival benefit (relative risk, 0.80, 95% upper confidence limit, 1.03; P = 0.071, one-tailed test). The addition of cisplatin to ifosfamide appears to offer a small improvement in progression-free survival over ifosfamide alone in the management of advanced carcinosarcoma of the uterus; the added toxicity may not justify the use of this combination.",
"We compared concomitant cisplatin and irradiation with radiotherapy alone as adjuvant treatment for stage III or IV head and neck cancer. After undergoing surgery with curative intent, 167 patients were randomly assigned to receive radiotherapy alone (66 Gy over a period of 6 1/2 weeks) and 167 to receive the same radiotherapy regimen combined with 100 mg of cisplatin per square meter of body-surface area on days 1, 22, and 43 of the radiotherapy regimen. After a median follow-up of 60 months, the rate of progression-free survival was significantly higher in the combined-therapy group than in the group given radiotherapy alone (P=0.04 by the log-rank test; hazard ratio for disease progression, 0.75; 95 percent confidence interval, 0.56 to 0.99), with 5-year Kaplan-Meier estimates of progression-free survival of 47 percent and 36 percent, respectively. The overall survival rate was also significantly higher in the combined-therapy group than in the radiotherapy group (P=0.02 by the log-rank test; hazard ratio for death, 0.70; 95 percent confidence interval, 0.52 to 0.95), with five-year Kaplan-Meier estimates of overall survival of 53 percent and 40 percent, respectively. The cumulative incidence of local or regional relapses was significantly lower in the combined-therapy group (P=0.007). The estimated five-year cumulative incidence of local or regional relapses (considering death from other causes as a competing risk) was 31 percent after radiotherapy and 18 percent after combined therapy. Severe (grade 3 or higher) adverse effects were more frequent after combined therapy (41 percent) than after radiotherapy (21 percent, P=0.001); the types of severe mucosal adverse effects were similar in the two groups, as was the incidence of late adverse effects. Postoperative concurrent administration of high-dose cisplatin with radiotherapy is more efficacious than radiotherapy alone in patients with locally advanced head and neck cancer and does not cause an undue number of late complications.",
"To report results of a randomized phase II trial (Radiation Therapy Oncology Group RTOG-0234) examining concurrent chemoradiotherapy and cetuximab in the postoperative treatment of patients with squamous cell carcinoma of the head and neck (SCCHN) with high-risk pathologic features. Eligibility required pathologic stage III to IV SCCHN with gross total resection showing positive margins and/or extracapsular nodal extension and/or two or more nodal metastases. Patients were randomly assigned to 60 Gy radiation with cetuximab once per week plus either cisplatin 30 mg/m(2) or docetaxel 15 mg/m(2) once per week. Between April 2004 and December 2006, 238 patients were enrolled. With a median follow-up of 4.4 years, 2-year overall survival (OS) was 69% for the cisplatin arm and 79% for the docetaxel arm; 2-year disease-free survival (DFS) was 57% and 66%, respectively. Patients with p16-positive oropharynx tumors showed markedly improved survival outcome relative to patients with p16-negative oropharynx tumors. Grade 3 to 4 myelosuppression was observed in 28% of patients in the cisplatin arm and 14% in the docetaxel arm; mucositis was observed in 56% and 54%, respectively. DFS in this study was compared with that in the chemoradiotherapy arm of the RTOG-9501 trial (Phase III Intergroup Trial of Surgery Followed by Radiotherapy Versus Radiochemotherapy for Resectable High Risk Squamous Cell Carcinoma of the Head and Neck), which had a hazard ratio of 0.76 for the cisplatin arm versus control (P = .05) and 0.69 for the docetaxel arm versus control (P = .01), reflecting absolute improvement in 2-year DFS of 2.5% and 11.1%, respectively. The delivery of postoperative chemoradiotherapy and cetuximab to patients with SCCHN is feasible and tolerated with predictable toxicity. The docetaxel regimen shows favorable outcome with improved DFS and OS relative to historical controls and has commenced formal testing in a phase II/III trial.",
"The activity of docetaxel (Taxotere) as a single agent (overall response rates, 24%-45%) in the treatment of patients with recurrent squamous cell cancer of the head and neck has resulted in the investigation of docetaxel-based doublet and triplet combinations in both the recurrent and neoadjuvant settings. When combined with cisplatin, with or without fluorouracil (5-FU), in the treatment of recurrent disease, response rates of 33% to 44% have been observed for docetaxel, with median survival ranging from 9.6 to 11 months. In the neoadjuvant setting, response rates have been typically greater than 90%, with promising disease-free and overall survival results. Randomized trials are now under way to assess the value of docetaxel-based therapy relative to that of the standard cisplatin/5-FU combination in both the neoadjuvant and recurrent settings. Preclinical data indicate that docetaxel is a potent radiosensitizer and its initial evaluation with concurrent radiation in patients with locally advanced unresectable squamous cell cancer of the head and neck suggests feasibility. Phase II evaluation of this approach is in progress.",
"For rare outcomes, meta-analysis of randomized trials may be the only way to obtain reliable evidence of the effects of healthcare interventions. However, many methods of meta-analysis are based on large sample approximations, and may be unsuitable when events are rare. Through simulation, we evaluated the performance of 12 methods for pooling rare events, considering estimability, bias, coverage and statistical power. Simulations were based on data sets from three case studies with between five and 19 trials, using baseline event rates between 0.1 and 10 per cent and risk ratios of 1, 0.75, 0.5 and 0.2. We found that most of the commonly used meta-analytical methods were biased when data were sparse. The bias was greatest in inverse variance and DerSimonian and Laird odds ratio and risk difference methods, and the Mantel-Haenszel (MH) odds ratio method using a 0.5 zero-cell correction. Risk difference meta-analytical methods tended to show conservative confidence interval coverage and low statistical power at low event rates. At event rates below 1 per cent the Peto one-step odds ratio method was the least biased and most powerful method, and provided the best confidence interval coverage, provided there was no substantial imbalance between treatment and control group sizes within trials, and treatment effects were not exceptionally large. In other circumstances the MH OR without zero-cell corrections, logistic regression and the exact method performed similarly to each other, and were less biased than the Peto method."
] |
Operational recommendations for deliberative dialogue workshops in Africa
|
Policy decisions do not always take into account research results, and there is still little research being conducted on interventions that promote their use, particularly in Africa. To promote the use of research evidence in Africa, deliberative dialogue workshops are increasingly recommended as a means to establish evidence-informed dialogue among multiple stakeholders engaged in policy decision-making. In this paper, we reflect on our experiences of conducting national workshops in six African countries, and we propose operational recommendations for those wishing to organise deliberative dialogue. Our reflective and cross-sectional analysis of six national deliberative dialogue workshops in which we participated shows there are many specific challenges that should be taken into account when organising such encounters. In conclusion, we offer operational recommendations, drawn from our experience, to guide the preparation and conduct of deliberative workshops.
|
[
"Flaws in the design, conduct, analysis, and reporting of randomised trials can cause the effect of an intervention to be underestimated or overestimated. The Cochrane Collaboration’s tool for assessing risk of bias aims to make the process clearer and more accurate",
"In Burkina Faso, malaria remains the primary cause of healthcare use, morbidity and child mortality. Therefore, efforts are needed to support the knowledge transfer and application of the results of numerous studies to better formulate and implement programs in the fight against the malaria pandemic. To this end, a 2-day dissemination workshop was held to share the most recent results produced by a multidisciplinary research team. The objective of the present study was to evaluate the workshop and the policy briefs distributed there, the effects these produced on research results use and the processes that facilitated, or not, the application of the knowledge transmitted. A mixed-methods design was used. The data were drawn from a quantitative evaluation questionnaire completed after the workshop (n = 25/31) and qualitative interviews conducted with the researchers and various actors who attended the workshop (n = 11) and with participants in working groups (n = 40) that later analysed the policy briefs distributed at the workshop. The participants recognised the quality of the research results presented, but felt that more needed to be done to adapt the researchers' language and improve the functioning of the workshop. The potential effects of the workshop were rather limited. Effects were mainly at two levels: individual (e.g. acquisition of new knowledge, personal awareness raising) and local (e.g. change of practice in a local non-governmental organisation). Most participants perceived the utility of the research results, but several reported that their narrow decisional power limited their ability to apply this knowledge. This study showed the importance of workshops to inform key actors of research results and the need to undertake several different activities to increase the chances that the knowledge will be applied. Several recommendations are proposed to improve knowledge translation approaches in the West African context, including organising working and discussion groups, developing an action plan at the end of the workshop and offering support to participants after the workshop, among others.",
"David Moher and colleagues introduce PRISMA, an update of the QUOROM guidelines for reporting systematic reviews and meta-analyses",
"We completed a scoping review on the barriers and facilitators to use of systematic reviews by health care managers and policy makers, including consideration of format and content, to develop recommendations for systematic review authors and to inform research efforts to develop and test formats for systematic reviews that may optimise their uptake. We used the Arksey and O'Malley approach for our scoping review. Electronic databases (e.g., MEDLINE, EMBASE, PsycInfo) were searched from inception until September 2014. Any study that identified barriers or facilitators (including format and content features) to uptake of systematic reviews by health care managers and policy makers/analysts was eligible for inclusion. Two reviewers independently screened the literature results and abstracted data from the relevant studies. The identified barriers and facilitators were charted using a barriers and facilitators taxonomy for implementing clinical practice guidelines by clinicians. We identified useful information for authors of systematic reviews to inform their preparation of reviews including providing one-page summaries with key messages, tailored to the relevant audience. Moreover, partnerships between researchers and policy makers/managers to facilitate the conduct and use of systematic reviews should be considered to enhance relevance of reviews and thereby influence uptake. Systematic review authors can consider our results when publishing their systematic reviews. These strategies should be rigorously evaluated to determine impact on use of reviews in decision-making.",
"Despite wide promulgation, clinical practice guidelines have had limited effect on changing physician behavior. Little is known about the process and factors involved in changing physician practices in response to guidelines. To review barriers to physician adherence to clinical practice guidelines. We searched the MEDLINE, Educational Resources Information Center (ERIC), and HealthSTAR databases (January 1966 to January 1998); bibliographies; textbooks on health behavior or public health; and references supplied by experts to find English-language article titles that describe barriers to guideline adherence. Of 5658 articles initially identified, we selected 76 published studies describing at least 1 barrier to adherence to clinical practice guidelines, practice parameters, clinical policies, or national consensus statements. One investigator screened titles to identify candidate articles, then 2 investigators independently reviewed the texts to exclude articles that did not match the criteria. Differences were resolved by consensus with a third investigator. Two investigators organized barriers to adherence into a framework according to their effect on physician knowledge, attitudes, or behavior. This organization was validated by 3 additional investigators. The 76 articles included 120 different surveys investigating 293 potential barriers to physician guideline adherence, including awareness (n = 46), familiarity(n = 31), agreement (n = 33), self-efficacy (n = 19), outcome expectancy (n = 8), ability to overcome the inertia of previous practice (n = 14), and absence of external barriers to perform recommendations (n = 34). The majority of surveys (70 [58%] of 120) examined only 1 type of barrier. Studies on improving physician guideline adherence may not be generalizable, since barriers in one setting may not be present in another. Our review offers a differential diagnosis for why physicians do not follow practice guidelines, as well as a rational approach toward improving guideline adherence and a framework for future research.",
"Policymaking is a highly complex process that is often difficult to predict or influence. Most of the scholarship examining the role of research evidence in policymaking has focused narrowly on characteristics of the evidence and the interactions between scientists and government officials. The real-life context in which policymakers are situated and make decisions also is crucial to the development of evidence-informed policy. This qualitative study expands on other studies of research utilization at the state level through interviews with twenty-eight state legislators and administrators about their real-life experiences incorporating evidence into policymaking. The interviews were coded inductively into the following categories: (1) the important or controversial issue or problem being addressed, (2) the information that was used, (3) facilitators, and (4) hindrances. Hindrances to evidence-informed policymaking included institutional features; characteristics of the evidence supply, such as research quantity, quality, accessibility, and usability; and competing sources of influence, such as interest groups. The policymakers identified a number of facilitators to the use of evidence, including linking research to concrete impacts, costs, and benefits; reframing policy issues to fit the research; training to use evidence-based skills; and developing research venues and collaborative relationships in order to generate relevant evidence. Certain hindrances to the incorporation of research into policy, like limited budgets, are systemic and not readily altered. However, some of the barriers and facilitators of evidence-informed health policymaking are amenable to change. Policymakers could benefit from evidence-based skills training to help them identify and evaluate high-quality information. Researchers and policymakers thus could collaborate to develop networks for generating and sharing relevant evidence for policy.",
"Abstract-In Burkina Faso, inadequate interaction among researchers, decision makers, and practitioners, together with low use of research results, impedes the development of health policies and interventions to improve equity. A knowledge translation strategy was implemented as part of a research program. The broker and his team promoted links between actors (health agents, nongovernmental organizations, public administration, policy makers, researchers), provided them with research results related to their needs, and supported them in applying this knowledge in their practices. The strategy was first implemented in Kaya District, Burkina Faso. To increase impact on population health, the strategy included widening the sphere of action through collaboration with the Ministry of Health. The broker was affiliated with a public health consulting firm in the capital, Ouagadougou, and supported by Canadian experts and a senior Burkinabè broker. Evaluation shows that research use increased at the local level among health mutuals, regional nongovernmental organizations, and health professionals in Kaya, but the objective of reaching Ministry of Health decision makers was not achieved. Results highlight the need for better training in knowledge transfer for both local and international researchers and proper identification of the gateways to reach high level decision makers. This ambitious strategy encountered several obstacles: difficult access to decision makers, poor team communication, and broker's nonconducive working environment. Future brokering strategies should analyze the political situation in depth to determine when and how to approach national and regional decision makers; invest time and effort in developing different actors' (including researchers') knowledge transfer skills; and ensure sufficient and good quality communications and resources within the team.",
"The DECIDE framework was developed to support evidence-informed health system decisions through evidence summaries tailored to health policymakers. The objective of this study was to determine policymakers' perceptions regarding the criteria in the DECIDE framework and how best to summarise and present evidence to support health system decisions. We conducted an online survey of a diverse group of stakeholders with health system decision experience from 15 countries and the World Health Organization. We asked about perceptions of criteria relevant to making health system decisions, use of evidence, grading systems, and evidence summaries. We received 112 responses (70% response rate). Most respondents had healthcare (85%) and research (79%) experience. They (99%) indicated that systematic consideration of the available evidence would help to improve health system decision-making processes and supported the use of evidence from other countries (94%) and grading systems (81%). All ten criteria in the DECIDE framework were rated as important in the decision-making process. Respondents had divergent views regarding whether the same (38%) or different (45%) grading systems should be used across different types of health decisions. All components of our evidence summary were rated as important by over 90% of respondents. Survey respondents were supportive of the DECIDE framework for health system decisions and the use of succinct summaries of the estimated size of effects and the quality of evidence. It is uncertain whether the findings of this survey represent the views of policymakers with little or no healthcare and research experience.",
"Systematic reviews have the potential to inform clinical decisions, yet little is known about the impact of interventions on increasing the use of systematic reviews in clinical decision-making. To systematically review the evidence on the impact of interventions for seeking, appraising, and applying evidence from systematic reviews in decision-making by clinicians. Medline, EMBASE, CINAHL, Cochrane Central Register of Controlled Trials, and LISA were searched from the earliest date available until July 2009. Two independent reviewers selected studies for inclusion if the intervention intended to increase seeking, appraising, or applying evidence from systematic reviews by a clinician. Information about the study population, features of each intervention, methods used to measure the use of systematic reviews and those used to measure professional performance or health care outcomes, existence and use of statistical tests, study outcomes, and comparative data were extracted. A total of 8,104 titles and abstracts were reviewed, leading to retrieval of 189 full-text articles for assessment; five of these studies met all inclusion criteria. All five studies reported on professional performance behavior; none reported on patient health outcomes. One study reported positive outcomes in improving preventive care. Three studies focused on obstetrical care, with two reporting no impact on professional practice change, and one study reporting increases in the use of prophylactic oxytocin and episiotomy. One study found no improvement in the sealant rate of newly erupted molars among dentists in Scotland. The small number of studies available for examination indicates the difficulty in summarizing and identifying key aspects in successful strategies that encourage clinicians to use systematic reviews in decision-making. Other concerns lay in selective reporting and lack of blinding during data collection. The limited empirical data render the strength of evidence weak for the effectiveness and types of interventions that encourage clinicians to use systematic reviews in clinical decision making.",
"Healthcare executives report that it is difficult to access the research literature and once found, it is frequently not relevant. A study was conducted to explore ways in which healthcare executives, enrolled in the EXTRA program, used a virtual desktop environment. Despite some design and function limitations, the desktop was perceived positively by most participants and was effective in supporting evidence-informed practice and decision making."
] |
Molecular alterations associated with smoking in lung cancer cells
|
Diverse molecular alterations associated with smoking in normal and precursor lung cancer cells have been reported, yet their role in lung cancer etiology remains unclear. A prominent example is hypomethylation of the aryl hydrocarbon-receptor repressor (AHRR) locus, which is observed in blood and squamous epithelial cells of smokers, but not in lung cancer.
|
[
"Infiltrating stromal and immune cells form the major fraction of normal cells in tumour tissue and not only perturb the tumour signal in molecular studies but also have an important role in cancer biology. Here we describe 'Estimation of STromal and Immune cells in MAlignant Tumours using Expression data' (ESTIMATE)--a method that uses gene expression signatures to infer the fraction of stromal and immune cells in tumour samples. ESTIMATE scores correlate with DNA copy number-based tumour purity across samples from 11 different tumour types, profiled on Agilent, Affymetrix platforms or based on RNA sequencing and available through The Cancer Genome Atlas. The prediction accuracy is further corroborated using 3,809 transcriptional profiles available elsewhere in the public domain. The ESTIMATE method allows consideration of tumour-associated normal cells in genomic and transcriptomic studies. An R-library is available on https://sourceforge.net/projects/estimateproject/.",
"Smoking is the leading cause of preventable death worldwide. Hundreds of millions of individuals still smoke, affecting their health as well as that of their peers, family and offspring. Smoking is a well-established prime risk factor for chronic obstructive pulmonary disease and hampers the response to treatment in asthma and chronic obstructive pulmonary disease. In the present paper, new concepts are discussed with respect to pathology, treatment, smoking cessation and tobacco control. Recommendations for future directions are given.",
"Tobacco smoking is responsible for over 90% of lung cancer cases, and yet the precise molecular alterations induced by smoking in lung that develop into cancer and impact survival have remained obscure. We performed gene expression analysis using HG-U133A Affymetrix chips on 135 fresh frozen tissue samples of adenocarcinoma and paired noninvolved lung tissue from current, former and never smokers, with biochemically validated smoking information. ANOVA analysis adjusted for potential confounders, multiple testing procedure, Gene Set Enrichment Analysis, and GO-functional classification were conducted for gene selection. Results were confirmed in independent adenocarcinoma and non-tumor tissues from two studies. We identified a gene expression signature characteristic of smoking that includes cell cycle genes, particularly those involved in the mitotic spindle formation (e.g., NEK2, TTK, PRC1). Expression of these genes strongly differentiated both smokers from non-smokers in lung tumors and early stage tumor tissue from non-tumor tissue (p<0.001 and fold-change >1.5, for each comparison), consistent with an important role for this pathway in lung carcinogenesis induced by smoking. These changes persisted many years after smoking cessation. NEK2 (p<0.001) and TTK (p = 0.002) expression in the noninvolved lung tissue was also associated with a 3-fold increased risk of mortality from lung adenocarcinoma in smokers. Our work provides insight into the smoking-related mechanisms of lung neoplasia, and shows that the very mitotic genes known to be involved in cancer development are induced by smoking and affect survival. These genes are candidate targets for chemoprevention and treatment of lung cancer in smokers.",
"Lung squamous cell carcinoma is a common type of lung cancer, causing approximately 400,000 deaths per year worldwide. Genomic alterations in squamous cell lung cancers have not been comprehensively characterized, and no molecularly targeted agents have been specifically developed for its treatment. As part of The Cancer Genome Atlas, here we profile 178 lung squamous cell carcinomas to provide a comprehensive landscape of genomic and epigenomic alterations. We show that the tumour type is characterized by complex genomic alterations, with a mean of 360 exonic mutations, 165 genomic rearrangements, and 323 segments of copy number alteration per tumour. We find statistically recurrent mutations in 11 genes, including mutation of TP53 in nearly all specimens. Previously unreported loss-of-function mutations are seen in the HLA-A class I major histocompatibility gene. Significantly altered pathways included NFE2L2 and KEAP1 in 34%, squamous differentiation genes in 44%, phosphatidylinositol-3-OH kinase pathway genes in 47%, and CDKN2A and RB1 in 72% of tumours. We identified a potential therapeutic target in most tumours, offering new avenues of investigation for the treatment of squamous cell lung cancers."
] |
Conceptus implantation to the maternal endometrium
|
Processes of conceptus implantation and placentation, unique to mammalian reproduction, have been extensively studied. It was once thought that processes of these events varied greatly, notably between invasive and noninvasive modes of implantation and/or placentation. Regardless of the mode of implantation, however, physiological and biochemical processes in conceptus implantation to the maternal endometrium including the kinds of gene expression and their products are now considered not to differ so much. Recent progress has identified that in addition to the hormones, cytokines, proteases and cell adhesion molecules classically characterized, epithelial-mesenchymal transition, molecules related to lymphocyte homing, the expression of endogenous retroviruses and possibly exosomes are all required for the progression of conceptus implantation to placentation. In this review, therefore, new findings related to these events are integrated into the context of conceptus implantation to the maternal endometrium.
|
[
"The definitive demonstration of a role for a recently acquired gene is a difficult task, requiring exhaustive genetic investigations and functional analysis. The situation is indeed much more complicated when facing multicopy gene families, because most or portions of the gene are conserved among the hundred copies of the family. This is the case for the ERVWE1 locus of the human endogenous retrovirus W family (HERV-W), which encodes an envelope glycoprotein (syncytin) likely involved in trophoblast differentiation. Here we describe, in 155 individuals, the positional conservation of this locus and the preservation of the envelope ORF. Sequencing of the critical elements of the ERVWE1 provirus showed a striking conservation among the 48 alleles of 24 individuals, including the LTR elements involved in the transcriptional machinery, the splice sites involved in the maturation of subgenomic Env mRNA, and the Env ORF. The functionality and tissue specificity of the 5' LTR were demonstrated, as well as the fusogenic activity of the envelope polymorphic variants. Such functions were also shown to be preserved in the orthologous loci isolated from chimpanzee, gorilla, orangutan, and gibbon. This functional preservation among humans and during evolution strongly argued for the involvement of this recently acquired retroviral envelope glycoprotein in hominoid placental physiology.",
"The diversity of placental structures in Eutherian mammals is such that drawing generalizations from the definitive forms is problematic. There are always areas of reduced interhaemal distance whether the placenta is epitheliochorial, synepitheliochorial, endotheliochorial or haemochorial. However, the thinning may be achieved by different means. The presence of a haemophagous area as an iron transport facilitator is generally associated with endotheliochorial placentae but is also found in sheep and goats (synepitheliochorial) and in tenrecs and hyaenas (haemochorial). Although similar chorioallantoic placentae are found within families, structure begins to diverge at the ordinal level and there is little correlation at the supraordinal level of phylogeny. Differences in formation and function of the yolk sac provide additional variation. There would appear to be considerable adaptive pressure for development or retention of the haemochorial type of chorioallantoic placenta. This type of placenta has several possible drawbacks including more ready passage of fetal cells to the maternal organism and, should the haemochorial condition be achieved early, oxidative stress. At any rate no animal larger than the human and gorilla has this type of placenta. The endotheliochorial condition is found in animals as large as the bears, manatee and elephants. In addition to the ungulates, the epitheliochorial condition is present in the largest animals with the longest gestation periods, the whales. Considering the length of time since the early stages of mammalian evolution, it is probable that few unmodified structural features are present in any currently surviving mammal. Nevertheless, more complete studies of divergent types of mammalian placenta should help our understanding of mammalian interrelationships as well as placental function.",
"Syncytins are envelope genes of retroviral origin that have been co-opted by the host to mediate a specialized function in placentation. Two of these genes have already been identified in primates, as well as two distinct, non orthologous genes in rodents. Here we identified within the rabbit Oryctolagus cuniculus-which belongs to the lagomorpha order- an envelope (env) gene of retroviral origin with the characteristic features of a bona fide syncytin, that we named syncytin-Ory1. An in silico search for full-length env genes with an uninterrupted open reading frame within the rabbit genome first identified two candidate genes that were tested for their specific expression in the placenta by quantitative RT-PCR of RNA isolated from a large set of tissues. This resulted in the identification of an env gene with placenta-specific expression and belonging to a family of endogenous retroelements present at a limited copy number in the rabbit genome. Functional characterization of the identified placenta-expressed env gene after cloning in a CMV-driven expression vector and transient transfection experiments, demonstrated both fusogenic activity in an ex vivo cell-cell fusion assay and infectivity of pseudotypes. The receptor for the rabbit syncytin-Ory1 was found to be the same as that for human syncytin-1, i.e. the previously identified ASCT2 transporter. This was demonstrated by a co-culture fusion assay between hamster A23 cells transduced with an expression vector for ASCT2 and A23 cells transduced with syncytin-Ory1. Finally, in situ hybridization of rabbit placenta sections with a syncytin-Ory1 probe revealed specific expression at the level of the junctional zone between the placental lobe and the maternal decidua, where the invading syncytial fetal tissue contacts the maternal decidua to form the labyrinth, consistent with a role in the formation of the syncytiotrophoblast. The syncytin-Ory1 gene is found in Leporidae but not in Ochotonidae, and should therefore have entered the lagomorpha order 12-30 million years ago. The identification of a novel syncytin gene within a third order of mammals displaying syncytiotrophoblast formation during placentation strongly supports the notion that on several occasions retroviral infections have resulted in the independent capture of genes that have been positively selected for a convergent physiological role.",
"Syncytin is an envelope protein of the human endogenous retrovirus family W (HERV-W). Syncytin is specifically expressed in the human placenta and mediates trophoblast cell fusion into the multinucleated syncytiotrophoblast layer. It is a polypeptide of 538 amino acids and is predicted to be posttranslationally cleaved into a surface (SU) subunit and a transmembrane (TM) subunit. Functional characterization of syncytin protein can aid understanding of the molecular mechanism underlying syncytin-mediated cell fusion. In this report, we studied the structure-function relationship of syncytin in 293T and HeLa cells transiently expressing wild-type syncytin or syncytin mutants generated by linker scanning and deletion mutagenesis. Of the 22 linker-inserted mutants, mutants InS51, InV139, InE156, InS493, InA506, and InL529 were fusogenic, suggesting that regions around amino acids S51, V139, and E156 in the SU subunit and S493, A506, and L529 in the cytoplasmic domain (CTM) of syncytin are flexible in conformation. Of the 17 deletion mutants, nine mutants with deletions in the region from amino acids 479 to 538 were fusogenic. The deletion mutant DelI480, containing only the first four amino acid residues in the cytoplasmic domain, had enhanced fusogenic activity in comparison with the wild-type. In addition, two heptad repeat regions (HRA and B) were defined in the TM subunit of syncytin. A peptide inhibitor derived from the C-terminal heptad repeat region (HRB) was shown to potently inhibit syncytin-mediated cell fusion. Our results suggest that the cytoplasmic domain of syncytin is not essential for syncytin-mediated fusion but may play a regulatory role, and an intramolecular interaction between HRA and B is involved in the fusion process.",
"The preimplantation embryo floats freely within the oviduct and is capable of developing into a blastocyst independently of the maternal reproductive tract. While establishment of the trophoblast lineage is dependent on expression of developmental regulatory genes, further differentiation leading to blastocyst implantation in the uterus requires external cues emanating from the microenvironment. Recent studies suggest that trophoblast differentiation requires intracellular signaling initiated by uterine-derived growth factors and integrin-binding components of the extracellular matrix. The progression of trophoblast development from the early blastocyst stage through the onset of implantation appears to be largely independent of new gene expression. Instead, extrinsic signals direct the sequential trafficking of cell surface receptors to orchestrate the developmental program that initiates blastocyst implantation. The dependence on external cues could coordinate embryonic activities with the developing uterine endometrium. Biochemical events that regulate trophoblast adhesion to fibronectin are presented to illustrate a developmental strategy employed by the peri-implantation blastocyst.",
"Implantation sites from rats were studied on days 6, 7, and 8 of pregnancy to determine the sequence of events in the formation of blood spaces in the trophoblast that is part of the parietal wall of the yolk sac placenta and to determine how trophoblast gains access to maternal blood. The maternal blood flowing through these spaces is the source of nutrients that reach the embryo via the visceral endoderm. Tissues were prepared for light microscopy, scanning electron microscopy, and transmission electron microscopy. Trophoblast blood spaces are derived from the lateral intercellular spaces of trophoblast cells and are present in a collapsed condition until day 8, when maternal vessels are tapped by trophoblast. These spaces then contain circulating maternal blood, and trophoblast cells reflect adaptations for metabolic exchange including thinning of trophoblast covering Reichert's membrane and the appearance of numerous fenestrations, with and without diaphragms, in the areas where trophoblast is attenuated. Between days 6 and 7 decidual cells appear to form a barrier between the maternal circulation and trophoblast. On day 7, however, decidual cell processes penetrate the residual uterine luminal epithelial basal lamina, and then the decidual cells that are juxtaposed to trophoblast undergo degradative changes that resemble apoptosis. There is condensation of cytoplasmic contents, fragmentation of the cells, and phagocytosis of the fragments by trophoblast. Some decidual cells are interposed between endothelial cells in the walls of maternal vessels as early as day 7. Trophoblast may gain access to the maternal vessels by replacing decidual cells or by direct imposition of trophoblast cell processes between endothelial cells.",
"Menangle virus is a novel paramyxovirus isolated in Australia in 1997, but its classification position has not yet been finally settled. Here by using a computational program, LearnCoil-VMF, we determined the heptad repeat (HR) regions (HR1 and HR2) of Menangle virus F protein. Subsequently the HR1 and HR2 peptides were expressed as a single chain (named 2-Helix) connected by a six amino-acid linker as a GST fusion protein with an E. coli in vitro expression system. The GST-removed purified 2-Helix protein could form a stable trimer in vitro judging by gel-filtration and chemical cross-linking. CD spectra showed that the 2-Helix protein had a high percentage of alpha-helix and was very thermo-stable. Crystals of the 2-Helix protein preparations have been obtained in many conditions with hanging-drop diffusion method. These results indicated that Menangle virus has the common features of the fusion protein for other paramyxoviruses and should adopt a similar fusion mechanism to other members. As the HR regions of Menangle virus F protein could form stable six-helix bundle coiled coil structure, they should be used as drug target for the design of fusion inhibitors, as successfully used for other parmyxoviruses. This is especially relevant to such a newly emergent virus with zoonotic potentials."
] |
Roles of spectrin in heart function and disease
|
In the heart, pathways that transduce extracellular environmental cues (e.g. mechanical force, inflammatory stress) into electrical and/or chemical signals at the cellular level are critical for the organ-level response to chronic biomechanical/neurohumoral stress. Specifically, a diverse array of membrane-bound receptors and stretch-activated proteins converge on a network of intracellular signaling cascades that control gene expression, protein translation, degradation and/or regulation. These cellular reprogramming events ultimately lead to changes in cell excitability, growth, proliferation, and/or survival. Areas covered: The actin/spectrin cytoskeleton has emerged as having important roles in not only providing structural support for organelle function but also in serving as a signaling 'superhighway,' linking signaling events at/near the membrane to distal cellular domains (e.g. nucleus, mitochondria). Furthermore, recent work suggests that the integrity of the actin/spectrin cytoskeleton is critical for canonical signaling of pathways involved in cellular response to stress. This review discusses these emerging roles for spectrin and consider implications for heart function and disease. Expert commentary: Despite growth in our understanding of the broader roles for spectrins in cardiac myocytes and other metazoan cells, there remain important unanswered questions, the answers to which may point the way to new therapies for human cardiac disease patients.
|
[
"Ion channel clustering at the axon initial segment (AIS) and nodes of Ranvier has been suggested to be a key evolutionary innovation that enabled the development of the complex vertebrate nervous system. This innovation epitomizes a signature feature of neurons, namely polarity. The mechanisms that establish neuronal polarity, channel clustering and axon-dendrite identity during development are becoming clearer. However, much less is known about how polarity is maintained throughout life. Here, I review the role of the AIS in the development and maintenance of neuronal polarity and discuss how disrupted polarity may be a common component of many diseases and injuries that affect the nervous system.",
"We report identification of an ankyrin-B-based macromolecular complex of Na/K ATPase (alpha 1 and alpha 2 isoforms), Na/Ca exchanger 1, and InsP3 receptor that is localized in cardiomyocyte T-tubules in discrete microdomains distinct from classic dihydropyridine receptor/ryanodine receptor \"dyads.\" E1425G mutation of ankyrin-B, which causes human cardiac arrhythmia, also blocks binding of ankyrin-B to all three components of the complex. The ankyrin-B complex is markedly reduced in adult ankyrin-B(+/-) cardiomyocytes, which may explain elevated [Ca2+]i transients in these cells. Thus, loss of the ankyrin-B complex provides a molecular basis for cardiac arrhythmia in humans and mice. T-tubule-associated ankyrin-B, Na/Ca exchanger, and Na/K ATPase are not present in skeletal muscle, where ankyrin-B is expressed at 10-fold lower levels than in heart. Ankyrin-B also is not abundantly expressed in smooth muscle. We propose that the ankyrin-B-based complex is a specialized adaptation of cardiomyocytes with a role for cytosolic Ca2+ modulation.",
"The cardiac cytoskeleton plays key roles in maintaining myocyte structural integrity in health and disease. In fact, human mutations in cardiac cytoskeletal elements are tightly linked to cardiac pathologies, including myopathies, aortopathies, and dystrophies. Conversely, the link between cytoskeletal protein dysfunction and cardiac electric activity is not well understood and often overlooked in the cardiac arrhythmia field. Here, we uncover a new mechanism for the regulation of cardiac membrane excitability. We report that βII spectrin, an actin-associated molecule, is essential for the posttranslational targeting and localization of critical membrane proteins in heart. βII spectrin recruits ankyrin-B to the cardiac dyad, and a novel human mutation in the ankyrin-B gene disrupts the ankyrin-B/βII spectrin interaction, leading to severe human arrhythmia phenotypes. Mice lacking cardiac βII spectrin display lethal arrhythmias, aberrant electric and calcium handling phenotypes, and abnormal expression/localization of cardiac membrane proteins. Mechanistically, βII spectrin regulates the localization of cytoskeletal and plasma membrane/sarcoplasmic reticulum protein complexes, including the Na/Ca exchanger, ryanodine receptor 2, ankyrin-B, actin, and αII spectrin. Finally, we observe accelerated heart failure phenotypes in βII spectrin-deficient mice. Our findings identify βII spectrin as critical for normal myocyte electric activity, link this molecule to human disease, and provide new insight into the mechanisms underlying cardiac myocyte biology.",
"Non-erythroid alpha spectrin (αIISp) is a structural protein which we have shown is present in the nucleus of human cells. It interacts with a number of nuclear proteins such as actin, lamin, emerin, chromatin remodeling factors, and DNA repair proteins. αIISp's interaction with DNA repair proteins has been extensively studied. We have demonstrated that nuclear αIISp is critical in DNA interstrand cross-link (ICL) repair in S phase, in both genomic (non-telomeric) and telomeric DNA, and in maintenance of genomic stability following ICL damage to DNA. We have proposed that αIISp acts as a scaffold aiding to recruit repair proteins to sites of damage. This involvement of αIISp in ICL repair and telomere maintenance after ICL damage represents new and critical functions for αIISp. These studies have led to development of a model for the role of αIISp in DNA ICL repair. They have been aided by examination of cells from patients with Fanconi anemia (FA), a repair-deficient genetic disorder in which a deficiency in αIISp leads to defective ICL repair in genomic and telomeric DNA, telomere dysfunction, and chromosome instability following DNA ICL damage. We have shown that loss of αIISp in FA cells is due to increased breakdown by the protease, µ-calpain. Importantly, we have demonstrated that this deficiency can be corrected by knockdown of µ-calpain and restoring αIISp levels to normal. This corrects a number of the phenotypic deficiencies in FA after ICL damage. These studies suggest a new and unexplored direction for therapeutically restoring genomic stability in FA cells and for correcting numerous phenotypic deficiencies occurring after ICL damage. Developing a more in-depth understanding of the importance of the interaction of αIISp with other nuclear proteins could significantly enhance our knowledge of the consequences of loss of αIISp on critical nuclear processes.",
"Voltage-gated Na(v) channels are required for normal electrical activity in neurons, skeletal muscle, and cardiomyocytes. In the heart, Na(v)1.5 is the predominant Na(v) channel, and Na(v)1.5-dependent activity regulates rapid upstroke of the cardiac action potential. Na(v)1.5 activity requires precise localization at specialized cardiomyocyte membrane domains. However, the molecular mechanisms underlying Na(v) channel trafficking in the heart are unknown. In this paper, we demonstrate that ankyrin-G is required for Na(v)1.5 targeting in the heart. Cardiomyocytes with reduced ankyrin-G display reduced Na(v)1.5 expression, abnormal Na(v)1.5 membrane targeting, and reduced Na(+) channel current density. We define the structural requirements on ankyrin-G for Na(v)1.5 interactions and demonstrate that loss of Na(v)1.5 targeting is caused by the loss of direct Na(v)1.5-ankyrin-G interaction. These data are the first report of a cellular pathway required for Na(v) channel trafficking in the heart and suggest that ankyrin-G is critical for cardiac depolarization and Na(v) channel organization in multiple excitable tissues.",
"In the heart, cytoplasmic actin networks are thought to have important roles in mechanical support, myofibrillogenesis, and ion channel function. However, subcellular localization of cytoplasmic actin isoforms and proteins involved in the modulation of the cytoplasmic actin networks are elusive. Mena and VASP are important regulators of actin dynamics. Due to the lethal phenotype of mice with combined deficiency in Mena and VASP, however, distinct cardiac roles of the proteins remain speculative. In the present study, we analyzed the physiological functions of Mena and VASP in the heart and also investigated the role of the proteins in the organization of cytoplasmic actin networks. We generated a mouse model, which simultaneously lacks Mena and VASP in the heart. Mena/VASP double-deficiency induced dilated cardiomyopathy and conduction abnormalities. In wild-type mice, Mena and VASP specifically interacted with a distinct αII-Spectrin splice variant (SH3i), which is in cardiomyocytes exclusively localized at Z- and intercalated discs. At Z- and intercalated discs, Mena and β-actin localized to the edges of the sarcomeres, where the thin filaments are anchored. In Mena/VASP double-deficient mice, β-actin networks were disrupted and the integrity of Z- and intercalated discs was markedly impaired. Together, our data suggest that Mena, VASP, and αII-Spectrin assemble cardiac multi-protein complexes, which regulate cytoplasmic actin networks. Conversely, Mena/VASP deficiency results in disrupted β-actin assembly, Z- and intercalated disc malformation, and induces dilated cardiomyopathy and conduction abnormalities.",
"Ankyrin, the membrane attachment protein for human erythrocyte spectrin, is tightly linked in a 1:1 molar ratio with band 3 in detergent extracts of spectrin-depleted membranes. Ankyrin-linked band 3, which represents 10--15% of the total band 3, spans the membrane, and is nearly identical to the major band 3 by peptide analysis. Spectrin binds to solubilised ankyrin-linked band 3, but not to free band 3. A portion of band 3 remains firmly associated with detergent-extracted cytoskeletal proteins. It is concluded that a fraction of band 3 is attached to the erythrocyte cytoskeleton through association with ankyrin, which in turn is bound to spectrin.",
"Atrial fibrillation (AF) is the most common cardiac arrhythmia, affecting >2 million patients in the United States alone. Despite decades of research, surprisingly little is known regarding the molecular pathways underlying the pathogenesis of AF. ANK2 encodes ankyrin-B, a multifunctional adapter molecule implicated in membrane targeting of ion channels, transporters, and signaling molecules in excitable cells. In the present study, we report early-onset AF in patients harboring loss-of-function mutations in ANK2. In mice, we show that ankyrin-B deficiency results in atrial electrophysiological dysfunction and increased susceptibility to AF. Moreover, ankyrin-B(+/-) atrial myocytes display shortened action potentials, consistent with human AF. Ankyrin-B is expressed in atrial myocytes, and we demonstrate its requirement for the membrane targeting and function of a subgroup of voltage-gated Ca(2+) channels (Ca(v)1.3) responsible for low voltage-activated L-type Ca(2+) current. Ankyrin-B is associated directly with Ca(v)1.3, and this interaction is regulated by a short, highly conserved motif specific to Ca(v)1.3. Moreover, loss of ankyrin-B in atrial myocytes results in decreased Ca(v)1.3 expression, membrane localization, and function sufficient to produce shortened atrial action potentials and arrhythmias. Finally, we demonstrate reduced ankyrin-B expression in atrial samples of patients with documented AF, further supporting an association between ankyrin-B and AF. These findings support that reduced ankyrin-B expression or mutations in ANK2 are associated with AF. Additionally, our data demonstrate a novel pathway for ankyrin-B-dependent regulation of Ca(v)1.3 channel membrane targeting and regulation in atrial myocytes.",
"The membrane-associated cytoskeleton of the cardiac muscle cell is emerging as an important element in the maintenance of normal cell functioning. Recently it was shown that when proteins (betaII-spectrin, muscle Lim-only protein, ankyrin-B, ankyrin-G) of this system are defective or deficient, cardiac malfunction ensues. It is well-established that the spectrin cytoskeleton is associated with the plasma membrane, but it was only lately demonstrated that its components also lie on internal cell membranes. This is particularly apparent in muscle cells of the heart which contain specialised intracellular membrane compartments particular to this cell type such as the sarcoplasmic reticulum and T-tubules. Cardiomyocytes are subjected to constant mechanical stress. Since their mechanics are controlled through coordination of calcium fluxes mediated via cell membrane-based assemblies, it is imperative that these essential elements withstand the displacement forces of contraction. Cardiomyocyte spectrin locates the multifunctional spectrin/actin-binding and membrane-binding component, protein 4.1, and they act together on the plasma membrane as well as on internal membranes. We have found that cardiac protein 4.1 links to the calcium handling apparatus whilst spectrins connect with the sarcomeric contractile elements of the cell. Overall this assembly fulfils roles in stabilising cardiomyocyte cell membranes and in coordinating the macromolecular protein accumulations which regulate and accomplish cardiac molecular crosstalk, whilst at the same time enabling the muscle cells to resist extreme forces of contraction.",
"The use of biomarkers of brain injury in pediatric neurocritical care has been explored for at least 15 years. Two general lines of research on biomarkers in pediatric brain injury have been pursued: (1) studies of \"bio-mediators\" in cerebrospinal fluid (CSF) of children after traumatic brain injury (TBI) to explore the components of the secondary injury cascades in an attempt to identify potential therapeutic targets and (2) studies of the release of structural proteins into the CSF, serum, or urine in order to diagnose, monitor, and/or prognosticate in patients with TBI or other pediatric neurocritical care conditions. Unique age-related differences in brain biology, disease processes, and clinical applications mandate the development and testing of brain injury bio-mediators and biomarkers specifically in pediatric neurocritical care applications. Finally, although much of the early work on biomarkers of brain injury in pediatrics has focused on TBI, new applications are emerging across a wide range of conditions specifically for pediatric neurocritical care including abusive head trauma, cardiopulmonary arrest, septic shock, extracorporeal membrane oxygenation, hydrocephalus, and cardiac surgery. The potential scope of the utility of biomarkers in pediatric neurocritical care is thus also discussed."
] |
Long non protein coding RNAs in Neurospora crassa
|
Long non protein coding RNAs (lncRNAs) have been identified in many different organisms and cell types. Emerging examples emphasize the biological importance of these RNA species but their regulation and functions remain poorly understood. In the filamentous fungus Neurospora crassa, the annotation and characterization of lncRNAs is incomplete.
|
[
"An increasing number of eukaryotic genes are being found to have naturally occurring antisense transcripts. Here we study the extent of antisense transcription in the human genome by analyzing the public databases of expressed sequences using a set of computational tools designed to identify sense-antisense transcriptional units on opposite DNA strands of the same genomic locus. The resulting data set of 2,667 sense-antisense pairs was evaluated by microarrays containing strand-specific oligonucleotide probes derived from the region of overlap. Verification of specific cases by northern blot analysis with strand-specific riboprobes proved transcription from both DNA strands. We conclude that > or =60% of this data set, or approximately 1,600 predicted sense-antisense transcriptional units, are transcribed from both DNA strands. This indicates that the occurrence of antisense transcription, usually regarded as infrequent, is a very common phenomenon in the human genome. Therefore, antisense modulation of gene expression in human cells may be a common regulatory mechanism.",
"We describe an open source, portable, JavaScript-based genome browser, JBrowse, that can be used to navigate genome annotations over the web. JBrowse helps preserve the user's sense of location by avoiding discontinuous transitions, instead offering smoothly animated panning, zooming, navigation, and track selection. Unlike most existing genome browsers, where the genome is rendered into images on the webserver and the role of the client is restricted to displaying those images, JBrowse distributes work between the server and client and therefore uses significantly less server overhead than previous genome browsers. We report benchmark results empirically comparing server- and client-side rendering strategies, review the architecture and design considerations of JBrowse, and describe a simple wiki plug-in that allows users to upload and share annotation tracks.",
"A few years ago, FlyBase undertook to design a new database schema to store Drosophila data. It would fully integrate genomic sequence and annotation data with bibliographic, genetic, phenotypic and molecular data from the literature representing a distillation of the first 100 years of research on this major animal model system. In developing this new integrated schema, FlyBase also made a commitment to ensure that its design was generic, extensible and available as open source, so that it could be employed as the core schema of any model organism data repository, thereby avoiding redundant software development and potentially increasing interoperability. Our question was whether we could create a relational database schema that would be successfully reused. Chado is a relational database schema now being used to manage biological knowledge for a wide variety of organisms, from human to pathogens, especially the classes of information that directly or indirectly can be associated with genome sequences or the primary RNA and protein products encoded by a genome. Biological databases that conform to this schema can interoperate with one another, and with application software from the Generic Model Organism Database (GMOD) toolkit. Chado is distinctive because its design is driven by ontologies. The use of ontologies (or controlled vocabularies) is ubiquitous across the schema, as they are used as a means of typing entities. The Chado schema is partitioned into integrated subschemas (modules), each encapsulating a different biological domain, and each described using representations in appropriate ontologies. To illustrate this methodology, we describe here the Chado modules used for describing genomic sequences. GMOD is a collaboration of several model organism database groups, including FlyBase, to develop a set of open-source software for managing model organism data. The Chado schema is freely distributed under the terms of the Artistic License (http://www.opensource.org/licenses/artistic-license.php) from GMOD (www.gmod.org).",
"Affymetrix exon arrays aim to target every known and predicted exon in the human, mouse or rat genomes, and have reporters that extend beyond protein coding regions to other areas of the transcribed genome. This combination of increased coverage and precision is important because a substantial proportion of protein coding genes are predicted to be alternatively spliced, and because many non-coding genes are known also to be of biological significance. In order to fully exploit these arrays, it is necessary to associate each reporter on the array with the features of the genome it is targeting, and to relate these to gene and genome structure. X:Map is a genome annotation database that provides this information. Data can be browsed using a novel Google-maps based interface, and analysed and further visualized through an associated BioConductor package. The database can be found at http://xmap.picr.man.ac.uk.",
"The mouse (Mus musculus) is the premier animal model for understanding human disease and development. Here we show that a comprehensive understanding of mouse biology is only possible with the availability of a finished, high-quality genome assembly. The finished clone-based assembly of the mouse strain C57BL/6J reported here has over 175,000 fewer gaps and over 139 Mb more of novel sequence, compared with the earlier MGSCv3 draft genome assembly. In a comprehensive analysis of this revised genome sequence, we are now able to define 20,210 protein-coding genes, over a thousand more than predicted in the human genome (19,042 genes). In addition, we identified 439 long, non-protein-coding RNAs with evidence for transcribed orthologs in human. We analyzed the complex and repetitive landscape of 267 Mb of sequence that was missing or misassembled in the previously published assembly, and we provide insights into the reasons for its resistance to sequencing and assembly by whole-genome shotgun approaches. Duplicated regions within newly assembled sequence tend to be of more recent ancestry than duplicates in the published draft, correcting our initial understanding of recent evolution on the mouse lineage. These duplicates appear to be largely composed of sequence regions containing transposable elements and duplicated protein-coding genes; of these, some may be fixed in the mouse population, but at least 40% of segmentally duplicated sequences are copy number variable even among laboratory mouse strains. Mouse lineage-specific regions contain 3,767 genes drawn mainly from rapidly-changing gene families associated with reproductive functions. The finished mouse genome assembly, therefore, greatly improves our understanding of rodent-specific biology and allows the delineation of ancestral biological functions that are shared with human from derived functions that are not.",
"Tumour suppressor genes (TSGs) inhibiting normal cellular growth are frequently silenced epigenetically in cancer. DNA methylation is commonly associated with TSG silencing, yet mutations in the DNA methylation initiation and recognition machinery in carcinogenesis are unknown. An intriguing possible mechanism for gene regulation involves widespread non-coding RNAs such as microRNA, Piwi-interacting RNA and antisense RNAs. Widespread sense-antisense transcripts have been systematically identified in mammalian cells, and global transcriptome analysis shows that up to 70% of transcripts have antisense partners and that perturbation of antisense RNA can alter the expression of the sense gene. For example, it has been shown that an antisense transcript not naturally occurring but induced by genetic mutation leads to gene silencing and DNA methylation, causing thalassaemia in a patient. Here we show that many TSGs have nearby antisense RNAs, and we focus on the role of one RNA in silencing p15, a cyclin-dependent kinase inhibitor implicated in leukaemia. We found an inverse relation between p15 antisense (p15AS) and p15 sense expression in leukaemia. A p15AS expression construct induced p15 silencing in cis and in trans through heterochromatin formation but not DNA methylation; the silencing persisted after p15AS was turned off, although methylation and heterochromatin inhibitors reversed this process. The p15AS-induced silencing was Dicer-independent. Expression of exogenous p15AS in mouse embryonic stem cells caused p15 silencing and increased growth, through heterochromatin formation, as well as DNA methylation after differentiation of the embryonic stem cells. Thus, natural antisense RNA may be a trigger for heterochromatin formation and DNA methylation in TSG silencing in tumorigenesis."
] |
Clustering of Complement Receptor 1 (CR1) in the Erythrocyte Membrane
|
Clustering of Complement Receptor 1 (CR1) in the erythrocyte membrane is important for immune-complex transfer and clearance. CR1 contains the Knops blood group antigens, including the antithetical pairs Swain-Langley 1 and 2 (Sl1 and Sl2) and McCoy a and b (McC
|
[
"The E C3b/C4b receptor (CR1) has been shown to rapidly bind large complement-fixing immune complexes (IC) both in vivo and in vitro. It has been proposed that E (RBC) CR1 act as a shuttle mechanism, binding circulating IC and transporting them to tissue macrophages, thereby preventing their deposition in target tissues. In this study we have established an in vitro model system with which to study the transfer of model IC from CR1 on the RBC surface to phagocytic cells. Aggregated IgG (AHG) was opsonized with C3b, bound to RBC CR1, and the binding of these RBC-bound IC by a human monocyte cell line (U937 cells) was examined. U937 binding of AHG from the RBC surface was complete within 2 min, whereas binding of the same AHG from solution required 30 to 60 min. Despite the difference in kinetics of binding, the total amount of IC bound by U937 cells at equilibrium was the same for RBC-bound AHG and for AHG in solution. The transfer of AHG from the RBC to the U937 cell did not require exogenous factor I and was not accompanied by binding of RBC to U937 cells or by erythrophagocytosis. Our data lend support to the hypothesis that binding of IC to RBC CR1 may facilitate the clearance of IC from the circulation by enhancing their uptake by phagocytic cells.",
"Vesicles released from human E by Ca(2+)-loading, ATP-depletion, or storage are enriched in several glycosylphosphatidylinositol-anchored proteins such as acetylcholinesterase (AchE) and decay-accelerating factor (DAF). As a result of this, the remaining E are depleted of these proteins. We analyzed whether vesiculation induced by ATP-depletion in vitro was also responsible for a loss of C receptor 1 (CR1), which is a transmembrane protein arranged predominantly in small clusters. ATP-depleted E had lost 15.4% to 33.9% of their CR1. This loss was similar to that of AchE and DAF. The released vesicles contained CR1. The number of CR1 per band 3 protein was 1.7 to 2.7 that in the original E, indicating an enrichment of CR1 in vesicles. This enrichment was similar to that observed for AchE and DAF (1.83- and 2.6-fold, respectively). The capacity of the vesicles and the ATP-depleted E to bind C3b-coated immune complexes correlated with the CR1 number, suggesting that there was no preferential loss of CR1 clusters. Vesicles released from human E during C attack also contained CR1. In conclusion, in vitro aging induced by ATP-depletion is responsible not only for a loss of glycosylphosphatidylinositol-anchored proteins, but also of CR1. Whether vesiculation explains the loss of CR1 from aging E in vivo and from E of patients with SLE or AIDS remains to be studied.",
"Malaria remains one of the world's deadliest diseases. Plasmodium falciparum is responsible for the most severe and lethal form of human malaria. P. falciparum's life cycle involves two obligate hosts: human and mosquito. From initial entry into these hosts, malaria parasites face the onslaught of the first line of host defence, the complement system. In this review, we discuss the complex interaction between complement and malaria infection in terms of hosts immune responses, parasite survival and pathogenesis of severe forms of malaria. We will focus on the role of complement receptor 1 and its associated polymorphisms in malaria immune complex clearance, as a mediator of parasite rosetting and as an entry receptor for P. falciparum invasion. Complement evasion strategies of P. falciparum parasites will also be highlighted. The sexual forms of the malaria parasites recruit the soluble human complement regulator Factor H to evade complement-mediated killing within the mosquito host. A novel evasion strategy is the deployment of parasite organelles to divert complement attack from infective blood stage parasites. Finally we outline the future challenge to understand the implications of these exploitation mechanisms in the interplay between successful infection of the host and pathogenesis observed in severe malaria.",
"Complement receptor 1 (CR1) on primate red blood cells (RBC) binds most complement-fixing immune complexes in the circulation. It has been postulated that by binding them, RBC keep immune complexes in the intravascular space and deliver them to the tissue macrophages of the mononuclear phagocyte system. We have developed an in vitro model to study the transfer of RBC-bound immune complexes (heat-aggregated IgG and DNA-anti-DNA) to phagocytic cells (human monocytes). Transfer of immune complexes from RBC to monocytes occurred significantly more rapidly than monocyte uptake of the same immune complexes from solution. In the transfer process, complex-bearing RBC were not bound or sequestered by the monocytes. To define the monocyte receptors involved in binding immune complexes from the RBC surface, monocyte receptors were blocked with MoAbs (anti-CR1, anti-FcRII) or EDTA (to block CR3). Monocyte binding of immune complexes primarily used CR1 with a small contribution from FcRII, and with little or no contribution from CR3 and FcRI. Uptake of immune complexes from solution employed the same monocyte receptors as binding of complexes from the RBC surface. Immune complexes in solution bound to RBC and to monocytes with equally high avidity (approximately 1 x 10(11) l/M), but monocytes expressed a 15-20-fold greater number of immune complex binding sites. We propose that immune complexes distribute between RBC and monocytes according to the binding capacity of these cells, such that at equal or high RBC/monocyte ratios as would be seen in the circulation immune complexes bind to RBC, but at low RBC/monocyte ratios (as would be seen in the sinusoidal circulation of the liver and spleen), most immune complexes bind to monocytes. To define the pathway by which immune complexes move from RBC to monocytes, their release from RBC CR1 was examined. Under various conditions, the dissociation rate was extremely slow, and did not increase with the addition of monocyte supernatants. To examine whether factor I-mediated processing of immune complexes enhances binding of immune complexes to monocytes, RBC-bound complexes were released with factor I, and binding of these 'processed' immune complexes to monocytes was examined. Monocyte binding of these processed immune complexes was slower than of control ones; furthermore, performance of transfer experiments at 4 degrees C, which significantly shows enzymatic processes, did not decrease the rate of immune complex transfer from RBC to monocytes.(ABSTRACT TRUNCATED AT 400 WORDS)",
"Human CR1 is widely distributed in the circulation as a surface receptor as well as in soluble form in the plasma. It mediates a variety of functions that include phagocytosis and regulation of the complement cascade. This receptor has been cloned and the primary structure reveals that the cell-bound molecule is an integral membrane protein with typical transmembrane and cytoplasmic domains. Its extracellular portion is composed entirely of 30 short consensus repeats (SCR) each having 60 to 70 amino acids. This type of motif is the common structural element of the superfamily of complement regulatory and receptor proteins on chromosome 1. The amino-terminal 28 SCR of CR1 is uniquely organized into four tandem long homologous repeats (LHR) with sequence homologies among corresponding SCR as high as 99%. Each LHR encodes approximately 45 kD and each except the one that is proximal to the cell surface contains a separate binding site for C3b or C4b. Analysis of the genomic structure of CR1 reveals that these LHR are results of intragenic duplication of 20- to 30-kb segments of DNA. The structural allotypes of CR1 that vary in the lengths of the polypeptides are encoded by alleles that contain different numbers of LHR. Their predicted structures would have different numbers of C3b binding sites, perhaps resulting in molecules with different capacities to bind immune complexes.",
"The complex interactions between the human host and the Plasmodium falciparum parasite and the factors influencing severity of disease are still not fully understood. Human single nucleotide polymorphisms SNPs associated with Knops blood group system; carried by complement receptor 1 may be associated with the pathology of P. falciparum malaria, and susceptibility to disease. The objective of this study was to determine the genotype and haplotype frequencies of the SNPs defining the Knops blood group antigens; Kna/b, McCoya/b, Swain-Langley1/2 and KCAM+/- in Ghanaian patients with malaria and determine possible associations between these polymorphisms and the severity of the disease. Study participants were patients (n = 267) admitted to the emergency room at the Department of Child Health, Korle-Bu Teaching Hospital, Accra, Ghana during the malaria season from June to August in 1995, 1996 and 1997, classified as uncomplicated malaria (n = 89), severe anaemia (n = 57) and cerebral malaria (n = 121) and controls who did not have a detectable Plasmodium infection or were symptomless carriers of the parasite (n = 275). The frequencies were determined using a post-PCR ligation detection reaction-fluorescent microsphere assay, developed to detect the SNPs defining the antigens. Chi-square/Fisher's exact test and logistic regression models were used to analyse the data. As expected, high frequencies of the alleles Kna, McCb, Sl2 and KCAM- were found in the Ghanaian population. Apart from small significant differences between the groups at the Sl locus, no significant allelic or genotypic differences were found between the controls and the disease groups or between the disease groups. The polymorphisms define eight different haplotypes H1(2.4%), H2(9.4%), H3(59.8%), H4(0%), H5(25.2%), H6(0.33%), H7(2.8%) and H8(0%). Investigating these haplotypes, no significant differences between any of the groups were found. The results confirm earlier findings of high frequencies of certain CR1 alleles in Africa; and shed more light on earlier conflicting findings; the alleles McCb, Sl2, Knb and KCAM- or combined haplotypes do not seem to confer any protective advantage against malaria infection or resulting disease severity. Based on these findings, in a very well-characterized population, malaria does not seem to be the selective force on these alleles.",
"We used soluble, C-fixing antibody/dsDNA IC to investigate immune complex (IC) handling and erythrocyte (E)-to-phagocyte transfer in chimpanzees. IC bound efficiently to chimpanzee E in vitro and showed minimal release with further in vitro incubation in the presence of serum in EDTA (less than or equal to 15% within 1 h). These IC also bound rapidly to E in vivo (70-80% binding within 1 min) and did not show detectable release from E in the peripheral circulation after infusion in vivo (less than or equal to 2% within 1 h). Despite such slow C-mediated release of IC from E, IC were rapidly stripped from E by the mononuclear phagocyte system (T50 for E-IC1500 = 5 min) without sequestration of E. Treatment of the chimpanzees with the anti-Fc gamma RIII MAb 3G8 impaired the clearance of infused IC. This effect was most evident on the fraction of IC500 which did not bind to E and which presumably had captured less C3b (pre-MAb 3G8 T50: 45 min vs. post-MAb 3G8 T50: 180 min). With IC bound in vitro to E before infusion, anti-Fc gamma RIII MAb treatment led to significant amounts of non-E bound IC detectable in the circulation. Thus, the anti-Fc gamma RIII MAb appeared to interfere with the ability of fixed tissue mononuclear phagocytes to take up/or retain IC after their release from E. Both rapid stripping of IC from E, despite slow complement-mediated release of IC from E in the peripheral circulation, and blockade of IC clearance with anti-Fc gamma RIII MAb indicate that the interaction of IC with the fixed tissue phagocyte involves qualitatively different mechanisms than the interaction of IC with E. Fc gamma receptors appear to play an important role in the transfer and retention of IC by the phagocyte."
] |
Nonspecific phospholipase C genes from Gossypium hirsutum
|
Nonspecific phospholipase C (NPC), which belongs to a phospholipase C subtype, is a class of phospholipases that hydrolyzes the primary membrane phospholipids, such as phosphatidylcholine, to yield sn-1, 2-diacylglycerol and a phosphorylated head-group. NPC plays multiple physiological roles in lipid metabolism and signaling in plants. To fully understand the putative roles of NPC genes in upland cotton, we cloned NPC genes from Gossypium hirsutum and carried out structural, expression and evolutionary analysis.
|
[
"The PROSITE database consists of a large collection of biologically meaningful signatures that are described as patterns or profiles. Each signature is linked to documentation that provides useful biological information on the protein family, domain or functional site identified by the signature. The PROSITE web page has been redesigned and several tools have been implemented to help the user discover new conserved regions in their own proteins and to visualize domain arrangements. We also introduced the facility to search PDB with a PROSITE entry or a user's pattern and visualize matched positions on 3D structures. The latest version of PROSITE (release 18.17 of November 30, 2003) contains 1676 entries. The database is accessible at http://www.expasy.org/prosite/.",
"ScanProsite--http://www.expasy.org/tools/scanprosite/--is a new and improved version of the web-based tool for detecting PROSITE signature matches in protein sequences. For a number of PROSITE profiles, the tool now makes use of ProRules--context-dependent annotation templates--to detect functional and structural intra-domain residues. The detection of those features enhances the power of function prediction based on profiles. Both user-defined sequences and sequences from the UniProt Knowledgebase can be matched against custom patterns, or against PROSITE signatures. To improve response times, matches of sequences from UniProtKB against PROSITE signatures are now retrieved from a pre-computed match database. Several output modes are available including simple text views and a rich mode providing an interactive match and feature viewer with a graphical representation of results."
] |
Structure and function of Zika virus.
|
The emergence of Zika virus (ZIKV) as a major public health threat has focused research on understanding virus biology and developing a suite of strategies for disease intervention. Recent advances in cryoelectron microscopy have accelerated structure-function studies of flaviviruses and of ZIKV in particular. Structures of the mature and immature ZIKV have demonstrated its similarity with other known flaviviruses such as dengue and West Nile viruses. However, ZIKV's unique pathobiology demands an explanation of how its structure, although similar to its flavivirus relatives, is sufficiently unique to address questions of receptor specificity, transmission, and antigenicity. Progress in defining the immunodominant epitopes and how neutralizing antibodies bind to them will provide great insight as vaccines progress through clinical trials. Identification of host receptors will substantially illuminate the interesting ZIKV tropism and provide insights into pathogenesis. Although the answers to all of these questions are not yet available, rapid progress in combining structural biology with other techniques is revealing the similarities and the differences in virion structure and function between ZIKV and related flaviviruses.
|
[
"Zika virus is a member of the Flavivirus genus that had not been associated with severe disease in humans until the recent outbreaks, when it was linked to microcephaly in newborns in Brazil and to Guillain-Barré syndrome in adults in French Polynesia. Zika virus is related to dengue virus, and here we report that a subset of antibodies targeting a conformational epitope isolated from patients with dengue virus also potently neutralize Zika virus. The crystal structure of two of these antibodies in complex with the envelope protein of Zika virus reveals the details of a conserved epitope, which is also the site of interaction of the envelope protein dimer with the precursor membrane (prM) protein during virus maturation. Comparison of the Zika and dengue virus immunocomplexes provides a lead for rational, epitope-focused design of a universal vaccine capable of eliciting potent cross-neutralizing antibodies to protect simultaneously against both Zika and dengue virus infections.",
"To evaluate the potential role of Aedes albopictus (Skuse) as a vector of Zika virus (ZIKV), colonized mosquitoes of low generation number (≤ F5) from Brazil, Houston, and the Rio Grande Valley of Texas engorged on viremic mice infected with ZIKV strains originating from Senegal, Cambodia, Mexico, Brazil, or Puerto Rico. Vector competence was established by monitoring infection, dissemination, and transmission potential after 3, 7, and 14 days of extrinsic incubation. Positive saliva samples were assayed for infectious titer. Although all three mosquito populations were susceptible to all ZIKV strains, rates of infection, dissemination, and transmission differed among mosquito and virus strains. Aedes albopictus from Salvador, Brazil, were the least efficient vectors, demonstrating susceptibility to infection to two American strains of ZIKV but failing to shed virus in saliva. Mosquitoes from the Rio Grande Valley were the most efficient vectors and were capable of shedding all three tested ZIKV strains into saliva after 14 days of extrinsic incubation. In particular, ZIKV strain DakAR 41525 (Senegal 1954) was significantly more efficient at dissemination and saliva deposition than the others tested in Rio Grande mosquitoes. Overall, our data indicate that, while Ae. albopictus is capable of transmitting ZIKV, its competence is potentially dependent on geographic origin of both the mosquito population and the viral strain.",
"Previous binding studies of antibodies that recognized a partially or fully hidden epitope suggest that insect cell-derived dengue virus undergoes structural changes at an elevated temperature. This was confirmed by our cryo-electron microscopy images of dengue virus incubated at 37°C, where viruses change their surface from smooth to rough. Here we present the cryo-electron microscopy structures of dengue virus at 37°C. Image analysis showed four classes of particles. The three-dimensional (3D) map of one of these classes, representing half of the imaged virus population, shows that the E protein shell has expanded and there is a hole at the 3-fold vertices. Fitting E protein structures into the map suggests that all of the interdimeric and some intradimeric E protein interactions are weakened. The accessibility of some previously found cryptic epitopes on this class of particles is discussed.",
"The recent rapid spread of Zika virus and its unexpected linkage to birth defects and an autoimmune neurological syndrome have generated worldwide concern. Zika virus is a flavivirus like the dengue, yellow fever, and West Nile viruses. We present the 3.8 angstrom resolution structure of mature Zika virus, determined by cryo-electron microscopy (cryo-EM). The structure of Zika virus is similar to other known flavivirus structures, except for the ~10 amino acids that surround the Asn(154) glycosylation site in each of the 180 envelope glycoproteins that make up the icosahedral shell. The carbohydrate moiety associated with this residue, which is recognizable in the cryo-EM electron density, may function as an attachment site of the virus to host cells. This region varies not only among Zika virus strains but also in other flaviviruses, which suggests that differences in this region may influence virus transmission and disease.",
"We report that two laboratory colonies of Culex quinquefasciatus and Culex pipiens mosquitoes were experimentally unable to transmit ZIKV either up to 21 days post an infectious blood meal or up to 14 days post intrathoracic inoculation. Infectious viral particles were detected in bodies, heads or saliva by a plaque forming unit assay on Vero cells. We therefore consider it unlikely that Culex mosquitoes are involved in the rapid spread of ZIKV.",
"Zika virus (ZIKV) is an emerging arbovirus of the Flaviviridae family, which includes dengue, West Nile, yellow fever, and Japanese encephalitis viruses, that causes a mosquito-borne disease transmitted by the Aedes genus, with recent outbreaks in the South Pacific. Here we examine the importance of human skin in the entry of ZIKV and its contribution to the induction of antiviral immune responses. We show that human dermal fibroblasts, epidermal keratinocytes, and immature dendritic cells are permissive to the most recent ZIKV isolate, responsible for the epidemic in French Polynesia. Several entry and/or adhesion factors, including DC-SIGN, AXL, Tyro3, and, to a lesser extent, TIM-1, permitted ZIKV entry, with a major role for the TAM receptor AXL. The ZIKV permissiveness of human skin fibroblasts was confirmed by the use of a neutralizing antibody and specific RNA silencing. ZIKV induced the transcription of Toll-like receptor 3 (TLR3), RIG-I, and MDA5, as well as several interferon-stimulated genes, including OAS2, ISG15, and MX1, characterized by strongly enhanced beta interferon gene expression. ZIKV was found to be sensitive to the antiviral effects of both type I and type II interferons. Finally, infection of skin fibroblasts resulted in the formation of autophagosomes, whose presence was associated with enhanced viral replication, as shown by the use of Torin 1, a chemical inducer of autophagy, and the specific autophagy inhibitor 3-methyladenine. The results presented herein permit us to gain further insight into the biology of ZIKV and to devise strategies aiming to interfere with the pathology caused by this emerging flavivirus. Zika virus (ZIKV) is an arbovirus belonging to the Flaviviridae family. Vector-mediated transmission of ZIKV is initiated when a blood-feeding female Aedes mosquito injects the virus into the skin of its mammalian host, followed by infection of permissive cells via specific receptors. Indeed, skin immune cells, including dermal fibroblasts, epidermal keratinocytes, and immature dendritic cells, were all found to be permissive to ZIKV infection. The results also show a major role for the phosphatidylserine receptor AXL as a ZIKV entry receptor and for cellular autophagy in enhancing ZIKV replication in permissive cells. ZIKV replication leads to activation of an antiviral innate immune response and the production of type I interferons in infected cells. Taken together, these results provide the first general insights into the interaction between ZIKV and its mammalian host.",
"Zika virus (ZIKV) is an arthropod-borne virus (arbovirus) in the genus Flavivirus and the family Flaviviridae. ZIKV was first isolated from a nonhuman primate in 1947 and from mosquitoes in 1948 in Africa, and ZIKV infections in humans were sporadic for half a century before emerging in the Pacific and the Americas. ZIKV is usually transmitted by the bite of infected mosquitoes. The clinical presentation of Zika fever is nonspecific and can be misdiagnosed as other infectious diseases, especially those due to arboviruses such as dengue and chikungunya. ZIKV infection was associated with only mild illness prior to the large French Polynesian outbreak in 2013 and 2014, when severe neurological complications were reported, and the emergence in Brazil of a dramatic increase in severe congenital malformations (microcephaly) suspected to be associated with ZIKV. Laboratory diagnosis of Zika fever relies on virus isolation or detection of ZIKV-specific RNA. Serological diagnosis is complicated by cross-reactivity among members of the Flavivirus genus. The adaptation of ZIKV to an urban cycle involving humans and domestic mosquito vectors in tropical areas where dengue is endemic suggests that the incidence of ZIKV infections may be underestimated. There is a high potential for ZIKV emergence in urban centers in the tropics that are infested with competent mosquito vectors such as Aedes aegypti and Aedes albopictus."
] |
Photoinduced ligand dissociation of pyridine occurs much more readily
|
Photoinduced ligand dissociation of pyridine occurs much more readily in [Ru(tpy)(Me
|
[
"Identification of transient species is a necessary part of delineating the kinetics and mechanisms associated with chemical dynamics; when dealing with photo-induced processes, this can be an exceptionally challenging task due to the fact that spectra associated with excited state(s) sampled over the course of a photochemical event often cannot be uniquely identified nor readily calculated. Using Group 8 complexes of the general form [M(terpy)2](2+) and [M(bpy)3](2+) as a platform (where terpy is 2,2':6',2''-terpyridine and bpy is 2,2'-bipyridine), we demonstrate how spectroelectrochemical measurements can serve as an effective tool for identifying spectroscopic signatures of charge-transfer excited states of transition metal-based chromophores. Formulating the metal-to-ligand charge-transfer (MLCT) excited state(s) as M(3+)-L(-), the extent to which a linear combination of the spectra of the oxidized and reduced forms of the parent complexes can be used to simulate the characteristic absorptions of MLCT-based transient species is examined. Quantitative agreement is determined to be essentially unachievable due to the fact that certain transitions associated with the optically prepared excited states are either overcompensated for in the spectroelectrochemical data, or simply cannot be replicated through electrochemical means. Despite this limitation, it is shown through several illustrative examples that this approach can still be extremely useful as a qualitative if not semi-quantitative guide for interpreting time-resolved electronic absorption data of charge-transfer compounds, particularly in the ultrafast time domain.",
"The new complex [Ru(bpy)(4AP)(4)](2+) (1), where bpy = 2,2'-bipyridine and 4AP = 4-aminopyridine, undergoes selective photodissociation of two 4APs upon light excitation of the metal-ligand-to-ligand charge-transfer (MLLCT) band at 510 nm. The photoproducts of the reaction are mer-[Ru(bpy)(4AP)(3)(H(2)O)](2+) (2a) and trans-(4AP)[Ru(bpy)(4AP)(2)(H(2)O)(2)](2+) (3a). Photodissociation occurs in two consecutive steps with quantum yields of phi(1) = (6.1 +/- 1.0) x 10(-3) and phi(2) = (1.7 +/- 0.1) x 10(-4), respectively. Complex 1 was characterized by combined spectroscopic and theoretical techniques. EXAFS experiments at the Ru K-edge (22 117 eV) of 1 in an aqueous solution gave a Ru-N distance of 2.09 +/- 0.01 A. Photoproducts were characterized by electronic spectroscopy, 1D and 2D NMR, and mass spectrometry. Singlet and triplet excited states of 1 were studied by density functional theory (DFT) and time-dependent DFT for characterizing the optical properties of the complex. In the singlet state, (1)MC (metal-centered) dissociative states lie 0.65 eV above the main (1)MLLCT transition in the visible region of the UV-vis absorption spectrum. In the triplet state, the energy difference between these states is not reduced. However, potential energy curves of singlet and triplet excited states of 1 along the Ru-N(axial 4AP) and Ru-N(equatorial 4AP) stretching coordinates show that the release of the first 4AP may occur from the triplet state by mixing of (3)MLLCT and (3)MC dissociative states. This mixing is favored when the Ru-N(equatorial 4AP) bond is elongated, explaining the formation of the photoproduct 2a.",
"Metal complexes that release ligands upon photoexcitation are important tools for biological research and show great potential as highly specific therapeutics. Upon excitation with visible light, [Ru(TQA)(MeCN)2](2+) [TQA = tris(2-quinolinylmethyl)amine] exchanges one of the two acetonitriles (MeCNs), whereas [Ru(DPAbpy)MeCN](2+) [DPAbpy = N-(2,2'-bipyridin-6-yl)-N,N-bis(pyridin-2-ylmethyl)amine] does not release MeCN. Furthermore, [Ru(TQA)(MeCN)2](2+) is highly selective for release of the MeCN that is perpendicular to the plane of the two axial quinolines. Density functional theory calculations provide a clear explanation for the photodissociation behavior of these two complexes. Excitation by visible light and intersystem crossing leads to a six-coordinate (3)MLCT state. Dissociation of acetonitrile can occur after internal conversion to a dissociative (3)MC state, which has an occupied dσ* orbital that interacts in an antibonding fashion with acetonitrile. For [Ru(TQA)(MeCN)2](2+), the dissociative (3)MC state is lower than the (3)MLCT state. In contrast, the (3)MC state of [Ru(DPAbpy)MeCN](2+) that releases acetonitrile has an energy higher than that of the (3)MLCT state, indicating dissociation is unfavorable. These results are consistent with the experimental observations that efficient photodissociation of acetonitrile occurs for [Ru(TQA)(MeCN)2](2+) but not for [Ru(DPAbpy)MeCN](2+). For the release of the MeCN ligand in [Ru(TQA)(MeCN)2](2+) that is perpendicular to the axial quinoline rings, the (3)MLCT state has an occupied quinoline π* orbital that can interact with a dσ* Ru-NCCH3 antibonding orbital as the Ru-NCCH3 bond is stretched and the quinolines bend toward the departing acetonitrile. This reduces the barrier for the formation of the dissociative (3)MC state, leading to the selective photodissociation of this acetonitrile. By contrast, when the acetonitrile is in the plane of the quinolines or bpy, no interaction occurs between the ligand π* orbital and the dσ* Ru-NCCH3 orbital, resulting in high barriers for conversion to the corresponding (3)MC structures and no release of acetonitrile.",
"A series of four photodissociable Ru polypyridyl complexes of general formula [Ru(bpy)2L2](2+), where bpy = 2,2'-bipyridine and L = 4-aminopyridine (1), pyridine (2), butylamine (3), and gamma-aminobutyric acid (4), was studied by density functional theory (DFT) and time-dependent density functional theory (TDDFT). DFT calculations (B3LYP/LanL2DZ) were able to predict and elucidate singlet and triplet excited-state properties of 1-4 and describe the photodissociation mechanism of one monodentate ligand. All derivatives display a Ru --> bpy metal-to-ligand charge transfer (MLCT) absorption band in the visible spectrum and a corresponding emitting triplet (3)MLCT state (Ru --> bpy). 1-4 have three singlet metal-centered (MC) states 0.4 eV above the major (1)MLCT states. The energy gap between the MC states and lower-energy MLCT states is significantly diminished by intersystem crossing and consequent triplet formation. Relaxed potential energy surface scans along the Ru-L stretching coordinate were performed on singlet and triplet excited states for all derivatives employing DFT and TDDFT. Excited-state evolution along the reaction coordinate allowed identification and characterization of the triplet state responsible for the photodissociation process in 1-4; moreover, calculation showed that no singlet state is able to cause dissociation of monodentate ligands. Two antibonding MC orbitals contribute to the (3)MC state responsible for the release of one of the two monodentate ligands in each complex. Comparison of theoretical triplet excited-state energy diagrams from TDDFT and unrestricted Kohn-Sham data reveals the experimental photodissociation yields as well as other structural and spectroscopic features.",
"Ultrafast time-resolved infrared spectroscopy of [Ru(bpy)3]2+ (bpy = 2,2'-bipyridine), [Ru(mbpy)3]2+ (mbpy = 6-methyl-2,2'-bipyridine) and [Ru(mphen)3]2+ (mphen = 2-methyl-1,10'-phenanthroline) in deuterated acetonitrile serves to elucidate the evolution of the system following pulsed excitation into the 1MLCT band at 400 nm. While for [Ru(bpy)3]2+ no intermediate state can be evidenced for the relaxation of the corresponding 3MLCT state back to the ground state, for [Ru(mbpy)3]2+ and [Ru(mphen)3]2+ an intermediate state with a lifetime of about 400 ps is observed. The species associated IR difference spectra of this state are in good agreement with the calculated difference spectra of the lowest energy 3dd state using DFT. The calculated potential energy curves for all the complexes in the triplet manifold along the metal-ligand distance show that for [Ru(bpy)3]2+ the 3dd state is at a higher energy than the 3MLCT state and that there is a substantial barrier between the two minima. For [Ru(mbpy)3]2+ and [Ru(mphen)3]2+, the 3dd state is at a lower energy than the 3MLCT state."
] |
Preschoolers' nutrient intake before starting a nutrition and physical activity intervention programme
|
Nutrition in children has an important influence on health both in childhood and adulthood. Actions aimed at improving children's nutrition are essential, not only to the children and their families, but also to the whole society. The aim of the study was to present the results of nutrient intake before starting a nutrition and physical activity intervention programme, to investigate gender differences in nutrient intake and to discuss whether the preschoolers' nutrient intake is similar to the intake of their peers from other countries.
|
[
"Along with a dramatic rise in the rates of childhood obesity, obesity-related disorders, such as type 2 diabetes, hypertension, and obstructive sleep apnea, are seen with increasing frequency in children. As a consequence, overweight and obese children may use health care services more often than their normal weight peers. The aim of the current study was to assess health service use and costs across categories of weight status. Prospective cohort study using data from a population-based survey among grade 5 children in the Canadian province of Nova Scotia linked with administrative health data, using a combination of deterministic and probabalistic matching (n = 4 380). Total health care costs (physician and hospital costs), lifetime (up to age 14 years) physician costs and number of physician visits were assessed in a series of multiple regression models. There was a gradient for higher costs and utilization across the three weight groups. Total health care costs in the three years following the survey were 21% (95% CI: 2-43) higher in obese children compared with normal weight children. Obese children also had significantly higher lifetime physician costs and more physician visits than their normal weight peers. The health care cost trajectories of normal weight and obese children drift apart as early as 3 years of age. Interpretation. Obese children in the Canadian province of Nova Scotia have significantly higher health care costs and more physician visits and specialist referrals than their normal weight peers, highlighting the need for cost-effectiveness studies of obesity prevention programs.",
"Along with other countries having high and low-to-middle income, Mexico has experienced a substantial change in obesity rates. This rapid growth in obesity prevalence has led to high rates of obesity-related diseases and associated health-care costs. Micro-simulation is used to project future BMI trends. Additionally thirteen BMI-related diseases and health-care costs are estimated. The results are simulated for three hypothetical scenarios: no BMI reduction and BMI reductions of 1 % and 5 % across the population. Mexican Health and Nutrition Surveys 1999 and 2000, and Mexican National Health and Nutrition Survey 2006. Mexican adults. In 2010, 32 % of men and 26 % of women were normal weight. By 2050, the proportion of normal weight will decrease to 12 % and 9 % for males and females respectively, and more people will be obese than overweight. It is projected that by 2050 there will be 12 million cumulative incidence cases of diabetes and 8 million cumulative incidence cases of heart disease alone. For the thirteen diseases considered, costs of $US 806 million are estimated for 2010, projected to increase to $US 1·2 billion and $US 1·7 billion in 2030 and 2050 respectively. A 1 % reduction in BMI prevalence could save $US 43 million in health-care costs in 2030 and $US 85 million in 2050. Obesity rates are leading to a large health and economic burden. The projected numbers are high and Mexico should implement strong action to tackle obesity. Results presented here will be very helpful in planning and implementing policy interventions.",
"The quality of dietary fat in relation to cardiovascular disease forms the basis of the diet-heart hypothesis. Current recommendations on dietary fat now emphasise quality rather than quantity. The focus of this review is to summarise the results from prospective cohort studies on dietary fat and cardiovascular disease outcomes. Relatively few prospective cohort studies have found an association between dietary fat quality and cardiovascular disease, partly because of limitations in estimating dietary intake. Saturated and trans fatty acids have increased cardiovascular risk in several studies. Both n-6 and n-3 polyunsaturated fatty acids have been associated with lower cardiovascular risk. Within the n-6 series, linoleic acid seems to decrease cardiovascular risk. Within the n-3 series the long-chain fatty acids (eicosapentaenoic and docosahexaenoic acids) are associated with decreased risk for especially fatal coronary outcomes, whereas the role of alpha-linolenic acid is less clear. Dietary fat quality also influences the activity of enzymes involved in the desaturation of fatty acids in the body. Serum desaturase indices have been consistently associated with adverse cardiovascular outcomes. Data from metabolic and clinical studies reinforce findings from observational studies supporting recommendations to replace saturated and trans fat with unsaturated fat in the prevention of cardiovascular disease.",
"Studies on vitamin and mineral intakes in children are very important: firstly because of the high prevalence of diet-related diseases and secondly because of the widespread consumption of highly processed foods which are characterised by high energy content and low density of essential nutrients. Therefore, the purpose of this study was to analyse vitamin and mineral intakes in 6-year-old children from southern Poland. Vitamin and mineral intakes were estimated from a three-day food record in 120 children, 64 girls and 56 boys, aged 6 years. Nutrient densities were estimated as amounts per 1000 kcal (4185 kJ) of energy intake. Statistical analysis was carried out by means of the IBM SPSS Statistics computer programme, version 19. The studied population was divided according to gender. Intakes of folic acid (μg/1000 kcal) and vitamin C (mg, mg/1000 kcal) were significantly higher in girls. Nutrient densities for all vitamins were higher in girls, however, these results did not reach statistical significance. Intake of vitamin D was lower than EAR in all of the studied children. Intakes of sodium (mg) and zinc (mg) were significantly higher in boys. Intakes of the remaining minerals were higher in boys, however, these findings did not reach statistical significance. Nutrient densities for all minerals, except for sodium, zinc and manganese, were higher in girls. All of the studied children had sodium intakes above UL. Inadequate intakes of vitamin D, calcium and potassium in the studied 6-year-olds along with excessive sodium intake are the risk factors for developing osteoporosis and hypertension. To prevent these diseases in the studied children, educational programmes for both preschool staff and parents should be worked out and implemented.",
"The aim of this paper is to highlight the potential impact of costs associated with overweight and obesity for provincial policy and prevention initiatives. Prevalence-based cost-of-illness methodology was used to estimate the direct costs (hospital care, drugs, physician care, institutional care, additional costs) and indirect costs (short- and long-term disability, premature mortality) associated with excess weight for 22 health conditions. Total costs for each health condition were estimated using the Public Health Agency of Canada's Economic Burden of Illness database. Population attributable fractions (PAF) were also estimated using 2004 and 2005 CCHS data and current literature reviews. In 2005, the cost of excess weight in Alberta totaled $1.27 billion. The direct cost of excess weight was $630.1M (49.5%), the indirect cost $643.8M (50.5%). Excluding costs associated with premature mortality and caregiving, obesity accounted for 69.5% ($500.8M) of costs and overweight the remaining 30.5% ($220.2M). Among the 22 health conditions, coronary heart disease had the highest costs attributable to excess weight ($307.1 M), followed by osteoarthritis ($167.7M) and type 2 diabetes ($161.5M). The total cost of excess weight equated to 5.6% of the province's annual health care expenditures for 2005. While obesity costing research often focuses on the direct health care costs, this study reveals that the indirect costs of excess weight are also significant and can account for over half of the total costs. Interventions to reduce excess weight among Canadians have the potential to improve the health of the population while reducing provincial and national health care costs.",
"Public health decision makers not only consider health benefits but also economic implications when articulating and issuing lifestyle recommendations. Whereas various estimates exist for the economic burden of physical inactivity, excess body weight and smoking, estimates of the economic burden associated with our diet are rare. In the present study, we estimated the economic burden attributable to the inadequate consumption of vegetables and fruit in Canada. We accessed the Canadian Community Health Survey to assess the inadequacy in the consumption of vegetables and fruit and published meta-analyses to assemble risk estimates for chronic diseases. Based on these inadequacy and risk estimates, we calculated the population-attributable fraction and avoidable direct and indirect costs to society. Direct costs include those for hospital care, physician services and drugs in 2015. About 80 % of women and 89 % of men consume inadequate amounts of vegetables and fruit. We estimated this to result in an economic burden of $CAN 3·3 billion per year, of which 30·5 % is direct health-care costs and 69·5 % is indirect costs due to productivity losses. A modest 1 percentage point annual reduction in the prevalence of inadequate vegetables and fruit consumption over the next 20 years would avoid approximately $CAN 10·8 billion, and an increase of one serving of vegetables and fruit per day would avoid approximately $CAN 9·2 billion. Further investments in the promotion of vegetables and fruit will prevent chronic disease and substantially reduce direct and indirect health-care costs."
] |
Alternate-day fasting improves body composition, fat distribution, and adipokine profile
|
Indirect comparisons suggest that alternate-day fasting (ADF) may produce greater improvements in body composition, fat distribution, and/or the adipokine profile compared to daily calorie restriction (CR), but this has not been tested directly. In a pre-planned secondary analysis of a randomized controlled trial, we compared changes in the VAT:SAT ratio, FFM:total mass ratio, and the adipokine profile between ADF and CR.
|
[
"Obesity is associated with endothelial dysfunction that may contribute to the development of atherosclerosis. We studied whether weight reduction improves endothelial function in overweight individuals. Flow-mediated endothelium-dependent vasodilation of the brachial artery was measured in 67 adults (age: 46+/-7 years, body mass index: 35.2+/-5.4 kg/m2) before and after a 6-week weight reduction program induced by very-low-calorie diet (daily energy: 580 kcal/2.3 MJ). Caloric restriction reduced body weight from 101+/-18 to 90+/-17 kg. Flow-mediated vasodilation increased from 5.5%+/-3.7 to 8.8%+/-3.7% (P<0.0001). Nitrate-mediated vasodilation was not significantly affected. The improvement in flow-mediated dilation was associated with the reduction in plasma glucose concentration (P=0.0003). This relationship was independent of changes in weight, serum lipids, oxidized LDL, C-reactive protein, adiponectin, blood pressure, and insulin. Weight reduction with very-low-calorie diet improves flow-mediated vasodilation in obese individuals. This improvement is related to the reduction in plasma glucose concentration. These observations suggest that changes in glucose metabolism may determine endothelial vasodilatory function in obesity.",
"Persistent low-grade inflammation, as indicated by higher circulating levels of inflammatory mediators such as C-reactive protein, interleukin-6 and tumour necrosis factor-alpha, is a strong risk factor for several chronic diseases. There are data indicating that decreasing energy intake and increasing physical activity may be effective therapies for reducing overall inflammation. Evidence is strong that circulating levels of inflammatory markers are elevated with total and abdominal obesity, possibly owing to a higher secretion rate of cytokines by adipose tissue in obese people. Moreover, very-low-energy dietary weight loss reduces both circulating markers of inflammation and adipose-tissue cytokine production. Data from several large population-based cohorts show an inverse association between markers of systemic inflammation and physical activity or fitness status; small-scale intervention studies support that exercise training diminishes inflammation. Dietary weight loss plus exercise is likely more effective than weight reduction alone in reducing inflammation. To date, data from randomized, controlled trails designed to definitively test the effects of weight loss or exercise training, or both, on inflammation are limited. Future studies are required to define the amount of weight loss needed for clinically meaningful reductions of inflammation; in addition, fully powered and controlled studies are necessary to clarify the effect of exercise training on chronic, systemic inflammation.",
"Alternate-day fasting has become increasingly popular, yet, to date, no long-term randomized clinical trials have evaluated its efficacy. To compare the effects of alternate-day fasting vs daily calorie restriction on weight loss, weight maintenance, and risk indicators for cardiovascular disease. A single-center randomized clinical trial of obese adults (18 to 64 years of age; mean body mass index, 34) was conducted between October 1, 2011, and January 15, 2015, at an academic institution in Chicago, Illinois. Participants were randomized to 1 of 3 groups for 1 year: alternate-day fasting (25% of energy needs on fast days; 125% of energy needs on alternating \"feast days\"), calorie restriction (75% of energy needs every day), or a no-intervention control. The trial involved a 6-month weight-loss phase followed by a 6-month weight-maintenance phase. The primary outcome was change in body weight. Secondary outcomes were adherence to the dietary intervention and risk indicators for cardiovascular disease. Among the 100 participants (86 women and 14 men; mean [SD] age, 44 [11] years), the dropout rate was highest in the alternate-day fasting group (13 of 34 [38%]), vs the daily calorie restriction group (10 of 35 [29%]) and control group (8 of 31 [26%]). Mean weight loss was similar for participants in the alternate-day fasting group and those in the daily calorie restriction group at month 6 (-6.8% [95% CI, -9.1% to -4.5%] vs -6.8% [95% CI, -9.1% to -4.6%]) and month 12 (-6.0% [95% CI, -8.5% to -3.6%] vs -5.3% [95% CI, -7.6% to -3.0%]) relative to those in the control group. Participants in the alternate-day fasting group ate more than prescribed on fast days, and less than prescribed on feast days, while those in the daily calorie restriction group generally met their prescribed energy goals. There were no significant differences between the intervention groups in blood pressure, heart rate, triglycerides, fasting glucose, fasting insulin, insulin resistance, C-reactive protein, or homocysteine concentrations at month 6 or 12. Mean high-density lipoprotein cholesterol levels at month 6 significantly increased among the participants in the alternate-day fasting group (6.2 mg/dL [95% CI, 0.1-12.4 mg/dL]), but not at month 12 (1.0 mg/dL [95% CI, -5.9 to 7.8 mg/dL]), relative to those in the daily calorie restriction group. Mean low-density lipoprotein cholesterol levels were significantly elevated by month 12 among the participants in the alternate-day fasting group (11.5 mg/dL [95% CI, 1.9-21.1 mg/dL]) compared with those in the daily calorie restriction group. Alternate-day fasting did not produce superior adherence, weight loss, weight maintenance, or cardioprotection vs daily calorie restriction. clinicaltrials.gov Identifier: NCT00960505.",
"BACKGROUND: Obesity can have deleterious effects on insulin sensitivity leading to endothelial dysfunction. Whether alternate day fasting (ADF) can ameliorate insulin sensitivity in a way that improves endothelial function remains unknown. OBJECTIVE: This study examined the impact of ADF on endothelium dependent flow mediated dilation (FMD) in obese subjects with different degrees of insulin resistance. METHODS: Obese non-diabetic adults (n = 54) participated in an 8-week ADF protocol (25% energy intake \"fast day\", alternated with ad libitum intake \"feast day\"). Subjects were divided into tertiles according to degree of insulin resistance based on HOMA-IR (Homeostatic model assessment-Insulin resistance): tertile 1 (0.8-2.4), tertile 2 (2.5-3.6), tertile 3 (3.7-12.4). RESULTS: Body weight decreased (P < 0.001) by 4% in each tertile. Fat mass, lean mass, and visceral fat mass also decreased (P < 0.001) similarly in each tertile. After 8 weeks of ADF, FMD and adiponectin differed (P < 0.05) between tertile 1 (3±0%; 26±23%) versus tertile 3 (-3±0%; -13±10%). Changes in leptin did not differ between tertiles (tertile 1: -23±7%; tertile 2: -20±7%; tertile 3: -9±7%). Fasting glucose did not change in any tertile. Fasting insulin and HOMA-IR differed (P < 0.05) between tertile 1 (10±11%; 11±11%) versus tertile 3 (-27±8%; -30±9%). Plasma lipids, blood pressure and heart rate did not change in any tertile. CONCLUSION: Our data suggest that ADF may be effective for decreasing insulin resistance in insulin resistant subjects, but these changes have no effect on endothelial function."
] |
Axillary meristem branching in cereals
|
Inflorescence architecture is a key determinant of yield potential in many crops and is patterned by the organization and developmental fate of axillary meristems. In cereals, flowers and grain are borne from spikelets, which differentiate in the final iteration of axillary meristem branching. In
|
[
"Most angiosperms present flowers in inflorescences, which play roles in reproduction, primarily related to pollination, beyond those served by individual flowers alone. An inflorescence's overall reproductive contribution depends primarily on the three-dimensional arrangement of the floral canopy and its dynamics during its flowering period. These features depend in turn on characteristics of the underlying branching structure (scaffold) that supports and supplies water and nutrients to the floral canopy. This scaffold is produced by developmental algorithms that are genetically specified and hormonally mediated. Thus, the extensive inflorescence diversity evident among angiosperms evolves through changes in the developmental programmes that specify scaffold characteristics, which in turn modify canopy features that promote reproductive performance in a particular pollination and mating environment. Nevertheless, developmental and ecological aspects of inflorescences have typically been studied independently, limiting comprehensive understanding of the relations between inflorescence form, reproductive function, and evolution. This review fosters an integrated perspective on inflorescences by summarizing aspects of their development and pollination function that enable and guide inflorescence evolution and diversification. The architecture of flowering inflorescences comprises three related components: topology (branching patterns, flower number), geometry (phyllotaxis, internode and pedicel lengths, three-dimensional flower arrangement) and phenology (flower opening rate and longevity, dichogamy). Genetic and developmental evidence reveals that these components are largely subject to quantitative control. Consequently, inflorescence evolution proceeds along a multidimensional continuum. Nevertheless, some combinations of topology, geometry and phenology are represented more commonly than others, because they serve reproductive function particularly effectively. For wind-pollinated species, these combinations often represent compromise solutions to the conflicting physical influences on pollen removal, transport and deposition. For animal-pollinated species, dominant selective influences include the conflicting benefits of large displays for attracting pollinators and of small displays for limiting among-flower self-pollination. The variety of architectural components that comprise inflorescences enable diverse resolutions of these conflicts.",
"KNOTTED1 (KN1)-like homeobox (KNOX) transcription factors function in plant meristems, self-renewing structures consisting of stem cells and their immediate daughters. We defined the KN1 cistrome in maize inflorescences and found that KN1 binds to several thousand loci, including 643 genes that are modulated in one or multiple tissues. These KN1 direct targets are strongly enriched for transcription factors (including other homeobox genes) and genes participating in hormonal pathways, most significantly auxin, demonstrating that KN1 plays a key role in orchestrating the upper levels of a hierarchical gene regulatory network that impacts plant meristem identity and function.",
"Brassinosteroids (BRs) play crucial roles in plant growth and development. Previous studies have shown that BRs promote cell elongation in vegetative organs in several plant species, but their contribution to meristem homeostasis remains unexplored. Our analyses report that both loss- and gain-of-function BR-related mutants in Arabidopsis thaliana have reduced meristem size, indicating that balanced BR signalling is needed for the optimal root growth. In the BR-insensitive bri1-116 mutant, the expression pattern of the cell division markers CYCB1;1, ICK2/KRP2 and KNOLLE revealed that a decreased mitotic activity accounts for the reduced meristem size; accordingly, this defect could be overcome by the overexpression of CYCD3;1. The activity of the quiescent centre (QC) was low in the short roots of bri1-116, as reported by cell type-specific markers and differentiation phenotypes of distal stem cells. Conversely, plants treated with the most active BR, brassinolide, or mutants with enhanced BR signalling, such as bes1-D, show a premature cell cycle exit that results in early differentiation of meristematic cells, which also negatively influence meristem size and overall root growth. In the stem cell niche, BRs promote the QC renewal and differentiation of distal stem cells. Together, our results provide evidence that BRs play a regulatory role in the control of cell-cycle progression and differentiation in the Arabidopsis root meristem.",
"The plant hormone auxin plays a critical role in regulating various aspects of plant growth and development, and the spatial accumulation of auxin within organs, which is primarily attributable to local auxin biosynthesis and polar transport, is largely responsible for lateral organ morphogenesis and the establishment of plant architecture. Here, we show that three Arabidopsis INDETERMINATE DOMAIN (IDD) transcription factors, IDD14, IDD15, and IDD16, cooperatively regulate auxin biosynthesis and transport and thus aerial organ morphogenesis and gravitropic responses. Gain-of-function of each IDD gene in Arabidopsis results in small and transversally down-curled leaves, whereas loss-of-function of these IDD genes causes pleiotropic phenotypes in aerial organs and defects in gravitropic responses, including altered leaf shape, flower development, fertility, and plant architecture. Further analyses indicate that these IDD genes regulate spatial auxin accumulation by directly targeting YUCCA5 (YUC5), TRYPTOPHAN AMINOTRANSFERASE of ARABIDOPSIS1 (TAA1), and PIN-FORMED1 (PIN1) to promote auxin biosynthesis and transport. Moreover, mutation or ectopic expression of YUC suppresses the organ morphogenic phenotype and partially restores the gravitropic responses in gain- or loss-of-function idd mutants, respectively. Taken together, our results reveal that a subfamily of IDD transcription factors plays a critical role in the regulation of spatial auxin accumulation, thereby controlling organ morphogenesis and gravitropic responses in plants.",
"Phyllotaxis, the spatio-temporal pattern of organogenesis at the shoot apical meristem, emerges in large part from inhibitory fields consisting in auxin-depleted areas centered on organs. We recently demonstrated the existence of an additional hormone-based inhibitory field generated by Arabidopsis Histidine Phosphotransfer Protein 6 (AHP6), an inhibitor of cytokinin signaling. We have shown that the spatio-temporal distribution of AHP6 in the meristem is essential for optimizing the rhythmicity of organ initiation. Here, we further analyzed AHP6 expression using fluorescent whole mount mRNA in situ hybridization and demonstrate a precise control of AHP6 level and expression domain over time. While we previously showed a regulation of AHP6 directly downstream of auxin, we show here that AHP6 transcription is unlikely influenced by cytokinin distribution in the meristem. Finally, we provide evidence that cytokinins and auxin might act synergistically during organ initiation, providing a plausible explanation for how AHP6 regulates phyllotaxis.",
"The flower meristem identity genes floricaula (flo) and squamosa (squa) promote a change in phyllotaxy from spiral to whorled in Antirrhinum. To determine how this might be achieved, we have performed a combination of morphological, genetic, and expression analyses. Comparison of the phenotypes and RNA expression patterns of single and double mutants with the wild type showed that flo and squa act together to promote flower development but that flo is epistatic to squa with respect to early effects on phyllotaxy. We propose that a common process underlies the phyllotaxy of wildtype, flo, and squa meristem development but that the relative timing of primordium initiation or growth is altered. This process depends on two separable events: setting aside zones for potential primordium initiation and partitioning these zones into discrete primordia. Failure of the second event can lead to the formation of continuous double spirals, which are occasionally seen in flo mutants.",
"Inflorescence morphology is incredibly diverse. This diversity of form has been a fruitful source of inquiry for plant morphologists for more than a century. Work in the grasses (Poaceae), the tomato family (Solanaceae), and Arabidopsis thaliana (Brassicaceae) has led to a richer understanding of the molecular genetics underlying this diversity. The character of individual meristems, a combination of the number (determinacy) and nature (identity) of the products a meristem produces, is key in the development of plant form. A framework that describes inflorescence development in terms of shifting meristem identities has emerged and garnered empirical support in a number of model systems. We discuss this framework and highlight one important aspect of meristem identity that is often considered in isolation, phyllotaxis. Phyllotaxis refers to the arrangement of lateral organs around a central axis. The development and evolution of phyllotaxis in the inflorescence remains underexplored, but recent work analyzing early inflorescence development in the grasses identified an evolutionary shift in primary branch phyllotaxis in the Pooideae. We discuss the evidence for an intimate connection between meristem identity and phyllotaxis in both the inflorescence and vegetative shoot, and touch on what is known about the establishment of phyllotactic patterns in the meristem. Localized auxin maxima are instrumental in determining the position of lateral primordia. Upstream factors that regulate the position of these maxima remain unclear, and how phyllotactic patterns change over the course of a plant's lifetime and evolutionary time, is largely unknown. A more complete understanding of the molecular underpinnings of phyllotaxis and architectural diversity in inflorescences will require capitalizing on the extensive resources available in existing genetic systems, and developing new model systems that more fully represent the diversity of plant morphology.",
"In higher plants, determinate leaf primordia arise in regular patterns on the flanks of the indeterminate shoot apical meristem (SAM). The acquisition of leaf form is then a gradual process, involving the specification and growth of distinct domains within the three leaf axes. The recessive corkscrew1 (cks1) mutation of maize (Zea mays) disrupts both leaf initiation patterns in the SAM and domain specification within the mediolateral and proximodistal leaf axes. Specifically, cks1 mutant leaves exhibit multiple midribs and leaf sheath tissue differentiates in the blade domain. Such perturbations are a common feature of maize mutants that ectopically accumulate KNOTTED1-like homeobox (KNOX) proteins in leaf tissue. Consistent with this observation, at least two knox genes are ectopically expressed in cks1 mutant leaves. However, ectopic KNOX proteins cannot be detected. We therefore propose that CKS1 primarily functions within the SAM to establish boundaries between meristematic and leaf zones. Loss of gene function disrupts boundary formation, impacts phyllotactic patterns, and leads to aspects of indeterminate growth within leaf primordia. Because these perturbations arise independently of ectopic KNOX activity, the cks1 mutation defines a novel component of the developmental machinery that facilitates leaf-versus-shoot development in maize.",
"New cultivars with very erect leaves, which increase light capture for photosynthesis and nitrogen storage for grain filling, may have increased grain yields. Here we show that the erect leaf phenotype of a rice brassinosteroid-deficient mutant, osdwarf4-1, is associated with enhanced grain yields under conditions of dense planting, even without extra fertilizer. Molecular and biochemical studies reveal that two different cytochrome P450s, CYP90B2/OsDWARF4 and CYP724B1/D11, function redundantly in C-22 hydroxylation, the rate-limiting step of brassinosteroid biosynthesis. Therefore, despite the central role of brassinosteroids in plant growth and development, mutation of OsDWARF4 alone causes only limited defects in brassinosteroid biosynthesis and plant morphology. These results suggest that regulated genetic modulation of brassinosteroid biosynthesis can improve crops without the negative environmental effects of fertilizers.",
"The family of grasses encompasses the world's most important food, feed, and bioenergy crops, yet we are only now beginning to develop the genetic resources to explore the diversity of form and function that underlies economically important traits. Two emerging model systems, Brachypodium distachyon and Setaria viridis, promise to greatly accelerate the process of gene discovery in the grasses and to serve as bridges in the exploration of panicoid and pooid grasses, arguably two of the most important clades of plants from a food security perspective. We provide both a historical view of the development of plant model systems and highlight several recent reports that are providing these developing communities with the tools for gene discovery and pathway engineering."
] |
Role of the thyroid hormone-v3 axis in epithelial to mesenchymal transition in ovarian cancer
|
Ovarian cancer is a highly metastatic disease. The metastatic potential is enhanced by epithelial to mesenchymal transition (EMT) in which αvβ3 integrin plays a role. Thyroid hormones (L-thyroxine, T4, and 3,5,3'-triiodo-L-thyronine, T3) bind this integrin, and we hypothesized that the thyroid hormone-αvβ3 axis may be involved in EMT activity in ovarian cancer. The transcription (mRNA), protein abundance (westerns), and protein localization (fluorescence microscopy) of several EMT markers were studied in ovarian cancer cells (OVCAR-3, A2780, and SKOV-3) treated with 1 nM T3 or 100 nM T4 for 1-24 h. The protein levels of β-catenin, and its downstream targets, zeb-1, slug, and vimentin, were significantly induced by both hormones, while the effect on transcription was limited. The pre-incubation of the cells overnight with two integrin inhibitors, RGD (0.1-10 μM) or αvβ3 blocking antibody (1-100 ng/mL), prevented the induction of β-catenin by T3 and zeb-1 by T4, indicating direct integrin involvement. The transcription of the mesenchymal markers, β-catenin, zeb-1, slug/snail, vimentin, and n-cadherin was hardly affected by T3 and T4, while that of the epithelial markers, e-cadherin and zo-1, was inhibited. Our results suggest a novel role for the thyroid hormone-αvβ3 axis in EMT, with possible implications for ovarian cancer metastasis.
|
[
"Ovarian surface epithelial (OSE) cells express multiple nuclear hormone receptor genes, including those encoding thyroid hormone and estrogen receptors (TR and ER, respectively). Ovarian cancer is hormone-dependent, and epidemiological evidence links hyperthyroidism, inflammation of the ovarian surface, and increased risk of ovarian cancer. The objective of this study was to assess T3 action on human OSE cells in vitro, asking 1) is there evidence for (pre)receptor control, 2) is T3 inflammatory, and 3) does T3 affect ER expression? Immunohistochemical analysis of fixed human ovaries and in vitro analysis of human OSE primary cell cultures were performed. Twelve women aged 29-50 yr (median, 41 yr) undergoing elective gynecological surgery for nonmalignant conditions were studied. Messenger RNA transcripts for TRalpha1, TRalpha2, TRbeta1, and T3 activating deiodinase 2 and inactivating deiodinase 3 were present in primary OSE cell cultures by RT-PCR. TRalpha and TRbeta proteins were also localized to intact OSE by immunohistochemistry. Treatment of OSE cell cultures for 24 h with T3 caused dose-dependent mRNA expression of inflammation-associated genes: cyclooxygenase-2, matrix metalloproteinase-9, and 11betahydroxysteroid dehydrogenase type 1, determined by quantitative RT-PCR. Finally, treatment with T3 dose dependently stimulated ERalpha mRNA expression without affecting ERbeta1 or ERbeta2. The ovarian surface is a potential T3 target. T3 exerts direct inflammatory effects on OSE cell function in vitro. OSE cell responses to T3 include increased expression of ERalpha mRNA, which encodes the ER isoform most strongly associated with ovarian cancer. This could help explain suggested epidemiological links between hyperthyroidism and ovarian cancer.",
"The adenomatous polyposis coli gene (APC) is mutated in most colon cancers. The APC protein binds to the cellular adhesion molecule beta-catenin, which is a mammalian homolog of ARMADILLO, a component of the WINGLESS signaling pathway in Drosophila development. Here it is shown that when beta-catenin is present in excess, APC binds to another component of the WINGLESS pathway, glycogen synthase kinase 3beta (GSK3beta), a mammalian homolog of Drosophila ZESTE WHITE 3. APC was a good substrate for GSK3 beta in vitro, and the phosphorylation sites were mapped to the central region of APC. Binding of beta-catenin to this region was dependent on phosphorylation by GSK3 beta.",
"Ovarian cancer cells disseminate by implanting onto the peritoneal mesothelial cell surface of the abdominal cavity. A common feature of these peritoneal implants is the presence of tumor cell invasion into the submesothelial extracellular matrix (ECM). In view of the important role of integrins in ECM recognition and cell migration, we were interested in defining the pattern of integrin expression and function in ovarian cancer cell lines and primary tissue samples. The beta 1 integrin chain was expressed by CAOV-3, SKOV-3, OVCAR-3, and SW626 ovarian cancer cell lines, associated with expression of alpha 1, -2, -3, -5, and -6 chains. The alpha 4 chain was also expressed by two of the four lines. In addition to beta 1 integrins, the alpha v beta 3 integrin was also expressed, although there was no expression of beta 2, -4, and -7 chains. Immunoprecipitation of surface-labeled CAOV-3 cells with an anti-beta 1 antibody revealed a band at approximately 110-130 kDa consistent with the known molecular mass of the beta 1 chain, as well as several associated bands consistent with noncovalently linked integrin alpha chains. A similar pattern of beta 1 and alpha v beta 3 integrin expression was observed for primary ovarian cancer tissue samples. Ovarian cancer cell lines exhibited significant binding to collagen type I and laminin which was primarily mediated by beta 1 integrins. In contrast, ovarian cancer cell binding to fibronectin was mediated by both alpha 5 beta 1 and alpha v beta 3 integrins. Even though mesothelial cells were observed to express fibronectin mRNA and protein, binding of ovarian cancer cells to peritoneal mesothelium was not blocked by neutralizing antibodies to beta 1 or alpha v beta 3 integrins. These data suggest that functional integrins are commonly expressed by ovarian cancer cells, although they do not appear to mediate ovarian cancer cell implantation onto peritoneal mesothelium. The role that integrins play in the invasion of ovarian cancer cells into the submesothelial ECM deserves further investigation.",
"Aberrant activation of β-catenin in the nucleus has been implicated in a variety of human cancers, but the fate of nuclear β-catenin is unknown. Here we demonstrate that the tripartite motif-containing protein 33 (TRIM33), acting as an E3 ubiquitin ligase, reduces the abundance of nuclear β-catenin protein. TRIM33-mediated β-catenin is destabilized and is GSK-3β or β-TrCP independent. TRIM33 interacts with and ubiquitylates nuclear β-catenin. Moreover, protein kinase Cδ, which directly phosphorylates β-catenin at Ser715, is required for the TRIM33-β-catenin interaction. The function of TRIM33 in suppressing tumour cell proliferation and brain tumour development depends on TRIM33-promoted β-catenin degradation. In human glioblastoma specimens, endogenous TRIM33 levels are inversely correlated with β-catenin. In summary, our findings identify TRIM33 as a tumour suppressor that can abolish tumour cell proliferation and tumorigenesis by degrading nuclear β-catenin. This work suggests a new therapeutic strategy against human cancers caused by aberrant activation of β-catenin.",
"Epithelial-mesenchymal transition (EMT) has been suggested to contribute to tumor progression and acquisition of therapeutic resistance. To assess the clinical significance of EMT-associated proteins, we evaluated the expression of Snail and Slug, the key regulators of EMT, in the primary ovarian cancer samples (n = 103) by immunohistochemistry. Snail was differentially expressed according to the histologic subtype (P = 0.001) and was predominantly expressed in serous and endometrioid types. In the serous and endometrioid adenocarcinomas, the expression of Snail remained high across the stage and grade, suggesting its role in the early phase of carcinogenesis. However, the expression of Snail and Slug was not related to chemoresistance and poor prognosis and did not serve as independent predictive or prognostic marker.",
"Post-translational stabilization of beta-catenin through mutation of the adenomatous polyposis coli (APC) gene has been proposed as an early step in colorectal carcinogenesis. Beta-catenin may translocate from the cytoplasm to the nucleus, where it might serve as a transcriptional factor to stimulate tumour formation. We investigated intracellular localization of beta-catenin in sporadic colorectal adenomas and cancers as well as familial adenomatous polyposis (FAP). Nuclear over-expression of beta-catenin was observed in 35% (7/20) of intramucosal cancers and 42% (23/55) of invasive cancers but was not seen in any adenomas from sporadic or FAP cases. Cytoplasmic beta-catenin in adenomas was significantly higher than that of normal mucosa in both sporadic and FAP cases. The cytoplasmic intensity index of cancers was significantly higher than that of sporadic adenomas, but the index was not correlated with nuclear expression in cancers. These findings suggest that nuclear translocation of beta-catenin is involved in development of intramucosal cancer rather than adenoma, independent of APC mutations. Cytoplasmic accumulation of beta-catenin may occur in adenomas, but it remains to be determined whether this is a cause or a consequence of colorectal cancer.",
"A cell line representative of human high-grade serous ovarian cancer (HGSOC) should not only resemble its tumour of origin at the molecular level, but also demonstrate functional utility in pre-clinical investigations. Here, we report the integrated proteomic analysis of 26 ovarian cancer cell lines, HGSOC tumours, immortalized ovarian surface epithelial cells and fallopian tube epithelial cells via a single-run mass spectrometric workflow. The in-depth quantification of >10,000 proteins results in three distinct cell line categories: epithelial (group I), clear cell (group II) and mesenchymal (group III). We identify a 67-protein cell line signature, which separates our entire proteomic data set, as well as a confirmatory publicly available CPTAC/TCGA tumour proteome data set, into a predominantly epithelial and mesenchymal HGSOC tumour cluster. This proteomics-based epithelial/mesenchymal stratification of cell lines and human tumours indicates a possible origin of HGSOC either from the fallopian tube or from the ovarian surface epithelium.",
"Pituitary tumor transforming gene (PTTG) is a well-studied oncogene for its role in tumorigenesis and serves as a marker of malignancy in several cancer types including lung. In the present study, we defined the role of PTTG in actin cytoskeleton remodeling, cell migration and induction of epithelial mesenchymal transition (EMT) through the regulation of integrin α(V)β(3)-FAK (focal adhesion kinase) signaling pathway. Overexpression of PTTG through an adenovirus vector resulted in a significant increase in the expression of integrins α(V) and β(3), a process that was reversed with the downregulation of PTTG expression through the use of an adenovirus expressing PTTG-specific small interfering RNA (siRNA). Western blot analysis of cells infected with adenovirus PTTG cDNA resulted in increased FAK and enhanced expression of adhesion complex molecules paxillin, metavincullin, and talin. Furthermore, downstream signaling genes Rac1, RhoA, Cdc42 and DOCK180 showed upregulation upon PTTG overexpression. This process was dependent on integrin α(V), as blockage by antagonist echistatin (RGD peptide) or α(V)-specific siRNA resulted in a decrease in FAK and subsequent adhesion molecules. Actin cytoskeleton disruption was detected as a result of integrin-FAK signaling by PTTG as well as enhanced cell motility. Taken together, our results suggest for the first time an important role of PTTG in regulation of integrins α(V) and β(3) and adhesion-complex proteins leading to induction of EMT.",
"The alpha(v)beta(3) integrin is expressed on proliferating endothelial cells and some cancer cells, but its expression on ovarian cancer cells and its potential as a therapeutic target are unknown. In this study, expression of the alpha(v)beta(3) integrin on ovarian cancer cell lines and murine endothelial cells was tested, and the effect of a fully humanized monoclonal antibody against alpha(v)beta(3), Abegrin (etaracizumab), on cell invasion, viability, tumor growth, and the Akt pathway were examined in vitro and in vivo. We found that etaracizumab recognizes alpha(v)beta(3) on the ovarian cancer cell lines SKOV3ip1, HeyA8, and A2780ip2 (at low levels) but not on murine endothelial cells. Etaracizumab treatment decreased ovarian cancer proliferation and invasion. In vivo, tumor-bearing mice treated with etaracizumab alone gave variable results. There was no effect on A2780ip2 growth, but a 36% to 49% tumor weight reduction in the SKOV3ip1 and HeyA8 models was found (P < .05). However, combined etaracizumab and paclitaxel was superior to paclitaxel in the SKOV3ip1 and A2780ip2 models (by 51-73%, P < .001) but not in the HeyA8 model. Treatment with etaracizumab was then noted to decrease p-Akt and p-mTOR in SKOV3ip1, but not in HeyA8, which is Akt-independent. Tumors resected after therapy showed that etaracizumab treatment reduced the proliferating cell nuclear antigen index but not microvessel density. This study identifies tumor cell alpha(v)beta(3) integrin as an attractive target and defines the Akt pathway as a predictor of response to function-blocking antibody.",
"Thyroid hormones (T3 and T4) induce proliferation in multiple myeloma (MM) cell lines via the αvβ3 integrin-mitogen-activated protein kinase (MAPK) pathway. We further show in primary MM bone marrow (BM) samples (n = 9) induction of cell viability by 1 nM T3 (13%, p < 0.002) and more potently by 100 nM T4 (21-45%, p < 0.0002) and a quick (1 h) and long-lasting (24 h) pERK activation, which was inhibited in the presence of β3 but not β1 blocking antibodies. Involvement of the integrin was further shown by two disintegrins, Arg-Gly-Asp (RGD) and echistatin peptides, which occluded the effects of T3/T4 on viability, proliferating cell nuclear antigen (PCNA) (proliferation marker) and apoptotic gene expression. Lastly, T3/T4 significantly opposed bortezomib (25 nM) cytotoxicy, as confirmed by several methods. In summary, our results imply that endogenous thyroid hormones in myeloma are factors that may support cell growth, with relevance to bortezomib action."
] |
Antimicrobial activity of a leaf dichloromethane extract from the shea butter tree
|
Combining antibiotics with resistance reversing agents is a key strategy to overcome bacterial resistance. Upon screening antimicrobial activities of plants used in traditional medicine, we found that a leaf dichloromethane extract from the shea butter tree (
|
[
"Staphylococcus aureus is a gram-positive bacterium that colonises the skin and is present in the anterior nares in about 25-30% of healthy people. Dependent on its intrinsic virulence or the ability of the host to contain its opportunistic behaviour, S aureus can cause a range of diseases in man. The bacterium readily acquires resistance against all classes of antibiotics by one of two distinct mechanisms: mutation of an existing bacterial gene or horizontal transfer of a resistance gene from another bacterium. Several mobile genetic elements carrying exogenous antibiotic resistance genes might mediate resistance acquisition. Of all the resistance traits S aureus has acquired since the introduction of antimicrobial chemotherapy in the 1930s, meticillin resistance is clinically the most important, since a single genetic element confers resistance to the most commonly prescribed class of antimicrobials--the beta-lactam antibiotics, which include penicillins, cephalosporins, and carbapenems.",
"The degree of cross-linking of the peptidoglycan of Staphylococcus aureus H and mutants lacking penicillin-binding proteins 1 and 4 was studied. No major changes were observed in organisms lacking protein 1 whereas loss of protein 4 was accompanied by a marked reduction in the degree of cross-linking and the absence of a membrane-bound 'model' transpeptidase activity. A similar effect was achieved when cultures of the staphylococci were treated with the beta-lactam antibiotic cefoxitin. At low concentrations (0.05 microgram ml-1) cefoxitin shows highest affinity for protein 4 to which it appears to bind irreversibly. Treatment of the mutant lacking protein 4 with this concentration of the antibiotic did not affect the degree of cross-linkage. The possibility that the decrease in cross-linkage was a consequence of DD-carboxypeptidase activity on peptidoglycan precursors was investigated. Although both S. aureus H and the mutants possessed such activity it was insensitive to benzylpenicillin and cefoxitin and the role of this enzyme(s) in peptidoglycan biosynthesis remains unknown. We conclude that in vivo protein 4 acts as a transpeptidase involved in the secondary cross-linking of peptidoglycan and this activity is necessary to achieve the high degree of cross-linkage observed in the peptidoglycan of staphylococci.",
"A new cephalosporin with a highly reactive beta-lactam ring was found to give an immediate color change in the presence of beta-lactamases from many bacteria, including staphylococci, Bacillus species, Enterobacteriaceae, and Pseudomonas. The reaction is confined to organisms producing beta-lactamases, but it is sufficiently sensitive to indicate the presence of this enzyme is small amounts in strains previously considered not to produce it. The compound has an unusual ultraviolet spectrum, and the color change can be followed quantitatively by measuring changes in absorption which occur in the 380- to 500-nm region, where cephalosporins normally have no absorption. The development of color is thought to be a consequence of the beta-lactam ring being unusually highly conjugated with the 3-substituent. Although in the bacteria only beta-lactamases produce this color change, it was found that serum and tissues from experimental animals also rapidly produced the colored breakdown product, which was then excreted in the urine. The mechanism of the mammalian breakdown was considered to be different from that found in bacteria.",
"The essential function of penicillin-binding protein 2 (PBP2) in methicillin-susceptible Staphylococcus aureus RN4220 was clearly established by placing the pbp2 gene under control of the inducible P(spac) promoter; the resulting bacteria were unable to grow in the absence of inducer. In contrast, the deficit in PBP2 caused by inhibition of transcription of the pbp2 gene did not block growth of a methicillin-resistant S. aureus strain expressing the extra penicillin-binding protein PBP2A, a protein of extraspecies origin that is central to the mechanism of methicillin resistance. Several lines of evidence indicate that the essential function of PBP2 that can be compensated for by PBP2A is the transpeptidase activity. This provides direct genetic evidence that PBP2A has transpeptidase activity.",
"Perturbation of the Staphylococcus aureus cytoplasmic membrane (CM) is felt to play a key role in the microbicidal mechanism of many antimicrobial peptides (APs). However, it is not established whether membrane permeabilization (MP) alone is sufficient to kill susceptible staphylococci or if the cell wall (CW) and/or intracellular targets contribute to AP-induced lethality. We hypothesized that the relationships between MP and killing may differ for distinct APs. In this study, we investigated the association between AP-induced MP and lethality in S. aureus whole cells versus CW-free protoplasts, and in comparison to the MP of liposomes modeled after whole CMs in terms of phospholipid composition, fluidity and charge. Four APs with different structure-activity relationships were examined: thrombin-induced platelet microbicidal protein 1 (tPMP-1), human neutrophil protein 1 (hNP-1), gramicidin D, and polymyxin B. MP was quantified fluorometrically by calcein release. All APs tested, except polymyxin B, caused concentration-dependent MP and killing of whole cells, but not of protoplasts. The reduced AP susceptibility of protoplasts was associated with increased cardiolipin and lysyl-phosphatidylglycerol content and reduced fluidity of their CMs. However, liposomal MP induced by tPMP-1, hNP-1, and gramicidin D paralleled that of whole cells. Collectively, these results indicate that (i) structurally distinct APs likely exert their staphylocidal effects by differing mechanisms, (ii) MP is not the sole event leading to AP-induced staphylocidal activity, (iii) a complex interrelationship exists between the CM and CW in AP-induced killing, and (iv) liposomes modeled upon whole cell or protoplast CMs can recapitulate the respective susceptibilities to killing by distinct APs.",
"In a loss-of-viability screen using small molecules against methicillin-resistant Staphylococcus aureus (MRSA) strain USA300 with a sub-MIC of a β-lactam, we found a small molecule, designated DNAC-1, which potentiated the effect of oxacillin (i.e., the MIC of oxacillin decreased from 64 to 0.25 μg/ml). Fluorescence microscopy indicated a disruption in the membrane structures within 15 min of exposure to DNAC-1 at 2× MIC. This permeabilization was accompanied by a rapid loss of membrane potential, as monitored by use of the DiOC2 (3,3'-diethyloxacarbocyanine iodide) dye. Macromolecular analysis showed the inhibition of staphylococcal cell wall synthesis by DNAC-1. Transmission electron microscopy of treated MRSA USA300 cells revealed a slightly thicker cell wall, together with mesosome-like projections into the cytosol. The exposure of USA300 cells to DNAC-1 was associated with the mislocalization of FtsZ accompanied by the localization of penicillin-binding protein 2 (PBP2) and PBP4 away from the septum, as well as mild activation of the vraRS-mediated cell wall stress response. However, DNAC-1 does not have any generalized toxicity toward mammalian host cells. DNAC-1 in combination with ceftriaxone is also effective against an assortment of Gram-negative pathogens. Using a murine subcutaneous coinjection model with 10(8) CFU of USA300 as a challenge inoculum, DNAC-1 alone or DNAC-1 with a sub-MIC of oxacillin resulted in a 6-log reduction in bacterial load and decreased abscess formation compared to the untreated control. We propose that DNAC-1, by exerting a bimodal effect on the cell membrane and cell wall, is a viable candidate in the development of combination therapy against many common bacterial pathogens."
] |
Toll-like receptors in human papilloma virus infection and HPV-induced cervical cancer
|
The main cause of cervical cancer is persistent infection with high-risk human papilloma virus (HR-HPV), but not all human papilloma virus (HPV) infections lead to cervical cancer. The key factors that determine the outcome of HPV infection remain poorly understood, and how the host immune system protects against HPV infection is unclear. Toll-like receptors (TLRs) are a group of pattern recognition receptors present in the cytoplasm and cell membrane, and can specifically recognize pathogen-associated molecular patterns. As the key molecules of innate and acquired immunity, TLRs not only play important roles in the immune defense against infectious diseases, but also are involved in the occurrence and development of a variety of malignant tumors. In cervical cancer caused by HR-HPV infection, TLRs have been found to regulate the local immune microenvironment. The role of TLRs in HR-HPV infection and HPV-induced cervical cancer and its relationship with HPV vaccine are reviewed in this article.
|
[
"Toll-like receptors (TLRs) are a family of pattern recognition receptors that are activated by specific components of microbes and certain host molecules. They constitute the first line of defense against many pathogens and play a crucial role in the function of the innate immune system. Recently, TLRs were observed to influence the development of adaptive immune responses, presumably by activating antigen-presenting cells. This has important implications for our understanding of how the host tailors its immune response as a function of specific pathogen recognition. The present review discusses the recent studies that demonstrate the role of TLRs in the regulation of adaptive T-helper-1 (Th1) and Th2 responses, and the mechanisms by which the effects are carried out. Most studies have focused on the role of TLRs and components of their signaling pathways in the control of Th1-type immune responses, and on the implications for their use as antimicrobial agents, such as adjuvants in vaccines, or to treat or prevent the Th2-type dominated immune responses seen in allergies. TLR-deficient mice have been described and used to come to these conclusions. Although controversial, there is also evidence that TLRs may be important for Th2-type responses, possibly by augmenting the overall maturity of dendritic cells. A greater understanding of the processes by which TLRs regulate adaptive immunity may yield not only improved ways to treat infectious diseases but also new approaches to the treatment and prevention of allergic and certain autoimmune disorders.",
"Bacterial lipopolysaccharide (LPS) triggers innate immune responses through Toll-like receptor (TLR) 4, a member of the TLR family that participates in pathogen recognition. TLRs recruit a cytoplasmic protein, MyD88, upon pathogen recognition, mediating its function for immune responses. Two major pathways for LPS have been suggested in recent studies, which are referred to as MyD88-dependent and -independent pathways. We report in this study the characterization of the MyD88-independent pathway via TLR4. MyD88-deficient cells failed to produce inflammatory cytokines in response to LPS, whereas they responded to LPS by activating IFN-regulatory factor 3 as well as inducing the genes containing IFN-stimulated regulatory elements such as IP-10. In contrast, a lipopeptide that activates TLR2 had no ability to activate IFN-regulatory factor 3. The MyD88-independent pathway was also activated in cells lacking both MyD88 and TNFR-associated factor 6. Thus, TLR4 signaling is composed of at least two distinct pathways, a MyD88-dependent pathway that is critical to the induction of inflammatory cytokines and a MyD88/TNFR-associated factor 6-independent pathway that regulates induction of IP-10.",
"Toll-like receptor (TLR) can activate dendritic cells (DC) through common signaling pathways requiring a cytoplasmic adapter, MyD88. However, the signaling is differentially regulated among TLR family members. TLR4 can activate MyD88-deficient bone marrow-derived DC (BMDC), and lead to induction of IFN-inducible genes and up-regulation of co-stimulatory molecules such as CD40, implying that the MyD88-independent signaling pathway functions downstream of TLR4. Because these effects can also be induced by type I IFN, we have analyzed whether type I IFN is involved in TLR4-induced responses. In response to lipopolysaccharide (LPS), IFN-beta gene expression was augmented in both wild-type and MyD88-deficient BMDC. Expression of all IFN-inducible genes except immune-responsive gene 1 (IRG1) was abolished and CD40 up-regulation was decreased in LPS-stimulated BMDC lacking either IFN-alpha/beta receptor (IFN-alpha/betaR) or signal transducer and activator of transcription 1 (STAT-1). Similar to the LPS response, TLR9 signaling can also induce expression of IFN-beta and IFN-inducible genes, and up-regulation of CD40. However, all these effects were MyD88 dependent. Thus, in TLR4 signaling, IFN-beta expression can be induced either by the MyD88-dependent or -independent pathway, whereas, in TLR9 signaling, it is dependent on MyD88. In CpG DNA-stimulated DC, expression of IFN-inducible genes except IRG1 was dependent on type I IFN signaling as in LPS-stimulated DC. However, in contrast to TLR4 signaling, TLR9 signaling requires type I IFN signaling for CD40 up-regulation. Taken together, this study demonstrates differential involvement of type I IFN in TLR4- and TLR9-induced effects on DC.",
"Helicobacter pylori (H. pylori) is a gram-negative pathogen that is widespread all over the world, infecting more than 50% of the world's population. It is etiologically associated with non-atrophic and atrophic gastritis, peptic ulcer and shows a deep association with primary gastric B-cell lymphoma and gastric adenocarcinoma. Recently, the medical research focused on the modification of the gastric environment induced by H. pylori infection, possibly affecting the absorption of nutrients and drugs as well as the production of hormones strongly implicated in the regulation of appetite and growth. Interestingly, the absorption of iron and vitamin B12 is impaired by H. pylori infection, while infected subjects have lower basal and fasting serum levels of ghrelin and higher concentration of leptin compared to controls. Since leptin is an anorexigenic hormone, and ghrelin stimulates powerfully the release of growth hormone in humans, H. pylori infection may finally induce growth retardation if acquired very early in the childhood and in malnourished children. This review is focused on the nutritional effects of H. pylori infection, such as the reduced bioavailability or the malabsorbption of essential nutrients, and of gastrointestinal hormones, as well as on the relationship between H. pylori and the metabolic syndrome.",
"The imidazoquinoline compounds imiquimod and R-848 are low-molecular-weight immune response modifiers that can induce the synthesis of interferon-alpha and other cytokines in a variety of cell types. These compounds have potent anti-viral and anti-tumor properties; however, the mechanisms by which they exert their anti-viral activities remain unclear. Here we show that the imidazoquinolines activate immune cells via the Toll-like receptor 7 (TLR7)-MyD88-dependent signaling pathway. In response to the imidazoquinolines, neither MyD88- nor TLR7-deficient mice showed any inflammatory cytokine production by macrophages, proliferation of splenocytes or maturation of dendritic cells. Imidazoquinoline-induced signaling events were also abolished in both MyD88- and TLR7-deficient mice.",
"Helicobacter pylori (H. pylori) -induced gastric inflammation impacts the functions of leptin- and ghrelin-producing cells in the gastroduodenum. Inflammation resulting from H. pylori sensing via Toll-like receptors (TLRs) and the associated downstream signaling largely remain ambiguous. Here, we investigated the role of gut hormones, pro-inflammatory cytokines and single nucleotide polymorphisms (SNPs) associated with TLR 4p14 in H. pylori disease in 30 subjects with non-ulcer dyspepsia (NUD), 40 with peptic ulcer disease (PUD) and 15 with gastric cancer (GC) subjects positive and negative for H. pylori infection. The level of pro-inflammatory cytokines was directly proportional to the severity of gastritis, and disease status influenced the levels of gut hormones and pro-inflammatory cytokines. TLR-1 SNPs rs4833095 and TLR-10 SNPs rs10004195 and were directly associated with H. pylori disease, and were up-regulated in the presence of H. pylori in a genotype-independent manner. We concluded that TLR-1 rs4833095 and TLR10 rs10004195 confer susceptibility to development of gastroduodenal disease, especially GC in H.pylori disease.",
"Inflammation is a physiological process that repairs tissues in response to endogenous or exogenous aggressions. Nevertheless, a chronic state of inflammation may have detrimental consequences. Aging is associated with increased levels of circulating cytokines and proinflammatory markers. Aged-related changes in the immune system, known as immunosenescence, and increased secretion of cytokines by adipose tissue, represent the major causes of chronic inflammation. This phenomenon is known as \"inflamm-aging.\" High levels of interleukin (IL)-6, IL-1, tumor necrosis factor-α, and C-reactive protein are associated in the older subject with increased risk of morbidity and mortality. In particular, cohort studies have indicated TNF-α and IL-6 levels as markers of frailty. The low-grade inflammation characterizing the aging process notably concurs at the pathophysiological mechanisms underlying sarcopenia. In addition, proinflammatory cytokines (through a variety of mechanisms, such as platelet activation and endothelial activation) may play a major role in the risk of cardiovascular events. Dysregulation of the inflammatory pathway may also affect the central nervous system and be involved in the pathophysiological mechanisms of neurodegenerative disorders (eg, Alzheimer disease).The aim of the present review was to summarize different targets of the activity of proinflammatory cytokines implicated in the risk of pathological aging."
] |
Object Location Touchscreen Test (OLTT): A Novel and Ecologically Relevant Associative Memory Test
|
We present findings of a novel and ecologically relevant associative memory test, the Object Location Touchscreen Test (OLTT), which was posited as sensitive to early medial temporal lobe compromise associated with mild cognitive impairment (MCI).
|
[
"FSL (the FMRIB Software Library) is a comprehensive library of analysis tools for functional, structural and diffusion MRI brain imaging data, written mainly by members of the Analysis Group, FMRIB, Oxford. For this NeuroImage special issue on \"20 years of fMRI\" we have been asked to write about the history, developments and current status of FSL. We also include some descriptions of parts of FSL that are not well covered in the existing literature. We hope that some of this content might be of interest to users of FSL, and also maybe to new research groups considering creating, releasing and supporting new software packages for brain image analysis.",
"Neuropsychologists are developing more challenging and specific tests to detect early and subtle changes in cognition related to preclinical Alzheimer's disease (AD). The 16-item Face-Name Associative Memory Exam (FNAME-16) is a challenging paired associative memory test able to detect subtle memory changes associated with biomarker evidence of preclinical AD. However, as individuals progress along the AD trajectory, measures that are sensitive at the preclinical stage may become too challenging by the stage of Mild Cognitive Impairment (MCI). Our goal was to develop a modified version of the face-name and face-occupation paired associative memory task (FNAME-12) with fewer stimuli and additional learning trials suitable for use in MCI. We administered the FNAME-12A, an alternate version FNAME 12B, the original FNAME-16, and a series of other neuropsychological measures to 65 clinically normal (CN) older adults (aged 65 to 85) and a subsample characterized by MCI (n = 18). The FNAME-12 exhibited psychometric equivalence with the FNAME-16 (r = .77, p < .001) and was correlated with other measures of episodic and semantic memory. The alternate form, FNAME-12B, was highly correlated with FNAME-12A (r = .76, p < .001). Mean performance on the FNAME 12A, stratified by education, was generated. The task could be completed by our MCI group yet remained challenging in the CN group, providing evidence for its utility along the AD trajectory.",
"The First Key Symposium was held in Stockholm, Sweden, 2-5 September 2003. The aim of the symposium was to integrate clinical and epidemiological perspectives on the topic of Mild Cognitive Impairment (MCI). A multidisciplinary, international group of experts discussed the current status and future directions of MCI, with regard to clinical presentation, cognitive and functional assessment, and the role of neuroimaging, biomarkers and genetics. Agreement on new perspectives, as well as recommendations for management and future research were discussed by the international working group. The specific recommendations for the general MCI criteria include the following: (i) the person is neither normal nor demented; (ii) there is evidence of cognitive deterioration shown by either objectively measured decline over time and/or subjective report of decline by self and/or informant in conjunction with objective cognitive deficits; and (iii) activities of daily living are preserved and complex instrumental functions are either intact or minimally impaired.",
"The National Institute on Aging and the Alzheimer's Association charged a workgroup with the task of developing criteria for the symptomatic predementia phase of Alzheimer's disease (AD), referred to in this article as mild cognitive impairment due to AD. The workgroup developed the following two sets of criteria: (1) core clinical criteria that could be used by healthcare providers without access to advanced imaging techniques or cerebrospinal fluid analysis, and (2) research criteria that could be used in clinical research settings, including clinical trials. The second set of criteria incorporate the use of biomarkers based on imaging and cerebrospinal fluid measures. The final set of criteria for mild cognitive impairment due to AD has four levels of certainty, depending on the presence and nature of the biomarker findings. Considerable work is needed to validate the criteria that use biomarkers and to standardize biomarker analysis for use in community settings.",
"Remembering the location of objects in the environment is both important in everyday life and difficult for patients with amnestic mild cognitive impairment (aMCI), a clinical precursor to Alzheimer's disease. To test the hypothesis that memory impairment for object location in aMCI reflects hippocampal dysfunction, we used an event-related functional magnetic resonance imaging paradigm to compare patients with aMCI and healthy elderly controls (HEC) as they encoded 90 ecologically relevant object-location associations (OLAs). Two additional OLAs, repeated a total of 45 times, served as control stimuli. Memory for these OLAs was assessed following a 1-h delay. The groups were well matched on demographics and brain volumetrics. Behaviorally, HEC remembered significantly more OLAs than did aMCI patients. Activity differences were assessed by contrasting activation for successfully encoded Novel stimuli vs. Repeated stimuli. The HEC demonstrated activity within object-related (ventral visual stream), spatial location-related (dorsal visual stream), and feature binding-related cortical regions (hippocampus and other memory-related regions) as well as in frontal cortex and associated subcortical structures. Activity in most of these regions correlated with memory test performance. Although the aMCI patients demonstrated a similar activation pattern, the HEC showed significantly greater activity within each of these regions. Memory test performance in aMCI patients, in contrast to the HEC, was correlated with activity in regions involved in sensorimotor processing. We conclude that aMCI patients demonstrate widespread cerebral dysfunction, not limited to the hippocampus, and rely on encoding-related mechanisms that differ substantially from healthy individuals."
] |
Web-based and mobile applications to mitigate stress, burnout, depression, and suicide in healthcare professionals
|
Being a healthcare professional can be a uniquely rewarding calling. However, the demands of training and practice can lead to chronic distress and serious psychological, interpersonal, and personal health burdens. Although higher burnout, depression, and suicide rates have been reported in healthcare professionals, only a minority receive treatment. Concerns regarding confidentiality, stigma, potential career implications, and cost and time constraints are cited as key barriers. Web-based and mobile applications have been shown to mitigate stress, burnout, depression, and suicidal ideation among several populations and may circumvent these barriers. Here, we reviewed published data on such resources and selected a small sample that readily can be used by healthcare providers.
|
[
"In the United States, approximately 1 physician dies by suicide every day. Training physicians are at particularly high risk, with suicidal ideation increasing more than 4-fold during the first 3 months of internship year. Despite this increase, to our knowledge, very few efforts have been made to prevent the escalation of suicidal thoughts among training physicians. To assess the effectiveness of a web-based cognitive behavioral therapy (wCBT) program delivered prior to the start of internship year in the prevention of suicidal ideation in medical interns. A randomized clinical trial conducted at 2 university hospitals with 199 interns from multiple specialties during academic years 2009-2010 or 2011-2012. The current study was conducted from May 2009 to June 2010 and May 2011 to June 2012, and data were analyzed using intent-to-treat principles, including last observation carried forward. Interns were randomly assigned to 2 study groups (wCBT and attention-control group [ACG]), and completed study activities lasting 30 minutes each week for 4 weeks prior to starting internship year. Participants assigned to wCBT completed online CBT modules and those assigned to ACG received emails with general information about depression, suicidal thinking, and local mental health professionals. The Patient Health Questionnaire-9 was used to assess suicidal ideation (ie, \"thoughts that you would be better off dead or hurting yourself in some way\") prior to the start of intern year and at 3-month intervals throughout the year. A total of 62.2% of interns (199 of 320) agreed to take part in the study; 100 were assigned to the wCBT group and 99 to the ACG. During at least 1 point over the course of internship year, 12% of interns (12 of 100) assigned to wCBT endorsed suicidal ideation compared with 21.2% of interns (21 of 99) assigned to ACG. After adjusting for covariates identified a priori that have previously shown to increase the risk for suicidal ideation, interns assigned to wCBT were less likely to endorse suicidal ideation during internship year (relative risk, 0.40; 95% CI, 0.17- 0.91; P = .03) compared with those assigned to ACG. This study demonstrates that a free, easily accessible, brief wCBT program is associated with reduced likelihood of suicidal ideation among medical interns. Prevention programs with these characteristics could be easily disseminated to medical training programs across the country. anzctr.org.au Identifier: ACTRN12610000628044.",
"Mixed evidence has suggested that homozygous carriers of the short allele (s/s) of the serotonin transporter gene-linked polymorphic region (5-HTTLPR) may be at increased risk for depression, if they have also been exposed to early or current adversity/stress. We address this debate by examining the relation of a stressful early family environment, recent adversity/stress, and the 5-HTTLPR to depressive symptomatology in a normal sample. A nonclinical sample of 118 young adult men and women completed assessments of early family environment, recent stressful events, psychosocial resources, and psychological distress, including depressive symptomatology. The 5-HTTLPR was genotyped using a standard protocol with DNA extracted from oral fluid. A stressful early family environment was significantly related to depressive symptomatology. In addition, gene-by-environment (GxE) interactions were observed between the 5-HTTLPR and both early family environment and current adversity/stress. Individuals homozygous for the short allele had greater depressive symptomatology if they had experienced early or recent adversity but significantly less depressive symptomatology if they reported a supportive early environment or recent positive experiences, compared with participants with the s/l or l/l genotype. Early or current environment, in conjunction with the serotonin transporter polymorphism, predicts depressive symptomatology.",
"Healthcare providers are under increasing stress and work-related burnout has become common. Mindfulness-based interventions have a potential role in decreasing stress and burnout. The purpose of this study was to determine if a continuing education course based on mindfulness-based stress reduction could decrease burnout and improve mental well-being among healthcare providers, from different professions. This was a pre-post observational study conducted in a university medical center. A total of 93 healthcare providers, including physicians from multiple specialties, nurses, psychologists, and social workers who practiced in both university and community settings, participated. The intervention was a continuing education course based on mindfulness-based stress reduction that met 2.5 hours a week for 8 weeks plus a 7-hour retreat. The classes included training in four types of formal mindfulness practices, including the body scan, mindful movement, walking meditation and sitting meditation, as well as discussion focusing on the application of mindfulness at work. The course was offered 11 times over 6 years. The main outcome measures were work-related burnout as measured by the Maslach Burnout Inventory and self-perceived mental and physical well-being as measured by the SF-12v2. Maslach Burnout Inventory scores improved significantly from before to after the course for both physicians and other healthcare providers for the Emotional Exhaustion (p < 0.03), Depersonalization (p < 0.04), and Personal Accomplishment (p < 0.001) scales. Mental well-being measured by the SF12v2 also improved significantly (p < 0.001). There were no significant changes in the SF12v2 physical health scores. A continuing education course based on mindfulness-based stress reduction was associated with significant improvements in burnout scores and mental well-being for a broad range of healthcare providers.",
"Burnout is plaguing the culture of medicine and is linked to several primary causes including long work hours, increasingly burdensome documentation, and resource constraints. Beyond these, additional emotional stressors for physicians are involvement in an adverse event, especially one that involves a medical error, and malpractice litigation. The authors argue that it is imperative that health care institutions devote resources to programs that support physician well-being and resilience. Doing so after adverse and other emotionally stressful events, such as the death of a colleague or caring for victims of a mass trauma, is crucial as clinicians are often at their most vulnerable during such times. To this end, the Center for Professionalism and Peer Support at Brigham and Women's Hospital redesigned the peer support program in 2009 to provide one-on-one peer support. The peer support program was one of the first of its kind; over 25 national and international programs have been modeled off of it. This Perspective describes the origin, structure, and basic workings of the peer support program, including important components for the peer support conversation (outreach call, invitation/opening, listening, reflecting, reframing, sense-making, coping, closing, and resources/referrals). The authors argue that creating a peer support program is one way forward, away from a culture of invulnerability, isolation, and shame and toward a culture that truly values a sense of shared organizational responsibility for clinician well-being and patient safety.",
"The authors replicated a program developed by UC San Diego, identified medical staff at risk for depression and suicide using a confidential online survey, and studied aspects of that program for 1 year. The authors used a 35-item, online assessment of stress and depression depression developed and licensed by the American Foundation for Suicide Prevention that aims to identify and suicide risk and facilitate access to mental health services. During 2013/2014, all 1864 UC Davis residents/fellows and faculty physicians received an invitation to take the survey and 158 responded (8% response rate). Most respondents were classified at either moderate (86 [59%]) or high risk for depression or suicide (54 [37%]). Seventeen individuals (11%) were referred for further evaluation or mental health treatment. Ten respondents consented to participate in the follow-up portion of the program. Five of the six who completed follow-up surveys reported symptom improvement and indicated the program should continue. This program has led to continued funding and a plan to repeat the Wellness Survey annually. Medical staff will be regularly reminded of its existence through educational interventions, as the institutional and professional culture gradually changes to promptly recognize and seek help for physicians' psychological distress.",
"To evaluate the efficacy of two internet interventions for community-dwelling individuals with symptoms of depression--a psychoeducation website offering information about depression and an interactive website offering cognitive behaviour therapy. Randomised controlled trial. Internet users in the community, in Canberra, Australia. 525 individuals with increased depressive symptoms recruited by survey and randomly allocated to a website offering information about depression (n = 166) or a cognitive behaviour therapy website (n = 182), or a control intervention using an attention placebo (n = 178). Change in depression, dysfunctional thoughts; knowledge of medical, psychological, and lifestyle treatments; and knowledge of cognitive behaviour therapy. Intention to treat analyses indicated that information about depression and interventions that used cognitive behaviour therapy and were delivered via the internet were more effective than a credible control intervention in reducing symptoms of depression in a community sample. For the intervention that delivered cognitive behaviour therapy the reduction in score on the depression scale of the Center for Epidemiologic Studies was 3.2 (95% confidence interval 0.9 to 5.4). For the \"depression literacy\" site (BluePages), the reduction was 3.0 (95% confidence interval 0.6 to 5.2). Cognitive behaviour therapy (MoodGYM) reduced dysfunctional thinking and increased knowledge of cognitive behaviour therapy. Depression literacy (BluePages) significantly improved participants' understanding of effective evidence based treatments for depression (P < 0.05). Both cognitive behaviour therapy and psychoeducation delivered via the internet are effective in reducing symptoms of depression.",
"To examine reductions in suicidal ideation among a sample of patients who were prescribed an internet cognitive behavior therapy (iCBT) course for depression. Effectiveness study within a quality assurance framework. Primary care. 299 patients who were prescribed an iCBT course for depression by primary care clinicians. Six lesson, fully automated cognitive behaviour therapy course delivered over the internet. suicidal ideation as measured by question 9 on the Patient Health Questionnaire (PHQ-9). Suicidal ideation was common (54%) among primary care patients prescribed iCBT treatment for depression but dropped to 30% post-treatment despite minimal clinician contact and the absence of an intervention focused on suicidal ideation. This reduction in suicidal ideation was evident regardless of sex and age. The findings do not support the exclusion of patients with significant suicidal ideation."
] |
Social network characteristics and health facility delivery in rural Ghana
|
Health facility births contribute to the prevention of maternal deaths. Although theoretical and empirical evidence suggest that social network characteristics influence facility delivery, examination of this relationship in sub-Saharan Africa is limited. We determined whether network structural and functional characteristics were associated with, or had an interactive effect on health facility delivery in rural Ghana.
|
[
"Almost 50% of women in low- and middle-income countries (LMICs) don't receive adequate antenatal care. Women's views can offer important insights into this problem. Qualitative studies exploring inadequate use of antenatal services have been undertaken in a range of countries, but the findings are not easily transferable. We aimed to inform the development of future antenatal care programmes through a synthesis of findings in all relevant qualitative studies. Using a predetermined search strategy, we identified robust qualitative studies reporting on the views and experiences of women in LMICs who received inadequate antenatal care. We used meta-ethnographic techniques to generate themes and a line-of-argument synthesis. We derived policy-relevant hypotheses from the findings. We included 21 papers representing the views of more than 1,230 women from 15 countries. Three key themes were identified: \"pregnancy as socially risky and physiologically healthy\", \"resource use and survival in conditions of extreme poverty\", and \"not getting it right the first time\". The line-of-argument synthesis describes a dissonance between programme design and cultural contexts that may restrict access and discourage return visits. We hypothesize that centralised, risk-focused antenatal care programmes may be at odds with the resources, beliefs, and experiences of pregnant women who underuse antenatal services. Our findings suggest that there may be a misalignment between current antenatal care provision and the social and cultural context of some women in LMICs. Antenatal care provision that is theoretically and contextually at odds with local contextual beliefs and experiences is likely to be underused, especially when attendance generates increased personal risks of lost family resources or physical danger during travel, when the promised care is not delivered because of resource constraints, and when women experience covert or overt abuse in care settings.",
"In Ghana, the site of this study, the maternal mortality ratio and under-five mortality rate remain high indicating the need to focus on maternal and child health programming. Ghana has high use of antenatal care (95%) but sub-optimum levels of institutional delivery (about 57%). Numerous barriers to institutional delivery exist including financial, physical, cognitive, organizational, and psychological and social. This study examines the psychological and social barriers to institutional delivery, namely women's decision-making autonomy and their perceptions about social support for institutional delivery in their community. This study uses cross-sectional data collected for the evaluation of the Maternal and Newborn Referrals Project of Project Fives Alive in Northern and Central districts of Ghana. In 2012 and 2013, a total of 2,527 women aged 15 to 49 were surveyed at baseline and midterm (half in 2012 and half in 2013). The analysis sample of 1,606 includes all women who had a birth three years prior to the survey date and who had no missing data. To determine the relationship between institutional delivery and the two key social barriers-women's decision-making autonomy and community perceptions of institutional delivery-we used multi-level logistic regression models, including cross-level interactions between community-level attitudes and individual-level autonomy. All analyses control for the clustered survey design by including robust standard errors in Stata 13 statistical software. The findings show that women who are more autonomous and who perceive positive attitudes toward facility delivery (among women, men and mothers-in-law) were more likely to deliver in a facility. Moreover, the interactions between autonomy and community-level perceptions of institutional delivery among men and mothers-in-law were significant, such that the effect of decision-making autonomy is more important for women who live in communities that are less supportive of institutional delivery compared to communities that are more supportive. This study builds upon prior work by using indicators that provide a more direct assessment of perceived community norms and women's decision-making autonomy. The findings lead to programmatic recommendations that go beyond individuals and engaging the broader network of people (husbands and mothers-in-law) that influence delivery behaviors.",
"Ghana is attracting global attention for efforts to provide health insurance to all citizens through the National Health Insurance Scheme (NHIS). With the program's strong emphasis on maternal and child health, an expectation of the program is that members will have increased use of relevant services. This paper uses qualitative and quantitative data from a baseline assessment for the Maternal and Newborn errals Evaluation from the Northern and Central Regions to describe women's experiences with the NHIS and to study associations between insurance and skilled facility delivery, antenatal care and early care-seeking for sick children. The assessment included a quantitative household survey (n = 1267 women), a quantitative community leader survey (n = 62), qualitative birth narratives with mothers (n = 20) and fathers (n = 18), key informant interviews with health care workers (n = 5) and focus groups (n = 3) with community leaders and stakeholders. The key independent variables for the quantitative analyses were health insurance coverage during the past three years (categorized as all three years, 1-2 years or no coverage) and health insurance during the exact time of pregnancy. Quantitative findings indicate that insurance coverage during the past three years and insurance during pregnancy were associated with greater use of facility delivery but not ANC. Respondents with insurance were also significantly more likely to indicate that an illness need not be severe for them to take a sick child for care. The NHIS does appear to enable pregnant women to access services and allow caregivers to seek care early for sick children, but both the quantitative and qualitative assessments also indicated that the poor and least educated were less likely to have insurance than their wealthier and more educated counterparts. Findings from the qualitative interviews uncovered specific challenges women faced regarding registration for the NHIS and other barriers such lack of understanding of who and what services were covered for free. Efforts should be undertaken so all individuals understand the NHIS policy including who is eligible for free services and what services are covered. Increasing access to health insurance will enable Ghana to further improve maternal and child health outcomes.",
"While the importance of antenatal care for maternal and child health continues to be debated, several researchers have documented its impact on intermediate variables affecting survival such as birth weight. These studies have also highlighted the problems of causality that are typically not taken into account when estimating the effects of antenatal care on skilled birth attendance. In this study, we revisit this relation in the rural areas of four countries: Ghana, Kenya, Uganda and Tanzania. Using a structural equation modeling approach that corrects for endogeneity, in all four countries we find that the usual simpler probit (or logit) models tend to underestimate the direct effect of antenatal care on skilled birth attendance. Furthermore, in two of the countries, this estimated effect is mediated by the range of services offered to women during antenatal care. These results suggest that governments and NGOs should place more importance on the role of antenatal care providers and on the services they offer, in efforts to promote skilled birth attendance.",
"Demographic phenomena occur within social contexts and, therefore, should be studied as social processes. However, how to conceptualize and measure the social worlds that individuals inhabit has been the subject of debate. Data from a study conducted in Mali in 1996-1997 are used to explore the social networks of Bamanan women and their impact on fertility decisions. Ordinary least-squares and logistic regression techniques are employed to examine the relationship between selected household and social network characteristics and two fertility measures: children ever born and ever-use of contraceptives. Household characteristics do not have a significant effect on either outcome, whereas network attributes do. The more prominently conjugal kin are represented in a network, the fewer children a woman has ever had; however, fertility increases if the husband or unrelated older women are part of the network. Ever-use of contraceptives is elevated if the woman participates in a credit scheme, and rises as the proportion of network members located outside the village increases; it declines sharply as the proportion of network members who are conjugal kin increases, and is significantly elevated if the woman's mother is present. Network effects on fertility are much more pronounced for women aged 30 or older than for younger women, and network effects on contraceptive use are markedly different for younger and older women. Programs should consider not only women's individual and household characteristics, but also their larger social networks. Additionally, programs should be designed for specific age-groups, given the different network effects on older and younger women.",
"While the most important factors associated with facility-based delivery (FBD) have been explored within individual countries in Africa, no systematic review has explored the factors associated with FBD across sub-Saharan Africa. A systematic search of the peer-reviewed literature was conducted to identify articles published in English from 1/1995-12/2011 that reported on original research conducted entirely or in part in sub-Saharan Africa and included a primary outcome variable of FBD, delivery location, or skilled birth attendance (SBA). Out of 1,168 citations identified, 65 met inclusion criteria. 62 of 65 were cross-sectional, and 58 of 65 relied upon household survey data. Fewer than two-thirds (43) included multivariate analyses. The factors associated with facility delivery were categorized as maternal, social, antenatal-related, facility-related, and macro-level factors. Maternal factors were the most commonly studied. This may be a result of the overwhelming reliance on household survey data - where maternal sociodemographic factors are likely to be well-represented and non-maternal factors may be less consistently and accurately represented. Multivariate analysis suggests that maternal education, parity / birth order, rural / urban residence, household wealth / socioeconomic status, distance to the nearest facility, and number of antenatal care visits were the factors most consistently associated with FBD. In conclusion, FBD is a complex issue that is influenced by characteristics of the pregnant woman herself, her immediate social circle, the community in which she lives, the facility that is closest to her, and context of the country in which she lives. Research to date has been dominated by analysis of cross-sectional household survey data. More research is needed that explores regional variability, examines longitudinal trends, and studies the impact of interventions to boost rates of facility delivery in sub-Saharan Africa.",
"Evidence from diverse settings suggests that women often have limited control over their own reproductive health decisions. To increase uptake of preventive services and behaviors, it is important to understand how intrafamilial power dynamics and the attitudes of women, their husband and their mother-in-law are associated with maternal health practices. In 317 households in two rural districts of central Mali, women who had given birth in the previous year, their husband and their mother-in-law each completed a survey gauging their attitudes toward constructs of gender, power and health. Bivariate and multivariable logistic regression analyses were conducted to identify associations with four maternal health outcomes: antenatal care frequency, antenatal care timing, institutional delivery and postnatal care. In multivariable analyses, the preferences and opinions of mothers-in-law were associated with the maternal health behaviors of their daughters-in-law. Women's own perceptions of their self-efficacy, the value of women in society and the quality of services at the local health facility were also independently associated with their preventive and health-seeking practices. Husbands' preferences and opinions were not associated with any outcome. Interventions focusing on women or couples may be insufficient to advance women's reproductive health in patriarchal societies such as Mali. Future research and programmatic efforts need to address gender norms and consider the influence of other family members, such as mothers-in-law."
] |
Pharmacokinetics of 3 sustained-release buprenorphine dosages in guinea pigs.
|
In guinea pigs, studies addressing the efficacy, safety, and pharmacokinetic profiles of different sustained-release buprenorphine (SRB) formulations are still in their infancy. Here we assessed the pharmacokinetic profiles of 3 SRB dosages (SR-LAB, ZooPharm; SRBLow, 0.15 mg/kg; SRBMedium, 0.3 mg/kg; and SRBHigh, 0.6 mg/kg) for 72 h after a single subcutaneous administration to 8 (4 male and 4 female) healthy guinea pigs. Body weight, fecal output, and cortisol levels were also monitored and the results compared with those of the sham group. Within the first h after administration, the maximal plasma concentration (Cmax) of the drug was 64.3 ± 9.2 ng/mL (males) and 71.3 ± 3.7 ng/mL (females) in the SRBHigh group; 11.5 ± 3.2 ng/mL (males) and 6.9 ± 0.9 ng/mL (females) in the SRBMedium group; and 2.3 ± 0.8 ng/mL (males) and 2.0 ± 0.5 ng/mL (females) in the SRBLow group. After 72 h, therapeutic levels of the drug (>1 ng/mL) were observed only in guinea pigs treated with SRBHigh (both sexes) and males treated with SRBMediu cm. Fecal output (quantity and distribution) and body weight were significantly lower in the SRB groups as compared with the sham group, and with the SRBHigh group showing larger reductions. Baseline levels of serum cortisol in healthy females (1440 ± 106 ng/mL) were significantly greater than in males (550 ± 66 ng/mL). But, independent of the sex, SRB administration significantly reduced those levels. In conclusion, the data indicate that all 3 SRB dosages can be safely used in guinea pigs. However, therapeutic levels of the drug were observed for at least 48 h only guinea pigs treated with SRBHigh and SRBMedium. Further investigation is needed to determine if these dosages can alleviate pain in guinea pigs.
|
[
"Guinea pigs possess several biological similarities to humans and are validated experimental animal models(1-3). However, the use of guinea pigs currently represents a relatively narrow area of research and descriptive data on specific methodology is correspondingly scarce. The anatomical features of guinea pigs are slightly different from other rodent models, hence modulation of sampling techniques to accommodate for species-specific differences, e.g., compared to mice and rats, are necessary to obtain sufficient and high quality samples. As both long and short term in vivo studies often require repeated blood sampling the choice of technique should be well considered in order to reduce stress and discomfort in the animals but also to ensure survival as well as compliance with requirements of sample size and accessibility. Venous blood samples can be obtained at a number of sites in guinea pigs e.g., the saphenous and jugular veins, each technique containing both advantages and disadvantages(4,5). Here, we present four different blood sampling techniques for either conscious or anaesthetized guinea pigs. The procedures are all non-terminal procedures provided that sample volumes and number of samples do not exceed guidelines for blood collection in laboratory animals(6). All the described methods have been thoroughly tested and applied for repeated in vivo blood sampling in studies within our research facility.",
"Bacterial lipoproteins are a functionally diverse class of membrane anchored proteins. Lipoproteins constitute nearly 2.5% of the Mycobacterium tuberculosis proteome. Inactivation of genes coding for individual lipoproteins results in attenuated phenotype of the mutants. LpqS is a lipoprotein highly conserved among slow growing pathogenic mycobacteria. Our previous study has shown that the lpqS gene deletion mutant of M. tuberculosis (MtbΔlpqS) poorly replicates in THP1-(human acute monocytic leukemia cell line) derived macrophagic cell line. In addition, guinea pigs, when infected with the mutant strain exhibited significantly reduced bacterial burden and pathological damage in the infected tissues in comparison with the parental strain infected group. Subsequently, we evaluated the protective efficacy of the mutant by immunization of guinea pigs through aerosol and subcutaneous routes. We observed that immunization of guinea pigs with MtbΔlpqS offered superior protection in lungs as compared to BCG. In addition, MtbΔlpqS also prevented the haematogenous spread of the disease which was evident from the significantly reduced splenic bacillary load compared to saline vaccinated animals. The gross pathological observations and the histopathological observations well corroborated the bacterial findings. We also observed that aerogenic route of immunization imparts superior protection compared to subcutaneous route of immunization. These findings well establishes the efficacy of M. tuberculosis mutant in imparting protection against pulmonary TB.",
"Ivermectin (IVM), an FDA approved anthelmintic agent, can significantly reduce ethanol intake in mice following acute administration. The current study evaluates the sustainability and safety of multiday IVM administration in reducing 10% v/v ethyl alcohol (10E) intake in mice at a dose shown to be safe in humans. We tested the effect of 10-day administration of IVM (3.0 mg/kg/day; intraperitoneally) on reducing 10E intake in C57BL/6J mice using a 24-h, two-bottle choice paradigm. On the 10th day of IVM administration, mice were sacrificed at 0, 0.5, 2, 8, 32, 48, and 72 h after injection. Brain tissue and plasma samples were collected and analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Analysis of variance (ANOVA) was used to assess the effect of 10-day IVM administration on 10E intake, 10E preference, water intake, and total fluid intake with Dunnett's multiple comparison post-hoc test. Individual Student's t-tests were also used to further quantify changes in these dependent variables. IVM significantly decreased 10E intake over a 9-day period (P<0.01). Pre-IVM 10E intake was 9.1±3.2 g/kg/24 h. Following the 9th day of IVM injections, intake dropped by almost 30% (P<0.05). IVM had no effect on total water intake or mouse weight throughout the study; however, there was a significant decrease in both preference for 10E (P<0.01) and total fluid intake (P<0.05). Multiday administration of IVM significantly reduces 10E intake and preference in animals without causing any apparent adverse effects at a dose shown to be safe in humans.",
"Our laboratory is investigating ivermectin (IVM) and other members of the avermectin family as new pharmaco-therapeutics to prevent and/or treat alcohol use disorders (AUDs). Earlier work found that IVM significantly reduced ethanol intake in mice and that this effect likely reflects IVM's ability to modulate ligand-gated ion channels. We hypothesized that structural modifications that enhance IVM's effects on key receptors and/or increase its brain concentration should improve its anti-alcohol efficacy. We tested this hypothesis by comparing the abilities of IVM and two other avermectins, abamectin (ABM) and selamectin (SEL), to reduce ethanol intake in mice, to alter modulation of GABAARs and P2X4Rs expressed in Xenopus oocytes and to increase their ability to penetrate the brain. IVM and ABM significantly reduced ethanol intake and antagonized the inhibitory effects of ethanol on P2X4R function. In contrast, SEL did not affect either measure, despite achieving higher brain concentrations than IVM and ABM. All three potentiated GABAAR function. These findings suggest that chemical structure and effects on receptor function play key roles in the ability of avermectins to reduce ethanol intake and that these factors are more important than brain penetration alone. The direct relationship between the effect of these avermectins on P2X4R function and ethanol intake suggest that the ability to antagonize ethanol-mediated inhibition of P2X4R function may be a good predictor of the potential of an avermectin to reduce ethanol intake and support the use of avermectins as a platform for developing novel drugs to prevent and/or treat AUDs."
] |
Mitochondrial inner NEET protein (MiNT) is a key player in the mitochondrial labile iron pathway
|
The NEET family is a relatively new class of three related [2Fe-2S] proteins (CISD1-3), important in human health and disease. While there has been growing interest in the homodimeric gene products of CISD1 (mitoNEET) and CISD2 (NAF-1), the importance of the inner mitochondrial CISD3 protein has only recently been recognized in cancer. The CISD3 gene encodes for a monomeric protein that contains two [2Fe-2S] CDGSH motifs, which we term mitochondrial inner NEET protein (MiNT). It folds with a pseudosymmetrical fold that provides a hydrophobic motif on one side and a relatively hydrophilic surface on the diametrically opposed surface. Interestingly, as shown by molecular dynamics simulation, the protein displays distinct asymmetrical backbone motions, unlike its homodimeric counterparts that face the cytosolic side of the outer mitochondrial membrane/endoplasmic reticulum (ER). However, like its counterparts, our biological studies indicate that knockdown of MiNT leads to increased accumulation of mitochondrial labile iron, as well as increased mitochondrial reactive oxygen production. Taken together, our study suggests that the MiNT protein functions in the same pathway as its homodimeric counterparts (mitoNEET and NAF-1), and could be a key player in this pathway within the mitochondria. As such, it represents a target for anticancer or antidiabetic drug development.
|
[
"Apoptosis involves mitochondrial steps such as the release of the apoptogenic factor cytochrome c which are effectively blocked by Bcl-2. Although Bcl-2 may have a direct action on the mitochondrial membrane, it also resides and functions on the endoplasmic reticulum (ER), and there is increasing evidence for a role of the ER in apoptosis regulation as well. Here we uncover a hitherto unrecognized, apoptotic crosstalk between the ER and mitochondria that is controlled by Bcl-2. After triggering massive ER dilation due to an inhibition of secretion, the drug brefeldin A (BFA) induces the release of cytochrome c from mitochondria in a caspase-8- and Bid-independent manner. This is followed by caspase-3 activation and DNA/nuclear fragmentation. Surprisingly, cytochrome c release by BFA is not only blocked by wild-type Bcl-2 but also by a Bcl-2 variant that is exclusively targeted to the ER (Bcl-2/cb5). Similar findings were obtained with tunicamycin, an agent interfering with N-linked glycosylations in the secretory system. Thus, apoptotic agents perturbing ER functions induce a novel crosstalk between the ER and mitochondria that can be interrupted by ER-based Bcl-2.",
"Iron-sulfur (Fe-S) proteins are key players in vital processes involving energy homeostasis and metabolism from the simplest to most complex organisms. We report a 1.5 A x-ray crystal structure of the first identified outer mitochondrial membrane Fe-S protein, mitoNEET. Two protomers intertwine to form a unique dimeric structure that constitutes a new fold to not only the approximately 650 reported Fe-S protein structures but also to all known proteins. We name this motif the NEET fold. The protomers form a two-domain structure: a beta-cap domain and a cluster-binding domain that coordinates two acid-labile 2Fe-2S clusters. Binding of pioglitazone, an insulin-sensitizing thiazolidinedione used in the treatment of type 2 diabetes, stabilizes the protein against 2Fe-2S cluster release. The biophysical properties of mitoNEET suggest that it may participate in a redox-sensitive signaling and/or in Fe-S cluster transfer.",
"MitoNEET is the only identified Fe-S protein localized to the outer mitochondrial membrane and a 1.5 Å resolution X-ray analysis has revealed a unique structure [Paddock et al. (2007), Proc. Natl Acad. Sci. USA, 104, 14342-14347]. The 2Fe-2S cluster is bound with a 3Cys-1His coordination which defines a new class of 2Fe-2S proteins. The hallmark feature of this class is the single noncysteine ligand His87, which when replaced by Cys decreases the redox potential (E(m)) by ∼300 mV and increases the stability of the cluster by around sixfold. Unexpectedly, the pH dependence of the lifetime of the 2Fe-2S cluster remains the same as in the wild-type protein. Here, the crystal structure of H87C mitoNEET was determined to 1.7 Å resolution (R factor = 18%) to investigate the structural basis of the changes in the properties of the 2Fe-2S cluster. In comparison to the wild type, structural changes are localized to the immediate vicinity of the cluster-binding region. Despite the increased stability, Cys87 displays two distinct conformations, with distances of 2.3 and 3.2 Å between the S(γ) and the outer Fe of the 2Fe-2S cluster. In addition, Lys55 exhibits multiple conformations in the H87C mutant protein. The structure and distinct characteristics of the H87C mutant provide a framework for further studies investigating the effects of mutation on the properties of the 2Fe-2S cluster in this new class of proteins.",
"Increasing evidence suggests that mitoNEET, a target of the type II diabetes drug pioglitazone, is a key regulator of energy metabolism in mitochondria. MitoNEET is anchored to the mitochondrial outer membrane via its N-terminal α helix domain and hosts a redox-active [2Fe-2S] cluster in its C-terminal cytosolic region. The mechanism by which mitoNEET regulates energy metabolism in mitochondria, however, is not fully understood. Previous studies have shown that mitoNEET specifically interacts with the reduced flavin mononucleotide (FMNH2) and that FMNH2 can quickly reduce the mitoNEET [2Fe-2S] clusters. Here we report that the reduced mitoNEET [2Fe-2S] clusters can be readily oxidized by oxygen. In the presence of FMN, NADH, and flavin reductase, which reduces FMN to FMNH2 using NADH as the electron donor, mitoNEET mediates oxidation of NADH with a concomitant reduction of oxygen. Ubiquinone-2, an analog of ubiquinone-10, can also oxidize the reduced mitoNEET [2Fe-2S] clusters under anaerobic or aerobic conditions. Compared with oxygen, ubiquinone-2 is more efficient in oxidizing the mitoNEET [2Fe-2S] clusters, suggesting that ubiquinone could be an intrinsic electron acceptor of the reduced mitoNEET [2Fe-2S] clusters in mitochondria. Pioglitazone or its analog NL-1 appears to inhibit the electron transfer activity of mitoNEET by forming a unique complex with mitoNEET and FMNH2 The results suggest that mitoNEET is a redox enzyme that may promote oxidation of NADH to facilitate enhanced glycolysis in the cytosol and that pioglitazone may regulate energy metabolism in mitochondria by inhibiting the electron transfer activity of mitoNEET.",
"The NEET proteins mitoNEET (mNT) and nutrient-deprivation autophagy factor-1 (NAF-1) are required for cancer cell proliferation and resistance to oxidative stress. NAF-1 and mNT are also implicated in a number of other human pathologies including diabetes, neurodegeneration and cardiovascular disease, as well as in development, differentiation and aging. Previous studies suggested that mNT and NAF-1 could function in the same pathway in mammalian cells, preventing the over-accumulation of iron and reactive oxygen species (ROS) in mitochondria. Nevertheless, it is unknown whether these two proteins directly interact in cells, and how they mediate their function. Here we demonstrate, using yeast two-hybrid, in vivo bimolecular fluorescence complementation (BiFC), direct coupling analysis (DCA), RNA-sequencing, ROS and iron imaging, and single and double shRNA lines with suppressed mNT, NAF-1 and mNT/NAF-1 expression, that mNT and NAF-1 directly interact in mammalian cells and could function in the same cellular pathway. We further show using an in vitro cluster transfer assay that mNT can transfer its clusters to NAF-1. Our study highlights the possibility that mNT and NAF-1 function as part of an iron-sulfur (2Fe-2S) cluster relay to maintain the levels of iron and Fe-S clusters under control in the mitochondria of mammalian cells, thereby preventing the activation of apoptosis and/or autophagy and supporting cellular proliferation.",
"The endoplasmic reticulum protein Miner1 is essential for health and longevity. Mis-splicing of CISD2, which codes for Miner1, is causative in Wolfram Syndrome 2 (WFS2) resulting in early onset optic atrophy, diabetes mellitus, deafness and decreased lifespan. In knock-out studies, disruption of CISD2 leads to accelerated aging, blindness and muscle atrophy. In this work, we characterized the soluble region of human Miner1 and solved its crystal structure to a resolution of 2.1 A (R-factor=17%). Although originally annotated as a zinc finger, we show that Miner1 is a homodimer harboring two redox-active 2Fe-2S clusters, indicating for the first time an association of a redox-active FeS protein with WFS2. Each 2Fe-2S cluster is bound by a rare Cys(3)-His motif within a 17 amino acid segment. Miner1 is the first functionally different protein that shares the NEET fold with its recently identified paralog mitoNEET, an outer mitochondrial membrane protein. We report the first measurement of the redox potentials (E(m)) of Miner1 and mitoNEET, showing that they are proton-coupled with E(m) approximately 0 mV at pH 7.5. Changes in the pH sensitivity of their cluster stabilities are attributed to significant differences in the electrostatic distribution and surfaces between the two proteins. The structural and biophysical results are discussed in relation to possible roles of Miner1 in cellular Fe-S management and redox reactions.",
"A challenge in molecular biology is to distinguish the key subset of residues that allow two-component signaling (TCS) proteins to recognize their correct signaling partner such that they can transiently bind and transfer signal, i.e., phosphoryl group. Detailed knowledge of this information would allow one to search sequence space for mutations that can be used to systematically tune the signal transmission between TCS partners as well as potentially encode a TCS protein to preferentially transfer signals to a nonpartner. Motivated by the notion that this detailed information is found in sequence data, we explore the sequence coevolution between signaling partners to better understand how mutations can positively or negatively alter their ability to transfer signal. Using direct coupling analysis for determining evolutionarily conserved protein-protein interactions, we apply a metric called the direct information score to quantify mutational changes in the interaction between TCS proteins and demonstrate that it accurately correlates with experimental mutagenesis studies probing the mutational change in measured in vitro phosphotransfer. Furthermore, by subtracting from our metric an appropriate null model corresponding to generic, conserved features in TCS signaling pairs, we can isolate the determinants that give rise to interaction specificity and recognition, which are variable among different TCS partners. Our methodology forms a potential framework for the rational design of TCS systems by allowing one to quickly search sequence space for mutations or even entirely new sequences that can increase or decrease our metric, as a proxy for increasing or decreasing phosphotransfer ability between TCS proteins."
] |
PIAS1 Regulates Epstein-Barr Virus Lylytic Replication
|
Epstein-Barr virus (EBV) in tumor cells is predominately in the latent phase, but the virus can undergo lytic reactivation in response to various stimuli. However, the cellular factors that control latency and lytic replication are poorly defined. In this study, we demonstrated that a cellular factor, PIAS1, restricts EBV lytic replication. PIAS1 depletion significantly facilitated EBV reactivation, while PIAS1 reconstitution had the opposite effect. Remarkably, we found that various lytic triggers promote caspase-dependent cleavage of PIAS1 to antagonize PIAS1-mediated restriction and that caspase inhibition suppresses EBV replication through blocking PIAS1 cleavage. We further demonstrated that a cleavage-resistant PIAS1 mutant suppresses EBV replication upon B cell receptor activation. Mechanistically, we demonstrated that PIAS1 acts as an inhibitor for transcription factors involved in lytic gene expression. Collectively, these results establish PIAS1 as a key regulator of EBV lytic replication and uncover a mechanism by which EBV exploits apoptotic caspases to antagonize PIAS1-mediated restriction.
|
[
"Interferon (IFN) activates the signal transducer and activator of transcription (STAT) pathway to regulate immune responses. The protein inhibitor of activated STAT (PIAS) family has been suggested to negatively regulate STAT signaling. To understand the physiological function of PIAS1, we generated Pias1(-/-) mice. Using PIAS1-deficient cells, we show that PIAS1 selectively regulates a subset of IFN-gamma- or IFN-beta-inducible genes by interfering with the recruitment of STAT1 to the gene promoter. The antiviral activity of IFN-gamma or IFN-beta was consistently enhanced by Pias1 disruption. Pias1(-/-) mice showed increased protection against pathogenic infection. Our data indicate that PIAS1 is a physiologically important negative regulator of STAT1 and suggest that PIAS1 is critical for the IFN-gamma- or IFN-beta-mediated innate immune responses.",
"Prostate cancer has emerged as the most frequent cancer amongst men in Europe, with incidence increasing rapidly over the past two decades. Incidence has been uniformly increasing in the 24 countries with comparable data available, although in a few countries with very high rates (Sweden, Finland and The Netherlands), incidence has begun to fall during the last 3-4 years. The highest prostate cancer mortality rates are in the Baltic region (Estonia, Latvia and Lithuania) and in Denmark, Norway and Sweden. Prostate cancer mortality has been decreasing in 13 of the 37 European countries considered - predominantly in higher-resource countries within each region - beginning in England and Wales (1992) and more recently in the Czech Republic (2004). There was considerable variability in the magnitude of the annual declines, varying from approximately 1% in Scotland (from 1994) to over 4% for the more recent declines in Hungary, France and the Czech Republic. There appears little relation between the extent of the increases in incidence (in the late 1990s) and the recent mortality declines. It remains unclear to what extent the increasing trends in incidence indicate true risk and how much is due to detection of latent disease. The decreasing mortality after 1990 may be attributable to improvements in treatment and to an effect of prostate specific antigen (PSA) testing. The increase in mortality observed in the Baltic region and in several Central and Eastern European countries appear to reflect a real increase in risk and requires further monitoring.",
"The large tegument proteins of herpesviruses contain N-terminal cysteine proteases with potent ubiquitin and NEDD8-specific deconjugase activities, but the function of the enzymes during virus replication remains largely unknown. Using as model BPLF1, the homologue encoded by Epstein-Barr virus (EBV), we found that induction of the productive virus cycle does not affect the total level of ubiquitin-conjugation but is accompanied by a BPLF1-dependent decrease of NEDD8-adducts and accumulation of free NEDD8. Expression of BPLF1 promotes cullin degradation and the stabilization of cullin-RING ligases (CRLs) substrates in the nucleus, while cytoplasmic CRLs and their substrates are not affected. The inactivation of nuclear CRLs is reversed by the N-terminus of CAND1, which inhibits the binding of BPLF1 to cullins and prevents efficient viral DNA replication. Targeting of the deneddylase activity to the nucleus is dependent on processing of the catalytic N-terminus by caspase-1. Inhibition of caspase-1 severely impairs viral DNA synthesis and the release of infectious virus, pointing a previously unrecognized role of the cellular response to danger signals triggered by EBV reactivation in promoting virus replication.",
"Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is a human gammaherpesvirus recognized as the principal causative agent of KS and primary effusion lymphoma (PEL). KSHV establishes persistent latent infection in B lymphocytes where viral gene expression is restricted, in part, by a cohesin-dependent chromosome conformation. Here, we show that endoplasmic reticulum (ER) stress induces a rapid, caspase-dependent cleavage of cohesin subunit RAD21. ER stress-induced cleavage of RAD21 correlated with a rapid and strong viral lytic transcriptional activation. This effect was observed in several KSHV positive PEL cells, but not in other B-cells or non-B-cell models of KSHV latency. The cleaved-RAD21 does not dissociate from viral genomes, nor disassemble from other components of the cohesin complex. However, RAD21 cleavage correlated with the disruption of the latency genome conformation as revealed by chromosome conformation capture (3C). Ectopic expression of C-terminal RAD21 cleaved form could partially induce KSHV lytic genes transcription in BCBLI cells, suggesting that ER-stress induced RAD21 cleavage was sufficient to induce KSHV reactivation from latency in PEL cells. Taken together our results reveal a novel aspect for control and maintenance of KSHV genome latency conformation mediated by stress-induced RAD21 cleavage. Our studies also suggest that RAD21 cleavage may be a general regulatory mechanism for rapid alteration of cellular chromosome conformation and cohesin-dependent transcription regulation.",
"Testing for correlations between different sets of genomic features is a fundamental task in genomics research. However, searching for overlaps between features with existing web-based methods is complicated by the massive datasets that are routinely produced with current sequencing technologies. Fast and flexible tools are therefore required to ask complex questions of these data in an efficient manner. This article introduces a new software suite for the comparison, manipulation and annotation of genomic features in Browser Extensible Data (BED) and General Feature Format (GFF) format. BEDTools also supports the comparison of sequence alignments in BAM format to both BED and GFF features. The tools are extremely efficient and allow the user to compare large datasets (e.g. next-generation sequencing data) with both public and custom genome annotation tracks. BEDTools can be combined with one another as well as with standard UNIX commands, thus facilitating routine genomics tasks as well as pipelines that can quickly answer intricate questions of large genomic datasets. BEDTools was written in C++. Source code and a comprehensive user manual are freely available at http://code.google.com/p/bedtools aaronquinlan@gmail.com; imh4y@virginia.edu Supplementary data are available at Bioinformatics online.",
"Androgens, acting through the androgen receptor (AR), are responsible for the development of the male phenotype during embryogenesis, the achievement of sexual maturation at puberty, and the maintenance of male reproductive function and behavior in adulthood. In addition, androgens affect a wide variety of nonreproductive tissues. Moreover, aberrant androgen action plays a critical role in multiple pathologies, including prostate cancer and androgen insensitivity syndromes. The formation of a productive AR transcriptional complex requires the functional and structural interaction of the AR with its coregulators. In the last decade, an overwhelming and ever increasing number of proteins have been proposed to possess AR coactivating or corepressing characteristics. Intriguingly, a vast diversity of functions has been ascribed to these proteins, indicating that a multitude of cellular functions and signals converge on the AR to regulate its function. The current review aims to provide an overview of the AR coregulator proteins identified to date and to propose a classification of these AR coregulator proteins according to the function(s) ascribed to them. Taken together, this approach will increase our understanding of the cellular pathways that converge on the AR to ensure an appropriate transcriptional response to androgens.",
"Androgen receptor (AR) is a ligand-activated transcription factor that plays a central role in the development and growth of prostate carcinoma. PIAS1 is an AR- and SUMO-interacting protein and a putative transcriptional coregulator overexpressed in prostate cancer. To study the importance of PIAS1 for the androgen-regulated transcriptome of VCaP prostate cancer cells, we silenced its expression by RNAi. Transcriptome analyses revealed that a subset of the AR-regulated genes is significantly influenced, either activated or repressed, by PIAS1 depletion. Interestingly, PIAS1 depletion also exposed a new set of genes to androgen regulation, suggesting that PIAS1 can mask distinct genomic loci from AR access. In keeping with gene expression data, silencing of PIAS1 attenuated VCaP cell proliferation. ChIP-seq analyses showed that PIAS1 interacts with AR at chromatin sites harboring also SUMO2/3 and surrounded by H3K4me2; androgen exposure increased the number of PIAS1-occupying sites, resulting in nearly complete overlap with AR chromatin binding events. PIAS1 interacted also with the pioneer factor FOXA1. Of note, PIAS1 depletion affected AR chromatin occupancy at binding sites enriched for HOXD13 and GATA motifs. Taken together, PIAS1 is a genuine chromatin-bound AR coregulator that functions in a target gene selective fashion to regulate prostate cancer cell growth."
] |
Synchronous clear cell and papillary renal cell carcinoma in the same kidney.
|
Clear cell renal cell carcinoma and papillary renal cell carcinoma are the most common types of renal tumors. However, coexistence of both tumors in the same kidney is a rare condition. We report a 56-year old male who was found to have ipsilateral synchronous clear cell and papillary renal cell carcinoma in the left kidney. Review of related literature is provided to estimate the prevalence of similar cases.
|
[
"Multifocal renal cell carcinoma (RCC) has been reported in 5-25% of cases worldwide. Although management of patients with multifocal RCC has not been clearly defined, presence of multifocal renal masses in one kidney and a normal contralateral kidney has often been considered a reason for performing radical nephrectomy. This study reviews the world literature to provide an accurate estimate of the prevalence of multifocal RCC and evaluates the oncologic outcomes of multifocal RCC after exclusion of patients with known hereditary and familial renal syndromes. A PubMed search of the literature was performed for articles in the English language using the following terms for the query: \"multifocal RCC,\" \"multifocality and RCC,\" \"multicentric RCC,\" or \"bilateral RCC.\" The references of the published articles were also reviewed for additional publications. Articles that did not specifically exclude patients with familial RCC or known hereditary RCC syndromes were excluded for estimation of multifocality prevalence and oncologic outcomes. After applying our exclusion criteria, nine articles were selected and form the basis of the current analysis. Weighted averages were used to calculate the prevalence of multifocality. Multifocal RCC was found in 6.8% of cases (373 of 5433 patients). Ipsilateral multifocality was found in 6.8% of cases. Bilateral multifocality was found in 11.7% of cases. Of all cases reported in this study, only 10% underwent partial nephrectomy. The rest of the study cohort underwent radical nephrectomy. The review of the literature showed that the use of nephron-sparing techniques in patients with multifocal disease did not compromise oncologic outcomes, despite the need for reoperation in certain cases. In conclusion, multifocal RCC remains a prevalent entity. Most clinicians still prefer to perform radical nephrectomies in these patients despite proven equivalent oncologic outcomes compared to nephron-sparing techniques. Urologists should be aware of these data when proposing treatment options to patients with multifocal RCC.",
"Pathological evaluation of renal tumors treated with ablative and observational therapy is often limited and renal tumor size may be the only prognostic index available. We established long-term survival in patients following partial nephrectomy according to tumor size. A retrospective review of our nephrectomy registry was performed to identify patients who underwent partial nephrectomy for localized (NX/N0/cM0) solid renal tumors 7 cm or less at our institution between 1970 and 2004. Overall, cancer specific, distant metastasis-free and local recurrence-free survival was estimated using the Kaplan-Meier method and stratified according to tumor size in all tumors treated and in patients with pathologically confirmed renal cell carcinoma. We identified 798 patients who underwent partial nephrectomy for a 7 cm or less renal tumor. Median patient age was 63.5 years and median tumor size was 3.0 cm. Renal cell carcinoma was present in 637 tumors (80%). Overall, cancer specific, metastasis-free and local recurrence-free survival significantly decreased with each 1 cm increase in size in all tumors treated and in those with pathologically confirmed renal cell carcinoma (each p <0.05). Partial nephrectomy is associated with durable cancer control in patients with renal tumors 7 cm or less, of which most represent renal cell carcinoma. Tumor size represents a valuable prognostic index in the absence of pathological evaluation of the entire tumor specimen. These results may be used for comparison against outcomes following ablative and observational therapy, for which tumor size is the only prognostic index available.",
"To evaluate the pathological concordance rate of multiple synchronous renal masses (MSRM) presumed to be sporadic and to analyze predictive factors of concordance. We identified from our institutional database patients with sporadic MSRM treated at our center between January 2000 and December 2015. All tumors were reviewed by a dedicated uropathologist. Pathological concordance rate was analyzed regarding clinical characteristics and preoperative imaging. We included 112 patients: 50 had unilateral synchronous renal masses and 62 bilateral synchronous renal masses. A total of 291 tumors were analyzed, with an average of 2.6 tumors per patient. Overall, the malignant concordance rate was 91.6%, the pathological concordance rate was 67.3% and the grade concordance rate was 62.5%. In univariate analysis, predictive factors of histological concordance were bilateral synchronous renal masses (odds ratio [OR] = 3.39; 95% CI: 1.06-10.8; P = 0.04), age<60 years (OR = 3.04; 95% CI: 1.2-7.7; P = 0.02) and ≥3 lesions (OR = 2.41; 95% CI: 1.03-5.68; P = 0.04). In multivariate analysis, age<60 remained significantly associated with histological concordance (OR = 3.84; 95% CI: 1.24-11.9; P = 0.02). The histological concordance rate of MSRM is low. Age at diagnosis <60 years, bilateral lesions and ≥3 tumors are predictive factors of histological concordance, but the pathological diagnosis remains difficult to predict. This heterogeneity is important to take into account, particularly when choosing the treatment upon the renal biopsy results from a single lesion.",
"We examined outcomes in patients with recurrent or de novo renal lesions treated with repeat partial nephrectomy on a solitary kidney. We reviewed the records of patients who underwent nephron sparing surgery at the National Cancer Institute from 1989 to 2008. Patients were included in analysis if they underwent repeat partial nephrectomy on a solitary kidney. Perioperative, functional and oncological outcomes were assessed. Functional outcomes were evaluated using the Modification of Diet in Renal Disease equation for the estimated glomerular filtration rate. Oncological efficacy was examined by the need for subsequent repeat renal surgery and the development of metastatic disease. A total of 25 patients were included in the analysis. A median of 4 tumors were resected. Median estimated blood loss was 2,400 ml and median operative time was 8.5 hours. Perioperative complications occurred in 52% of patients, including 1 death and the loss of 3 renal units. There was a decrease in the estimated glomerular filtration rate at followup visit 1 within 3 months after surgery but at 1-year followup the difference was not significant (p <0.01 and 0.12, respectively). Surgical intervention was recommended in 8 patients (38%) for recurrent or de novo tumors at a median of 36 months. The average metastasis-free survival rate in the cohort was 95% at 57 months (median 50, range 3 to 196). Repeat partial nephrectomy in patients with solitary kidney is a high risk alternative. The complication rate is high and there is a modest decrease in renal function but most patients remain free of dialysis with acceptable oncological outcomes at intermediate followup."
] |
Molecular dynamics simulation of trypsin-ligand binding under AMBER force field and polarized protein-specific charge force field combined the highly efficient interaction entropy method
|
Molecular dynamics (MD) simulation in the explicit water is performed to study the interaction mechanism of trypsin-ligand binding under the AMBER force field and polarized protein-specific charge (PPC) force field combined the new developed highly efficient interaction entropy (IE) method for calculation of entropy change. And the detailed analysis and comparison of the results of MD simulation for two trypsin-ligand systems show that the root-mean-square deviation (RMSD) of backbone atoms, B-factor, intra-protein and protein-ligand hydrogen bonds are more stable under PPC force field than AMBER force field. Our results demonstrate that the IE method is superior than the traditional normal mode (Nmode) method in the calculation of entropy change and the calculated binding free energy under the PPC force field combined with the IE method is more close to the experimental value than other three combinations (AMBER-Nmode, AMBER-IE and PPC-Nmode). And three critical hydrogen bonds between trypsin and ligand are broken under AMBER force field. However, they are well preserved under PPC force field. Detailed binding interactions of ligands with trypsin are further analyzed. The present work demonstrates that the polarized force field combined the highly efficient IE method is critical in MD simulation and free energy calculation.
|
[
"We present results of developing a methodology suitable for producing molecular mechanics force fields with explicit treatment of electrostatic polarization for proteins and other molecular system of biological interest. The technique allows simulation of realistic-size systems. Employing high-level ab initio data as a target for fitting allows us to avoid the problem of the lack of detailed experimental data. Using the fast and reliable quantum mechanical methods supplies robust fitting data for the resulting parameter sets. As a result, gas-phase many-body effects for dipeptides are captured within the average RMSD of 0.22 kcal/mol from their ab initio values, and conformational energies for the di- and tetrapeptides are reproduced within the average RMSD of 0.43 kcal/mol from their quantum mechanical counterparts. The latter is achieved in part because of application of a novel torsional fitting technique recently developed in our group, which has already been used to greatly improve accuracy of the peptide conformational equilibrium prediction with the OPLS-AA force field.1 Finally, we have employed the newly developed first-generation model in computing gas-phase conformations of real proteins, as well as in molecular dynamics studies of the systems. The results show that, although the overall accuracy is no better than what can be achieved with a fixed-charges model, the methodology produces robust results, permits reasonably low computational cost, and avoids other computational problems typical for polarizable force fields. It can be considered as a solid basis for building a more accurate and complete second-generation model.",
"A new scheme for direct linear-scaling quantum mechanical calculation of electron density of protein systems is developed. The new scheme gives much improved accuracy of electron density for proteins than the original MFCC (molecular fractionation with conjugate caps) approach in efficient linear-scaling calculation for protein systems. In this new approach, the error associated with each cut in the MFCC approach is estimated by computing the two neighboring amino acids in both cut and uncut calculations and is corrected. Numerical tests are performed on six oligopeptide taken from PDB (protein data bank), and the results show that the new scheme is efficient and accurate.",
"Ab initio quantum mechanical calculation of protein in solution is carried out to generate polarized protein-specific charge(s) (PPC) for molecular dynamics (MD) stimulation of protein. The quantum calculation of protein is made possible by developing a fragment-based quantum chemistry approach in combination with the implicit continuum solvent model. The computed electron density of protein is utilized to derive PPCs that represent the polarized electrostatic state of protein near the native structure. These PPCs are atom-centered like those in the standard force fields and are thus computationally attractive for molecular dynamics simulation of protein. Extensive MD simulations have been carried out to investigate the effect of electronic polarization on the structure and dynamics of thioredoxin. Our study shows that the dynamics of thioredoxin is stabilized by electronic polarization through explicit comparison between MD results using PPC and AMBER charges. In particular, MD free-energy calculation using PPCs accurately reproduced the experimental value of pK(a) shift for ionizable residue Asp(26) buried inside thioredoxin, whereas previous calculations using standard force fields overestimated pK(a) shift by twice as much. Accurate prediction of pK(a) shifts by rigorous MD free energy simulation for ionizable residues buried inside protein has been a significant challenge in computational biology for decades. This study presented strong evidence that electronic polarization of protein plays an important role in protein dynamics.",
"Electrostatic energies provide what is perhaps the most effective tool for structure-function correlation of biological molecules. This review considers the current state of simulations of electrostatic energies in macromolecules as well as the early developments of this field. We focus on the relationship between microscopic and macroscopic models, considering the convergence problems of the microscopic models and the fact that the dielectric 'constants' in semimacroscopic models depend on the definition and the specific treatment. The advances and the challenges in the field are illustrated considering a wide range of functional properties including pK(a)'s, redox potentials, ion and proton channels, enzyme catalysis, ligand binding and protein stability. We conclude by pointing out that, despite the current problems and the significant misunderstandings in the field, there is an overall progress that should lead eventually to quantitative descriptions of electrostatic effects in proteins and thus to quantitative descriptions of the function of proteins.",
"An overview is provided on the computation of free energy changes in solution using perturbation theory, overlap sampling, and related approximate methods. As a specific application, extensive results are provided for free energies of hydration of substituted benzenes using the OPLS-AA force field in explicit TIP4P water. For a similar amount of computer time, the double-wide sampling and overlap sampling methods yield very similar results in the free-energy perturbation calculations. With standard protocols, the average statistical uncertainty in computed differences in free energies of hydration is 0.1 - 0.2 kcal/mol. Application of the power-series expansion in the Peierls equation was also tested. Use of the first-order term is generally reliable, while inclusion of the slowly-convergent, second-order fluctuation term causes deterioration in the results for strongly hydrogen-bonded solutes.",
"Standard force fields used in biomolecular computing describe electrostatic interactions in terms of fixed, usually atom-centered, charges. Real physical systems, however, polarize substantially when placed in a high-dielectric medium such as water--or even when a strongly charged system approaches a neutral body in the gas phase. Such polarization strongly affects the geometry and energetics of molecular recognition. First introduced more than 20 years ago, polarizable force fields seek to account for appropriate variations in charge distribution with dielectric environment. Over the past five years, an accelerated pace of development of such force fields has taken place on systems ranging from liquid water to metalloenzymes. Noteworthy progress has been made in better understanding the capabilities and limitations of polarizable models for water and in the formulation and utilization of complete specifically parameterized polarizable force fields for peptides and proteins.",
"Efficient and reliable calculation of protein-ligand binding free energy is a grand challenge in computational biology and is of critical importance in drug design and many other molecular recognition problems. The main challenge lies in the calculation of entropic contribution to protein-ligand binding or interaction systems. In this report, we present a new interaction entropy method which is theoretically rigorous, computationally efficient, and numerically reliable for calculating entropic contribution to free energy in protein-ligand binding and other interaction processes. Drastically different from the widely employed but extremely expensive normal mode method for calculating entropy change in protein-ligand binding, the new method calculates the entropic component (interaction entropy or -TΔS) of the binding free energy directly from molecular dynamics simulation without any extra computational cost. Extensive study of over a dozen randomly selected protein-ligand binding systems demonstrated that this interaction entropy method is both computationally efficient and numerically reliable and is vastly superior to the standard normal mode approach. This interaction entropy paradigm introduces a novel and intuitive conceptual understanding of the entropic effect in protein-ligand binding and other general interaction systems as well as a practical method for highly efficient calculation of this effect.",
"A new method that incorporates the conductorlike polarizable continuum model (CPCM) with the recently developed molecular fractionation with conjugate caps (MFCC) approach is developed for ab initio calculation of electrostatic solvation energy of protein. The application of the MFCC method makes it practical to apply CPCM to calculate electrostatic solvation energy of protein or other macromolecules in solution. In this MFCC-CPCM method, calculation of protein solvation is divided into calculations of individual solvation energies of fragments (residues) embedded in a common cavity defined with respect to the entire protein. Besides computational efficiency, the current approach also provides additional information about contribution to protein solvation from specific fragments. Numerical studies are carried out to calculate solvation energies for a variety of peptides including alpha helices and beta sheets. Excellent agreement between the MFCC-CPCM result and those from the standard full system CPCM calculation is obtained. Finally, the MFCC-CPCM calculation is applied to several real proteins and the results are compared to classical molecular mechanics Poisson-Boltzmann (MM/PB) and quantum Divid-and-Conque Poisson-Boltzmann (D&C-PB) calculations. Large wave function distortion energy (solute polarization energy) is obtained from the quantum calculation which is missing in the classical calculation. The present study demonstrates that the MFCC-CPCM method is readily applicable to studying solvation of proteins.",
"The accuracy of biological simulations depends, in large part, on the treatment of electrostatics. Due to the availability of accurate experimental values, calculation of pKa provides stringent evaluation of computational methods. The generalized solvent boundary potential (GSBP) and Ewald summation electrostatic treatments were recently implemented for combined quantum mechanical and molecular mechanics (QM/MM) simulations by our group. These approaches were tested by calculating pKa shifts due to differences in electronic structure and electrostatic environment; the shifts were determined for a series of small molecules in solution, using various electrostatic treatments, and two residues (His 31, Lys 102) in the M102K T4-lysozyme mutant with large pKa shifts, using the GSBP approach. The calculations utilized a free energy perturbation scheme with the QM/MM potential function involving the self-consistent charge density functional tight binding (SCC-DFTB) and CHARMM as the QM and MM methods, respectively. The study of small molecules demonstrated that inconsistent electrostatic models produced results that were difficult to correct in a robust manner; by contrast, extended electrostatics, GSBP, and Ewald simulations produced consistent results once a bulk solvation contribution was carefully chosen. In addition to the electrostatic treatment, the pKa shifts were also sensitive to the level of the QM method and the scheme of treating QM/MM Coulombic interactions; however, simple perturbative corrections based on SCC-DFTB/CHARMM trajectories and higher level single point energy calculations were found to give satisfactory results. Combining all factors gave a root-mean-square difference of 0.7 pKa units for the relative pKa values of the small molecules compared to experiment. For the residues in the lysozyme, an accurate pKa shift was obtained for His 31 with multiple nanosecond simulations. For Lys 102, however, the pKa shift was estimated to be too large, even after more than 10 nanosecond simulations for each lambda window; the difficulty was due to the significant, but slow, reorganization of the protein and water structure when Lys 102 was protonated. The simulations support that Lys 102 is deprotonated in the X-ray structure and the protein is highly destabilized when this residue is protonated.",
"This article describes a number of algorithms that are designed to improve both the efficiency and accuracy of finite difference solutions to the Poisson-Boltzmann equation (the FDPB method) and to extend its range of application. The algorithms are incorporated in the DelPhi program. The first algorithm involves an efficient and accurate semianalytical method to map the molecular surface of a molecule onto a three-dimensional lattice. This method constitutes a significant improvement over existing methods in terms of its combination of speed and accuracy. The DelPhi program has also been expanded to allow the definition of geometrical objects such as spheres, cylinders, cones, and parallelepipeds, which can be used to describe a system that may also include a standard atomic level depiction of molecules. Each object can have a different dielectric constant and a different surface or volume charge distribution. The improved definition of the surface leads to increased precision in the numerical solutions of the PB equation that are obtained. A further improvement in the precision of solvation energy calculations is obtained from a procedure that calculates induced surface charges from the FDPB solutions and then uses these charges in the calculation of reaction field energies. The program allows for finite difference grids of large dimension; currently a maximum of 571(3) can be used on molecules containing several thousand atoms and charges. As described elsewhere, DelPhi can also treat mixed salt systems containing mono- and divalent ions and provide electrostatic free energies as defined by the nonlinear PB equation."
] |
Serum substance P as a simple biomarker for rheumatoid arthritis disease activity
|
The aim of the is study is to examine the role of serum substance P (SP) levels as a simple biomarker for rheumatoid arthritis (RA) disease activity, its correlation with other markers of disease activity, and with selected clinical parameters. The study comprised 90 RA patients and 24 healthy controls. RA activity was assessed by means of the disease activity 28-C-reactive protein (DAS28-CRP) index and ultrasound power Doppler (USPD) by the German ultrasound score based on seven joints. SP serum values were obtained by means of an ELISA commercial kit. Statistics were achieved by the Student's t test and Spearman correlation analysis with Bonferroni correction. As a group, RA patients had significantly increased levels of SP compared with healthy controls (p < 0.0001). SP levels correlated with DAS28-CRP (r = 0.5050, p < 0.0001), number of tender joints (NTJ, r = 0.4668, p < 0.0001), number of swollen joints (NSJ, r = 0.4439, p < 0.0001), visual analogue scale (VAS, r = 0.5131, p < 0.0001). However, SP did not correlate with CRP levels (r = 0.0468, p = 0.6613), nor with the USPD (r = 0.1740, p = 0.1009). Elevated serum SP is a common feature of RA patients, which also appears to correlate with clinical measurements of disease activity and with subjective clinical data (NTJ and VAS). Thus, although SP is higher in RA patients with high disease activity, it also detects subtle RA disease activity even in patients in apparent remission, which suggests its usefulness for therapeutic decisions.
|
[
"The tachykinins substance P (SP) and neurokinin A, released by the C-type primary afferent fibre terminals of the small dorsal root ganglion (DRG) neurons, play important roles in spinal nociception. By means of non-radioactive in situ hybridization and whole-cell recording, we showed that the small rat DRG neurons also express the NK-1 tachykinin receptor. In situ hybridization demonstrated that the positive neurons in rat DRG sections were mainly small cells (85.9%) with diameters less than 25 microm. The remaining positive neurons (14.1%) were cells with medium diameters between 26 and 40 microm. No positive large neurons (diameters > 40 microm) were observed. Expression in small DRG neurons (diameter < 21 microm) was confirmed by in situ hybridization of isolated cells, which were demonstrated to express NK-1 receptor mRNA at a very high frequency (> 90% of small DRG neurons) and therefore were subjected to whole-cell recording. In 57 of 61 cells recorded, SP or the selective NK-1 receptor agonist [Sar9, Met(O2)11]SP (Sar-SP, 1 or 2 microM) produced a delayed vibrating inward current (50-300 nA) with a long duration of 0.5-2 h. These currents were blocked by co-application of the NK-1 receptor antagonist L-668, 169 (1 microM), but were not affected by the NK-2 antagonist L-659, 877 (2 microM). Both current-clamp recording and cell-attached single-channel recording demonstrated that the long-lasting response was due to the opening of a channel with an inward current. Employment of non-Ca2+ and Ca2+ + choline solutions revealed that this channel might be a Ca2+-permeable, non-selective cation channel. The prolonged NK-1 tachykinin response exhibited extreme desensitization. This work suggests that presynaptic NK-1 autoreceptors may be present on the primary afferent terminals in the spinal cord, where they could contribute to the chronic pain and hyperalgesia.",
"Noxious stimuli activate small to medium-sized dorsal root ganglion (DRG) neurons. Intense noxious stimuli result in the release of substance P (SP) from the central terminals of these neurons. It binds to the neurokinin type 1 receptor (NK1r) and sensitises the dorsal horn neurons. SP is also released from the peripheral terminals leading to neurogenic inflammation. However, their individual contribution at spinal and peripheral levels to postincisional nociception has not been delineated as yet. Sprague-Dawley rats were administered different doses (3-100 μg) of an NK1r antagonist (L760735) by intrathecal (i.t.) route before hind paw incision. On the basis of its antinociceptive effect on guarding behaviour, the 30 μg dose was selected for further study. In different sets of animals, this was administered i.t. (postemptive) and intrawound (i.w.). Finally, in another group, drug (30 μg) was administered through both i.t and i.w. routes. The antinociceptive effect was assessed and compared. Expression of SP was examined in the spinal cord. Intrawound concentration of SP and inflammatory mediators was also evaluated. Postemptive i.t. administration significantly attenuated guarding and allodynia. Guarding was alone decreased after i.w. drug treatment. Combined drug administration further attenuated all nociceptive parameters, more so after postemptive treatment. Expression of SP in the spinal cord decreased post incision but increased in the paw tissue. Inflammatory mediators like the nerve growth factor also increased after incision. In conclusion, SP acting through the NK1r appears to be an important mediator of nociception, more so at the spinal level. These findings could have clinical relevance.",
"Substance P and its receptor (NK1) are thought to play an important role in pain and hyperalgesia. Here we have further examined this role by comparing the behavioural responses to intradermal capsaicin of mutant mice with a disruption of the NK1 receptor (NK1 KO) and wild-type (WT) mice. We have also evaluated the contribution of peripheral NK1 receptors to capsaicin-evoked behaviour by selective blockade of peripheral NK1 receptors in WT mice using a non-brain penetrant NK1 receptor antagonist. Injection of 6 microg capsaicin into the heel evoked paw licking with the same latency in WT and KO mice, but a significantly longer duration in WT mice. A higher dose (30 microg) evoked a similar duration of licking in both groups. There were no differences in mechanical sensitivity tested with von Frey hairs between WT and KO mice before capsaicin. Both capsaicin doses resulted in pronounced increases in responses to von Frey hairs (hyperalgesia) and novel responses to cotton wisps (allodynia) applied to the digits of the injected paw in WT mice, but no significant changes from baseline in KO mice. Selective blockade of peripheral NK1 receptors in WT mice resulted in a complete inhibition of capsaicin-evoked plasma extravasation, but the mechanical hyperalgesia induced by 30 microg capsaicin intraplantar was still significantly greater than that seen in KO mice. We conclude that the response to intradermal capsaicin is still present but abbreviated in mice lacking NK1 receptors, such that secondary hyperalgesia is not observed even after a high dose. Further, the lack of secondary hyperalgesia in NK1 KO mice is largely due to the loss of central rather than peripheral NK1 receptors. The phenotype of the NK1 KO mice is consistent with a loss of function of mechanically-insensitive nociceptors, and thus we propose that substance P may be expressed by this group of primary sensory neurones and required for their function.",
"Substance P (SP) signaling facilitates nociceptive sensitization in various inflammatory and chronic pain models and we postulated that SP signaling might also contribute to the development of post-incisional hyperalgesia. These studies used mice with a deletion of the pre-protachykinin A gene (ppt-A(-/-)) which codes for SP to determine the role of SP signaling in post-incisional pain and in the increased cytokine and nerve growth factor (NGF) expression observed in the incised skin. SP deficient ppt-A(-/-) mice displayed reduced mechanical allodynia and heat hyperalgesia compared to the wild-type (wt) mice at all post-incision time points, despite similar baseline values (p<0.001). Furthermore, the NK-1 receptor antagonist LY303870 attenuated mechanical allodynia produced by incision in the wt mice (p<0.001). Incision also up-regulated IL-6, TNF-alpha and KC levels but not IL-1beta after 2h in the wt mice skin. However, ppt-A(-/-) mice had more skin NGF levels 2h post-incision. Subcutaneous hind paw SP injection produced acute and transient elevations of IL-1beta, IL-6, and KC but modest elevations in TNF-alpha levels in the wt mice. Systemic LY303870 reversed the SP-induced elevations of these cytokines. Hind paw injection of IL-6 and NGF dose dependently produced less mechanical allodynia in the ppt-A(-/-) compared to wt mice. Additionally, SP produced mechanical allodynia in a dose-dependent fashion in wt mice. Therefore, SP supports nociceptive sensitization after hind paw incision and potentially participates directly in modulating the intensity of inflammatory response in peri-incisional tissue.",
"To explore whether changes in a composite (power Doppler/greyscale ultrasound (PDUS)) synovitis score, developed by the OMERACT-EULAR-Ultrasound Task Force, predict disease activity outcomes in rheumatoid arthritis (RA). Patients with RA who were methotrexate inadequate responders starting abatacept were evaluated. Individual joint PDUS scores were combined in the Global OMERACT-EULAR Synovitis Score (GLOESS) for metacarpophalangeal joints (MCPs) 2-5, all joints (22 paired) and a reduced (9 paired) joint set. The predictive value of changes in GLOESS at week 1-16 evaluations for clinical status and response (Disease Activity Score (DAS)28 (C reactive protein, CRP) <2.6; DAS28(CRP) ≤3.2; DAS28(CRP) ≥1.2 improvement) up to week 24, and correlations between DAS28 and GLOESS were assessed. Eighty-nine patients completed the 24-week treatment period. Changes in GLOESS (MCPs 2-5) from weeks 1 to 16 were unable to predict DAS28 outcomes up to week 24. However, significant improvements in GLOESS (MCPs 2-5) were observed at week 12 in patients with DAS28 ≥1.2 improvement at week 24 versus those who did not achieve that clinical response. In patients achieving DAS28 ≥1.2 improvement or DAS28 ≤3.2 at week 24, changes in GLOESS (22 and 9 paired joint sets) were greater in patients who already achieved DAS28 ≥1.2 at week 12 than in those who did not. No significant correlations were found between changes in DAS28 and GLOESS definitions at any time point. PDUS was not correlated with clinical status or response as measured by DAS28-derived criteria, and PDUS changes were not predictive of clinical outcome. The discrepancies require further exploration. NCT00767325; Results.",
"Substance P is a peptide mainly secreted by neurons and is involved in many biological processes, including nociception and inflammation. Animal models have provided insights into the biology of this peptide and offered compelling evidence for the importance of substance P in cell-to-cell communication by either paracrine or endocrine signaling. Substance P mediates interactions between neurons and immune cells, with nerve-derived substance P modulating immune cell proliferation rates and cytokine production. Intriguingly, some immune cells have also been found to secrete substance P, which hints at an integral role of substance P in the immune response. These communications play important functional roles in immunity including mobilization, proliferation and modulation of the activity of immune cells. This review summarizes current knowledge of substance P and its receptors, as well as its physiological and pathological roles. We focus on recent developments in the immunobiology of substance P and discuss the clinical implications of its ability to modulate the immune response.",
"Postoperative incident pain is not easily treated with opioids. Mechanical hyperalgesia induced by skin incision in rats is one of the animal models of postoperative incident pain. It is thought that mechanical hyperalgesia is maintained by the sensitization of spinal dorsal horn neurons. The NK-1 receptor, the opioid receptor like1 (ORL1) receptor, and cyclooxygenase (COX)-2 reportedly are involved in the development of spinal sensitization. In this study, we clarified the role of the NK-1 receptor, the ORL1 receptor, and COX-2 in the maintenance of mechanical hyperalgesia induced by skin incision. A 1-cm longitudinal incision was made through skin and fascia of the plantar aspect of the right foot in the rat. Four hours after the skin incision, significant mechanical hyperalgesia developed. An ORL1 receptor agonist (nociceptin), NK-1 receptor antagonists (CP-96,345 and FK888), and COX-2 inhibitors (NS398 and JTE522) were administered intrathecally 4 h after the skin incision. An ORL1 receptor agonist and NK-1 receptor antagonists, but not COX-2 inhibitors, significantly attenuated the level of mechanical hyperalgesia induced by the skin incision. These findings suggest that the spinal ORL1 receptor and the NK-1 receptor play an important role in maintaining the mechanical hyperalgesia induced by skin incision. Intrathecal injection of an NK-1 receptor antagonist and an ORL1 receptor agonist may be effective for the treatment of postoperative incident pain."
] |
Capsaicin and muco-cutaneous cancers
|
Capsaicin is the main pungent in chili peppers, one of the most commonly used spices in the world; its analgesic and anti-inflammatory properties have been proven in various cultures for centuries. It is a lipophilic substance belonging to the class of vanilloids and an agonist of the transient receptor potential vanilloid 1 receptor. Taking into consideration the complex neuro-immune impact of capsaicin and the potential link between inflammation and carcinogenesis, the effect of capsaicin on muco-cutaneous cancer has aroused a growing interest. The aim of this review is to look over the most recent data regarding the connection between capsaicin and muco-cutaneous cancers, with emphasis on melanoma and muco-cutaneous squamous cell carcinoma.
|
[
"To assess the efficacy, tolerability, and safety of NGX-4010, a high-concentration capsaicin dermal patch (capsaicin 640 microg/cm(2), 8%) in patients with postherpetic neuralgia (PHN). Patients were randomized to receive NGX-4010 or control patch in a 4-week, double-blind study. This was followed by an open-label extension phase (up to 48 weeks total) where patients could receive up to three additional treatments no sooner than 12 weeks after initial treatment. The primary efficacy variable was mean change from baseline in mean morning and evening numerical pain rating scale (NPRS) scores. During days 8-28 after the double-blind treatment, NGX-4010 patients had a mean change in NPRS scores from baseline of -32.7% compared with -4.4% for control patients (P = 0.003). Mean NPRS scores decreased from baseline during week 1 in both treatment groups, remained relatively stable through week 12 in NXG-4010 patients, but returned to near baseline during weeks 2-4 in controls. Mean change in NPRS scores from baseline during weeks 2-12 was -33.8% for NGX-4010 and +4.9% for control recipients. A similar decrease in NPRS scores from baseline was maintained with subsequent NGX-4010 treatments, regardless of the number of treatments received. Transient increases in application site pain were adequately managed with analgesics. No increases in application site reactions or adverse events were observed with repeated treatments. No patients discontinued the study due to an adverse event. NGX-4010 is a promising topical treatment for PHN patients, which appears to be tolerable, generally safe, and effective.",
"Non‑melanoma skin cancer (NMSC) is the most common form of cancer worldwide, comprising 95% of all cutaneous malignancies and approximately 40% of all cancers. In spite of intensive efforts aimed towards awareness campaigns and sun‑protective measures, epidemiological data indicate an increase in the incidence of NMSC. This category of skin cancers has many common environmental triggers. Arising primarily on sun‑exposed skin, it has been shown that ultraviolet radiation is, in the majority of cases, the main trigger involved in the pathogenesis of NMSC. Aside from the well‑known etiopathogenic factors, studies have indicated that several neuroactive factors are involved in the carcinogenesis of two of the most common types of NMSC, namely basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), with the exception of penile SCC, for which a paucity of specific data on their pathogenic role exists. The complex interaction between the peripheral nervous system and target cells in the skin appears to be mediated by locally released neuroendocrine factors, such as catecholamines, substance P, calcitonin gene‑related peptide and somatostatin, as well as neurohormones, such as proopiomelanocortin and its derived peptides, α‑melanocyte‑stimulating hormone and adrenocorticotropin. All these factors have been, at least at some point, a subject of debate regarding their precise role in the pathogenesis of NMSC. There is also a significant body of evidence indicating that psychological stress is a crucial impact factor influencing the course of skin cancers, including SCC and BCC. Numerous studies have suggested that neuroendocrine factor dysregulation, as observed in stress reactions, may be involved in tumorigenesis, accelerating the development and progression, and suppressing the regression of NMSC. Further studies are required in order to elucidate the exact mechanisms through which neuroactive molecules promote or inhibit cutaneous carcinogenesis, as this could lead to the development of more sophisticated and tailored treatment protocols, as well as open new perspectives in skin cancer research.",
"Although capsaicin (8-methyl-N-vanillyl-6-nonenamide), a pungent ingredient in a variety of red peppers of the genus Capsicum, has been shown to induce apoptotic cell death in many cancer cells, the exact mechanism of this action of capsaicin is not completely understood. In this study, we investigated the possible mediation of the NADPH oxidase-modulated production of reactive oxygen species (ROS) in the apoptotic mechanism of capsaicin in HepG2 human hepatoblastoma cells. Capsaicin induced apoptotic cell death in a time- and dose-dependent manner. Capsaicin at the concentration of inducing apoptosis also markedly increased the level of ROS. The capsaicin-induced generation of ROS and apoptosis was significantly suppressed by treatment with antioxidants, DPPD and tocopherol. In addition, inhibitors of NADPH oxidase, diphenylene iodonium, apocynin and neopterine, profoundly blocked the capsaicin-induced ROS generation and apoptosis. The expression of Rac1N17, a dominant negative mutant of Rac1, also significantly inhibited the capsaicin-induced apoptosis. Activation of nuclear factor-kappaB, a transcription factor essentially involved in ROS-induced apoptosis, was also observed by treatment with capsaicin. Collectively, these results suggest that the NADPH oxidase-mediated generation of ROS may be essentially involved in the mechanism of capsaicin-induced apoptosis in HepG2 cells. These results further suggest that capsaicin may be a valuable agent for the therapeutic intervention of human hepatomas.",
"The treatment of neuropathic pain is difficult. Oral pharmaceuticals have significant side effects, and treatment efficacy tends to be modest. The use of topical analgesics reduces the potential for systemic side effects and allows direct application of medications to the area of pain. The natural spicy substance, capsaicin, has historically been known for its topical use. Capsaicin, once applied to the skin, causes a brief initial sensitization followed by a prolonged desensitization of the local pain nerves. This occurs through stimulation of the transient receptor potential vanilloid-1 (TRPV1) expressing pain nerve fibers. While low-dose capsaicin has not resulted in good efficacy, the larger dose 8% topical capsaicin has had some of the best data currently available in the treatment of post-herpetic neuralgia (PHN) and other neuropathic conditions. This paper discusses the data currently existing for capsaicin 8% in the treatment of PHN. It further reviews data for the low-dose capsaicin products and the current status in the development of other capsaicinoids, e.g. resiniferotoxin, and high-concentration liquid capsaicin.",
"The limitations of current treatments for postherpetic neuralgia (PHN) have led to the investigation of localised, non-systemic alternatives. NGX-4010, a high-concentration (8%) capsaicin dermal patch, was developed to treat patients with neuropathic pain. We report the results of a randomised, double blind, 12-week study of the efficacy and safety of one application of NGX-4010 in patients with PHN. In this multicentre, double-blind, parallel-group trial, 402 patients were randomly assigned to one 60-min application of NGX-4010 (640 microg/cm(2) [8% capsaicin]) or a low-concentration capsaicin control patch (3.2 microg/cm(2) [0.04% capsaicin]). Patients were aged 18-90 years, had had postherpetic neuralgia for at least 6 months, and had an average baseline numeric pain rating scale (NPRS) score of 3 to 9. The primary efficacy endpoint was percentage change in NPRS score from baseline to weeks two to eight. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00115310. Patients who were randomly assigned to NGX-4010 (n=206) had a significantly greater reduction in pain during weeks two to eight than did patients who had the control patch (n=196). The mean changes in NPRS score were -29.6%vs -19.9% (difference -9.7%, 95% CI -15.47 to -3.95; p=0.001). 87 (42%) patients who received NGX-4010 and 63 (32%) controls had a 30% or greater reduction in mean NPRS score (odds ratio [OR] 1.56, 95% CI 1.03 to 2.37; p=0.03). The patients who had NGX-4010 had significant improvements in pain during weeks two to 12 (mean change in NPRS score -29.9%vs -20.4%, difference -9.5, -15.39 to -3.61; p=0.002). Transient blood pressure changes associated with changes in pain level were recorded on the day of treatment, and short-lasting erythema and pain at the site of application were common, self-limited, and generally mild to moderate in the NGX-4010 group and less frequent and severe in the controls. One 60-min application of NGX-4010 provided rapid and sustained pain relief in patients with postherpetic neuralgia. No adverse events were associated with treatment except for local reactions at the site of application and those related to treatment-associated pain."
] |
PNPLA3 polymorphism and liver fibrosis in Brazilian patients diagnosed with chronic hepatitis C
|
Prospective studies have shown that 80% of acute hepatitis C virus (HCV) cases progress to chronic infection; approximately 10-20% of patients with these conditions will develop liver cirrhosis within 2 to 3 decades, and 1-5% will develop liver cancer. Some studies have indicated that the rs738409 polymorphism of the PNPLA3 gene is associated with steatosis and the progression of advanced fibrosis. This study assessed the contribution of the PNPLA3 rs738409 polymorphism with regard to the steatosis and degree of liver fibrosis in Brazilian patients diagnosed with chronic hepatitis C.
|
[
"It has recently been suggested that the rs738409 G allele in PNPLA3, which encodes adiponutrin, is strongly associated with increased liver fat content in three different ethnic groups. The aims of the present study were as follows: (1) to try to replicate these findings in European individuals with quantitative measures of hepatic fat content; (2) to study whether the polymorphism influences hepatic and adipose tissue insulin sensitivity; and (3) to investigate whether PNPLA3 expression is altered in the human fatty liver. We genotyped 291 Finnish individuals in whom liver fat had been measured using proton magnetic resonance spectroscopy. Hepatic PNPLA3 expression was measured in 32 participants. Hepatic and adipose tissue insulin sensitivities were measured using a euglycaemic-hyperinsulinaemic (insulin infusion 0.3 mU kg(-1) min(-1)) clamp technique combined with infusion of [3-(3)H]glucose in 109 participants. The rs738409 G allele in PNPLA3 was associated with increased quantitative measures of liver fat content (p = 0.011) and serum aspartate aminotransferase concentrations (p = 0.002) independently of age, sex and BMI. Fasting serum insulin and hepatic and adipose tissue insulin sensitivity were related to liver fat content independently of genotype status. PNPLA3 mRNA expression in the liver was positively related to obesity (r = 0.62, p < 0.0001) and to liver fat content (r = 0.58, p = 0.025) in participants who were not morbidly obese (BMI < 40 kg/m(2)). A common variant in PNPLA3 increases the risk of hepatic steatosis in humans.",
"The recommended treatment for patients with chronic hepatitis C, pegylated interferon-alpha (PEG-IFN-alpha) plus ribavirin (RBV), does not provide sustained virologic response (SVR) in all patients. We report a genome-wide association study (GWAS) to null virological response (NVR) in the treatment of patients with hepatitis C virus (HCV) genotype 1 within a Japanese population. We found two SNPs near the gene IL28B on chromosome 19 to be strongly associated with NVR (rs12980275, P = 1.93 x 10(-13), and rs8099917, 3.11 x 10(-15)). We replicated these associations in an independent cohort (combined P values, 2.84 x 10(-27) (OR = 17.7; 95% CI = 10.0-31.3) and 2.68 x 10(-32) (OR = 27.1; 95% CI = 14.6-50.3), respectively). Compared to NVR, these SNPs were also associated with SVR (rs12980275, P = 3.99 x 10(-24), and rs8099917, P = 1.11 x 10(-27)). In further fine mapping of the region, seven SNPs (rs8105790, rs11881222, rs8103142, rs28416813, rs4803219, rs8099917 and rs7248668) located in the IL28B region showed the most significant associations (P = 5.52 x 10(-28)-2.68 x 10(-32); OR = 22.3-27.1). Real-time quantitative PCR assays in peripheral blood mononuclear cells showed lower IL28B expression levels in individuals carrying the minor alleles (P = 0.015).",
"The PNPLA3 I148M variant (rs738409) is robustly associated with hepatic steatosis. Intriguingly, initial findings in cohorts with a mean body mass index (BMI) of 30 kg m(-2) also suggested that it is associated with elevated liver enzymes but not with insulin resistance and dyslipidaemia. To determine whether the PNPLA3 variant alters the susceptibility of morbidly obese subjects to develop liver injury and metabolic sequelae. The study was carried out in 678 obese Italians (mean BMI = 41 kg m(-2)) who were genotyped for the I148M variant. All participants provided fasting blood samples and then underwent oral glucose tolerance tests. Indices of liver injury (alanine transaminase (ALT), aspartate transaminase (AST)), glucose tolerance and insulin resistance were measured. Markers of hepatic injury such as ALT and AST were significantly higher in carriers of the 148M allele (P = 2.2 x 10(-5) and 0.001, respectively). In all, 50% of 148M risk allele homozygotes had pathological levels of ALT (>40 U l(-1)) compared with 25% of 148I allele homozygotes (P = 0.005). Glucose tolerance and insulin sensitivity were similar in all three genotypes. Obese Southern Europeans carrying the 148M allele have increased indices of liver damage uncoupled from proxy measures of insulin resistance.",
"A single nucleotide polymorphism (rs12979860 C/T) 3kb upstream of the interleukin 28B (IL-28B) gene was shown to be associated with hepatitis C clearance. We verified whether this association also translates into a different genotype distribution at the end of the disease trajectory. A RFLP-PCR technique was used to genotype 412 patients with cirrhosis due to hepatitis C (n=199), hepatitis B (n=75), alcohol (n=110), and other causes (n=28), of whom 256 underwent liver transplantation (OLT). Hepatocellular carcinoma (HCC) was demonstrated in the native liver of 85 OLT patients, 52 with viral cirrhosis, and 33 with non-viral cirrhosis respectively. A group of 292 patients (235 HCV and 57 HBV positive) with mild chronic hepatitis and 344 healthy subjects served as controls. A significant difference (p=0.0005) was observed in IL-28B rs12979860 genotype frequencies between patients with viral cirrhosis (C/C=99, C/T=137, T/T=38) and those with non-viral cirrhosis (C/C=72, C/T=58, T/T=8). Patients with HCV related cirrhosis carried more frequently the T/T genotype in comparison to mild hepatitis C or HBV-related cirrhosis. IL-28B rs12979860 genotype frequencies were C/C=23, C/T=50, T/T=12 among OLT patients with cirrhosis complicated by HCC, and C/C=79, C/T=78, T/T=14 among patients with cirrhosis not complicated by HCC (p<0.005). IL-28B rs12979860 C/T polymorphism T allele is more prevalent in patients with viral cirrhosis due to HCV in comparison to other aetiologies and to patients with mild chronic hepatitis C. Among OLT patients, carriage of this allele seems to augment the risk of developing HCC.",
"Hepatitis C virus (HCV) infection is associated with the development of cirrhosis and hepatocellular carcinoma and is also related to fatty change of the liver. Variation in patatin-like phospholipase domain-containing 3 (PNPLA3) gene is associated with disease progression in nonalcoholic fatty liver disease (NAFLD). Recent reports have suggested that PNPLA3, IL28B and TLR4-associated single nucleotide polymorphisms (SNPs) may have an impact on hepatic steatosis or fibrosis in patients with chronic HCV infection. Four SNPs (PNPLA3 rs738409, TLR4 rs4986790, TLR4 rs4986791, IL28B rs8099917) were identified in Japanese patients infected with HCV. We examined the association between the distribution of these SNP alleles and fatty change of the liver or existence of hepatic cirrhosis diagnosed by ultrasonography, one of the widely accessible and easy-to-use methods. PNPLA3 rs738409 G-allele and IL28B rs 8099917 minor allele were found in 70.0% and 31.1%, respectively. These two TLR4 SNPs were uniform in Japanese. Fatty change of the liver developed independent of the abscence of hepatic cirrhosis on sonographic findings and younger age. Hepatic cirrhosis was associated with a higher aspartate aminotransferase/platelet ratio index (APRI), no fatty change of the liver, higher BMI and higher AFP levels. No association between PNPLA3 rs738409/IL28B rs8099917 genotypes and hepatic steatosis or liver fibrosis was observed. According to ultrasound examinations, no association between PNPLA3 rs738409 genotype and fatty change of the liver or hepatic cirrhosis was found in Japanese patients infected with HCV. Together, our results suggested that the mechanism of hepatic steatosis underlying HCV infection might differ from that of NAFLD and should be explored.",
"A common non-synonymous polymorphism, E167K, in transmembrane six superfamily member 2 (TM6SF2) gene has been recently associated with an increased hepatic triglyceride content, dyslipidemia and liver fibrosis in NAFLD patients. We investigated possible associations between the TM6SF2 variants and liver lesions in chronic hepatitis C. 148 consecutive patients with biopsy proven anti-HCV/HCV-RNA-positive chronic hepatitis, naive for antiviral therapy, were genotyped for TM6SF2 E167K and PNPLA3 I148M variants. The score of liver steatosis was higher in the 18 patients with TM6SF2 E167K variant (mean 1.9 ± 1.3) than in the 130 homozygotes for TM6SF2 167E allele (1.1 ± 1.1, P = 0.02), and the prevalence of a steatosis score ≥ 3 was 33.3% vs. 12.3% respectively (P = 0.02). No difference in necroinflammatory or fibrosis scores was found between the two groups. A general linear model identified as independent predictors of steatosis TM6SF2 E167K and PNPLA3 M148M variants and waist circumference (P = 0.0376, P = 0.0069 and P = 0.0273 respectively). This is the first demonstration that TM6SF2 E167K variant is an independent predictor of liver steatosis in chronic hepatitis C."
] |
Psychological functioning and peer relationship problems in gender diverse adolescents across Europe
|
Adolescents seeking professional help with their gender identity development often present with psychological difficulties. Existing literature on psychological functioning of gender diverse young people is limited and mostly bound to national chart reviews. This study examined the prevalence of psychological functioning and peer relationship problems in adolescents across four European specialist gender services (The Netherlands, Belgium, the UK, and Switzerland), using the Child Behavioural Checklist (CBCL) and the Youth Self-Report (YSR). Differences in psychological functioning and peer relationships were found in gender diverse adolescents across Europe. Overall, emotional and behavioural problems and peer relationship problems were most prevalent in adolescents from the UK, followed by Switzerland and Belgium. The least behavioural and emotional problems and peer relationship problems were reported by adolescents from The Netherlands. Across the four clinics, a similar pattern of gender differences was found. Birth-assigned girls showed more behavioural problems and externalising problems in the clinical range, as reported by their parents. According to self-report, internalising problems in the clinical range were more prevalent in adolescent birth-assigned boys. More research is needed to gain a better understanding of the difference in clinical presentations in gender diverse adolescents and to investigate what contextual factors that may contribute to this.
|
[
"To determine age and gender differences in health-related quality of life (HRQOL) in children and adolescents across 12 European countries using a newly developed HRQOL measure (KIDSCREEN). The KIDSCREEN-52 questionnaire was filled in by 21,590 children and adolescents aged 8-18 from 12 countries. We used multilevel regression analyses to model the hierarchical structure of the data. In addition, effect sizes were computed to test for gender differences within each age group. Children generally showed better HRQOL than adolescents (P < 0.001). While boys and girls had similar HRQOL at young age, girls' HRQOL declined more than boys' (P < 0.001) with increasing age, depending on the HRQOL scale. There was significant variation between countries both at the youngest age and for age trajectories. For the first time, gender and age differences in children's and adolescents' HRQOL across Europe were assessed using a comprehensive and standardised instrument. Gender and age differences exist for most HRQOL scales. Differences in HRQOL across Europe point to the importance of national contexts for youth's well-being.",
"A literature review of the associations between involvement in bullying and depression is presented. Many studies have demonstrated a concurrent association between involvement in bullying and depression in adolescent population samples. Not only victims but also bullies display increased risk of depression, although not all studies have confirmed this for the bullies. Retrospective studies among adults support the notion that victimization is followed by depression. Prospective follow-up studies have suggested both that victimization from bullying may be a risk factor for depression and that depression may predispose adolescents to bullying. Research among clinically referred adolescents is scarce but suggests that correlations between victimization from bullying and depression are likely to be similar in clinical and population samples. Adolescents who bully present with elevated numbers of psychiatric symptoms and psychiatric and social welfare treatment contacts.",
"The higher incidence of man-to-woman transsexuals compared to woman-to-man transsexuals varies markedly from country to country. This is the first survey of the sex ratio to be made in Germany. It covers 1785 patients who between 1964 and 1998 were diagnosed as transsexual at the four largest German centres offering treatment. From 1970 to 1994 the sex ratio remained constant at 2:1 in favour of man-to-woman transsexuals. Over the past 4 years, however, it has altered considerably and reached 1.2:1. Up to 1994 our results do not support the assumption that transsexualism is gradually becoming equally prevalent in both sexes. The drop in the sex ratio after 1994 can be explained either as a reduction of an overhang of male-to-female transsexuals or as an artificial phenomenon caused by recent developments in therapy and by the views of transsexuals' groups on the treatment they are offered.",
"The purpose of this study was to describe baseline characteristics of participants in a prospective observational study of transgender youth (aged 12-24 years) seeking care for gender dysphoria at a large, urban transgender youth clinic. Eligible participants presented consecutively for care at between February 2011 and June 2013 and completed a computer-assisted survey at their initial study visit. Physiologic data were abstracted from medical charts. Data were analyzed by descriptive statistics, with limited comparisons between transmasculine and transfeminine participants. A total of 101 youth were evaluated for physiologic parameters, 96 completed surveys assessing psychosocial parameters. About half (50.5%) of the youth were assigned a male sex at birth. Baseline physiologic values were within normal ranges for assigned sex at birth. Youth recognized gender incongruence at a mean age of 8.3 years (standard deviation = 4.5), yet disclosed to their family much later (mean = 17.1; standard deviation = 4.2). Gender dysphoria was high among all participants. Thirty-five percent of the participants reported depression symptoms in the clinical range. More than half of the youth reported having thought about suicide at least once in their lifetime, and nearly a third had made at least one attempt. Baseline physiologic parameters were within normal ranges for assigned sex at birth. Transgender youth are aware of the incongruence between their internal gender identity and their assigned sex at early ages. Prevalence of depression and suicidality demonstrates that youth may benefit from timely and appropriate intervention. Evaluation of these youth over time will help determine the impact of medical intervention and mental health therapy.",
"Adolescence is a period of life in which the sense of 'self' changes profoundly. Here, we review recent behavioural and neuroimaging studies on adolescent development of the self-concept. These studies have shown that adolescence is an important developmental period for the self and its supporting neural structures. Recent neuroimaging research has demonstrated that activity in brain regions associated with self-processing, including the medial prefrontal cortex, changes between early adolescence and adulthood. These studies indicate that neurocognitive development might contribute to behavioural phenomena characteristic of adolescence, such as heightened self-consciousness and susceptibility to peer influence. We attempt to integrate this recent neurocognitive research on adolescence with findings from developmental and social psychology.",
"Increasing numbers of adolescents present in adolescent gender identity services, desiring sex reassignment (SR). The aim of this study is to describe the adolescent applicants for legal and medical sex reassignment during the first two years of adolescent gender identity team in Finland, in terms of sociodemographic, psychiatric and gender identity related factors and adolescent development. Structured quantitative retrospective chart review and qualitative analysis of case files of all adolescent SR applicants who entered the assessment by the end of 2013. The number of referrals exceeded expectations in light of epidemiological knowledge. Natal girls were markedly overrepresented among applicants. Severe psychopathology preceding onset of gender dysphoria was common. Autism spectrum problems were very common. The findings do not fit the commonly accepted image of a gender dysphoric minor. Treatment guidelines need to consider gender dysphoria in minors in the context of severe psychopathology and developmental difficulties."
] |
Molecular mechanisms of NSC-743380-mediated apoptosis in acute myeloid leukemia cells
|
Our recent study showed that acute myeloid leukemia (AML) cells expressing SULT1A1 are highly sensitive to NSC-743380, a small molecule that inhibits STAT3 activity and induces SULT1A1-dependent apoptosis of various cancer cell lines. In this study, we characterized the molecular mechanisms of NSC-743380-mediated anti-leukemia activity in AML cell lines and antileukemia activity of NSC-743380 in patient-derived primary leukemia cells from AML patients. Our results showed that treatment with NSC-743380 triggered robust apoptosis in SULT1A1-positive AML cells. Treatment with NSC-743380 did not increase intracellular reactive oxygen species or change of STAT3 activity in AML cells, but did dramatically and rapidly decrease cFLIP expression. Proteomic analysis with reverse phase protein microarray revealed that treatment of U937 and THP-1 AML cells with NSC-743380 led to drastic and time-dependent suppression of phosphorylation of several key nodes in the PI3K/AKT/mTOR pathway, including AKT and mTOR. Moreover, primary AML cells expressed SULT1A1 were highly sensitive to treatment with NSC-743380, which was not affected by co-culture with bone marrow mesenchymal stem cells. Thus, our results provide proof-of-concept evidence that AML cells expressing SULT1A1 can be targeted by small molecules that induce apoptosis through inhibiting the expression or activities of multiple targets.
|
[
"The Philadelphia chromosome (Ph) is the most common cytogenetic abnormality associated with adult acute lymphoblastic leukemia (ALL). Before the advent of tyrosine kinase inhibitors (TKIs), Ph-positive ALL carried a dismal prognosis and was characterized by a poor response to most chemotherapy combinations, short remission durations, and poor survival rates. Outcomes for patients with Ph-positive ALL improved substantially with the introduction of TKIs, and the TKI imatinib induced complete remissions in >95% of patients with newly diagnosed Ph-positive ALL when it was combined with chemotherapy. However, imatinib resistance remains a problem in a substantial proportion of patients with Ph-positive ALL, and multiple molecular mechanisms that contribute to imatinib resistance have been identified. Second-generation TKIs (eg, dasatinib and nilotinib) have demonstrated promising efficacy in the treatment of imatinib-resistant, Ph-positive ALL. Future strategies for Ph-positive ALL include novel, molecularly targeted treatment modalities and further evaluations of TKIs in combination with established antileukemic agents. For this article, the authors reviewed past, current, and future treatment approaches for adult and elderly patients with Ph-positive ALL with a focus on TKIs and combined chemotherapeutic regimens.",
"Tyrosine kinase inhibitors (TKIs) are used as a frontline therapy for BCR-ABL(+) acute lymphoblastic leukemia (ALL). However, resistance to TKI therapy arises rapidly, and its underlying molecular mechanisms are poorly understood. In this study, we identified a novel cascade of events initiated by TKIs and traversing through mesenchymal stem cells (MSCs) to leukemic cells, leading to resistance. MSCs exposed to TKIs acquired a new functional status with the expression of genes encoding for chemo-attractants, adhesion molecules, and prosurvival growth factors, and this priming enabled leukemic cells to form clusters underneath the MSCs. This cluster formation was associated with the protection of ALL cells from therapy as leukemic cells switched from BCR-ABL signaling to IL-7R/Janus kinase signaling to survive in the MSC milieu. Our findings illustrate a novel perspective in the evolution of TKI resistance and provide insights for advancing the treatment of BCR-ABL(+) ALL.",
"Previous studies have suggested that the caspase 8 inhibitor FLIP is a promising anti-cancer therapeutic target. In this study, we characterised a novel FLIP-targeted antisense phosphorothioate oligonucleotide (AS PTO). FLIP AS and control PTOs were assessed in vitro in transient transfection experiments and in vivo using xenograft models in Balb/c nude mice. FLIP expression was assessed by QPCR and Western. Apoptosis induction was determined by flow cytometry and Western. Of 5 sequences generated, one potently down-regulated FLIP. AS PTO-mediated down-regulation of FLIP resulted in caspase 8 activation and apoptosis induction in non-small cell lung (NSCLC) cells but not in normal lung cells. Similar results were observed in colorectal and prostate cancer cells. Furthermore, the FLIP AS PTO sensitized cancer cells but not normal lung cells to apoptosis induced by rTRAIL. Moreover, the FLIP AS PTO enhanced chemotherapy-induced apoptosis in NSCLC cells. Importantly, compared to a control non-targeted PTO, intra-peritoneal delivery of FLIP AS PTO inhibited the growth of NSCLC xenografts and enhanced the in vivo antitumour effects of cisplatin. We have identified a novel FLIP-targeted AS PTO that has in vitro and in vivo activity and which therefore has potential for further pre-clinical development.",
"The intestinal epithelium generates a barrier that protects mammals from potentially harmful intestinal contents, such as pathogenic bacteria. Dysregulation of epithelial cell death has been implicated in barrier dysfunction and in the pathogenesis of intestinal inflammation. We investigated mechanisms of cell-death regulation in the intestinal epithelium of mice. Conditional knockout mice (either inducible or permanent) with deletion of cellular FLICE-inhibitory protein (cFlip) or caspase-8 in the intestinal epithelium were analyzed by histology and high-resolution endoscopy. We assessed the effects of cFlip or caspase-8 deficiency on intestinal homeostasis. Expression of cFlip in the intestinal epithelium was required for constitutive activation of caspase-8 under steady-state conditions. Intestinal expression of cFlip was required for development; disruption of the gene encoding cFlip from the intestinal epithelium (cFlip(fl/fl) VillinCre(+) mice) resulted in embryonic lethality. When cFlip was deleted from the intestinal epithelium of adult mice (cFlip(iΔIEC) mice), the animals died within a few days from severe tissue destruction, epithelial cell death, and intestinal inflammation. Death of cFlip-depleted intestinal epithelial cells was regulated extrinsically and required the presence of death receptor ligands, such as tumor necrosis factor-α and CD95 ligand, but was independent of receptor-interacting protein 3. cFlip deficiency was associated with strong up-regulation of caspase-8 and caspase-3 activity and excessive apoptosis in intestinal crypts. cFlip is required for intestinal tissue homeostasis in mice. It controls the level of activation of caspase-8 to promote survival of intestinal epithelial cells.",
"Celecoxib is a COX-2 inhibitor that reduces the risk of colon cancer. However, the basis for its cancer chemopreventive activity is not fully understood. In this study, we defined a mechanism of celecoxib action based on degradation of cellular FLICE-inhibitory protein (c-FLIP), a major regulator of the death receptor pathway of apoptosis. c-FLIP protein levels are regulated by ubiquitination and proteasome-mediated degradation. We found that celecoxib controlled c-FLIP ubiquitination through Akt-independent inhibition of glycogen synthase kinase-3 (GSK3), itself a candidate therapeutic target of interest in colon cancer. Celecoxib increased the levels of phosphorylated GSK3, including the α and β forms, even in cell lines, where phosphorylated Akt levels were not increased. Phosphoinositide 3-kinase inhibitors abrogated Akt phosphorylation as expected but had no effect on celecoxib-induced GSK3 phosphorylation. In contrast, protein kinase C (PKC) inhibitors abolished celecoxib-induced GSK3 phosphorylation, implying that celecoxib influenced GSK3 phosphorylation through a mechanism that relied upon PKC and not Akt. GSK3 blockade either by siRNA or kinase inhibitors was sufficient to attenuate c-FLIP levels. Combining celecoxib with GSK3 inhibition enhanced attenuation of c-FLIP and increased apoptosis. Proteasome inhibitor MG132 reversed the effects of GSK3 inhibition and increased c-FLIP ubiquitination, confirming that c-FLIP attenuation was mediated by proteasomal turnover as expected. Our findings reveal a novel mechanism through which the regulatory effects of c-FLIP on death receptor signaling are controlled by GSK3, which celecoxib acts at an upstream level to control independently of Akt.",
"The small molecule anticancer agent NSC-743380 modulates functions of multiple cancer-related pathways and is highly active in a subset of cancer cell lines in the NCI-60 cell line panel. It also has promising in vivo anticancer activity. However, the mechanisms underlying NSC-743380's selective anticancer activity remain uncharacterized. To determine biomarkers that may be used to identify responders to this novel anticancer agent, we performed correlation analysis on NSC-743380's anticancer activity and the gene expression levels in NCI-60 cell lines and characterized the functions of the top associated genes in NSC-743380-mediated anticancer activity. We found sulfotransferase SULT1A1 is causally associated with NSC-743380's anticancer activity. SULT1A1 was expressed in NSC-743380-sensitive cell lines but was undetectable in resistant cancer cells. Ectopic expression of SULT1A1 in NSC743380 resistant cancer cells dramatically sensitized the resistant cells to NSC-743380. Knockdown of the SULT1A1 in the NSC-743380 sensitive cancer cell line rendered it resistance to NSC-743380. The SULT1A1 protein levels in cell lysates from 18 leukemia cell lines reliably predicted the susceptibility of the cell lines to NSC-743380. Thus, expression of SULT1A1 in cancer cells is required for NSC-743380's anticancer activity and can be used as a biomarker for identification of NSC-743380 responders.",
"The bcr-abl oncogene, present in 95% of patients with chronic myelogenous leukemia (CML), has been implicated as the cause of this disease. A compound, designed to inhibit the Abl protein tyrosine kinase, was evaluated for its effects on cells containing the Bcr-Abl fusion protein. Cellular proliferation and tumor formation by Bcr-Abl-expressing cells were specifically inhibited by this compound. In colony-forming assays of peripheral blood or bone marrow from patients with CML, there was a 92-98% decrease in the number of bcr-abl colonies formed but no inhibition of normal colony formation. This compound may be useful in the treatment of bcr-abl-positive leukemias."
] |
The left parietal old/new effect does not correlate with recollection
|
A success story within neuroimaging has been the discovery of distinct neural correlates of episodic retrieval, providing insight into the processes that support memory for past life events. Here we focus on one commonly reported neural correlate, the left parietal old/new effect, a positive going modulation seen in event-related potential (ERP) data that is widely considered to index episodic recollection. Substantial evidence links changes in the size of the left parietal effect to changes in remembering, but the precise functional utility of the effect remains unclear. Here, using forced choice recognition of verbal stimuli, we present a novel population level test of the hypothesis that the magnitude of the left parietal effect correlates with memory performance. We recorded ERPs during old/new recognition, source accuracy and Remember/Know/Guess tasks in two large samples of healthy young adults, and successfully replicated existing within participant modulations of the magnitude of the left parietal effect with recollection. Critically, however, both datasets also show that across participants the magnitude of the left parietal effect does not correlate with behavioral measures of memory - including both subjective and objective estimates of recollection. We conclude that in these tasks, and across this healthy young adult population, the generators of the left parietal ERP effect do not index performance as expected. Taken together, these novel findings provide important constraints on the functional interpretation of the left parietal effect, suggesting that between group differences in the magnitude of old/new effects cannot always safely be used to infer differences in recollection.
|
[
"A functional polymorphism at the val66met locus in the BDNF gene has significant effects on the pro-form of the protein in intracellular trafficking and activity-dependent, but not constitutive, secretion. These differences are thought to underlie several findings in humans related to this polymorphism, including markers of neuronal viability, BOLD activation in medial temporal lobe regions, and some aspects of behavior. However, many important questions remain about the impact of BDNF on various mnemonic subprocesses at the behavioral level. In this study, we examined the impact of the val/met polymorphism in a verbal recognition memory paradigm involving manipulation of depth of encoding and differential delays for recall and analyses of hits for previously presented target words and correct rejections of foils. Twenty-four human val homozygous individuals and 24 met carrier individuals comprised the sample. All were healthy controls. IQ between the groups was equivalent. In the encoding phase of the study, words were presented and encoded either by a decision as to whether they were living or nonliving (\"deep\") or if they contained the letter \"A\" (shallow). After this phase, recognition was tested immediately, half an hour, and 24h later. BDNF genotype had significant effects on hits and discriminability (d'), accounting for at least 10% of the variance, but not on correct rejections or beta. BDNF did not interact with level of encoding, nor did it interact with delay. In sum, BDNF genotypes impacted \"hits\" in a recognition memory paradigm, findings consistent with the general notion that BDNF plays a prominent role in memory subprocesses thought to engage the medial temporal lobe.",
"Dual process theories posit that separate recollection and familiarity processes contribute to recognition memory. Previous research, testing recognition memory for words, indicates that event-related brain potentials (ERPs) can be used to dissociate recollection from familiarity. It has been hypothesized that the FN400 ERP old/new effect (300-500 ms) varies with stimulus familiarity, but the parietal ERP old/new effect (400-800 ms) varies with recollection. The results reported here are consistent with this hypothesis, extending it to the recognition of pictures when subjects had to discriminate between studied pictures, highly familiar lures (mirror-reversals of studied pictures), and new pictures. Furthermore, the parietal old/new effect showed significant recollection-related differences only for subjects with good behavioral discrimination between studied items and similar lures.",
"The electrophysiological correlates of recognition memory for new associations were investigated in two experiments. In both experiments subjects first studied unrelated word pairs. At test, they were presented with old words in the same pairing as at study (same pairs), old words in a different pairing from study (rearranged pairs), and pairs of new words. In Experiment 1 the test requirement was to discriminate between old and new pairs and, for any pair judged old, to then judge whether the pair was the same or rearranged. In Experiment 2 the requirement was merely to discriminate between old and new pairs. Event-related potentials (ERPs) were recorded for correctly classified same, rearranged and new pairs. The ERPs elicited by same pairs exhibited a similar pattern of effects in both experiments. Relative to the ERPs to new pairs, these effects took the form of sustained positive shifts with two distinct scalp maxima, over the left temporo-parietal and right frontal scalp respectively. ERPs to rearranged pairs showed effects which were similar in scalp topography, but markedly smaller in magnitude. This pattern of ERP effects closely resembles that found previously for test items defined as recollected on the basis of their attracting a successful source judgement. The findings therefore suggest that associative recognition memory shares some of the recollective processes that are engaged by the requirement to retrieve contextual information about a study episode. The findings from Experiment 2 indicate that the processes associated with the recollection of associated pairs are engaged regardless of whether the retrieval of associative information is an explicit task requirement.",
"We report an event-related potential (ERP) experiment of human recognition memory that explored the relation between conscious awareness and electrophysiological activity of the brain. We recorded ERPs from healthy adults while they made \"remember\" and \"know\" recognition judgments about previously seen words. These two kinds of judgments reflect \"autonoetic\" and \"noetic\" awareness, respectively. The ERP effects differed between the two kinds of awareness while they were similar for \"true\" and \"false\" recognition. Noetic awareness was associated with a temporoparietal positivity in the N400 range (325-600 ms) and a late (600-1,000 ms) frontocentral negativity, whereas autonoetic awareness was associated with a widespread, late, bifrontal and left parietotemporal (600-1000 ms) positivity. In the very late (1,300-1, 900 ms) time window, a right frontal positivity was observed for both remember and know judgments of both true and false targets. These results provide physiological evidence for two types of conscious awareness in episodic memory retrieval.",
"According to dual-process models, recognition memory is supported by distinct retrieval processes known as familiarity and recollection. Important evidence supporting the dual-process framework has come from studies using event-related brain potentials (ERPs). These studies have identified two topographically distinct ERP correlates of recognition memory--the \"parietal\" and \"mid-frontal\" old/new effects--that are dissociated by variables that selectively modulate recollection and familiarity, respectively. We evaluate the extent to which ERP data support dual-process models in light of the proposal that recollection is a continuous rather than a discrete memory process. We also examine the claim that the putative ERP index of familiarity is a reflection of implicit rather than explicit memory. We conclude that ERP findings continue to offer strong support for the dual-process perspective.",
"There is keen interest in what enables rememberers to differentiate true from false memories and which strategies are likely to be the most effective. This study measured electrical brain activity while healthy young adults performed a mnemonic discrimination task, deciding whether color pictures had been studied, were similar to studied pictures (lures), or were new. Between 500 and 800 ms post-stimulus, event-related potentials (ERPs) for correctly recognized studied pictures and falsely recognized lures compared to those for correctly rejected novel items had a left centroparietal scalp distribution. This was typical of the parietal old/new effect associated with recollection, and in line with previous evidence that similar lures may elicit false or phantom recollection as opposed to just familiarity. There was no evidence of a parietal effect for correctly rejected lures as would be expected if recall-to-reject was used. The ERP old/new effects for lures also varied with individual differences in performance. Parietal effects for falsely recognized lures were larger in better performers, who successfully rejected a greater number of lures as \"similar\". The better performers also showed more pronounced right frontocentral old/new effects between 800 and 1100 ms for correctly rejected and falsely recognized similar lures. The enhancement of false recollection in better performers implies false recognition of lures occurred only when more specific information was recovered about the study episodic. Together, the findings suggest reliance on recollection to decide that items were studied, supported by post-retrieval processing.",
"We used event-related functional magnetic resonance imaging (efMRI) to investigate brain regions showing differential responses as a function of confidence in an episodic word recognition task. Twelve healthy volunteers indicated whether their old-new judgments were made with high or low confidence. Hemodynamic responses associated with each judgment were modeled with an \"early\" and a \"late\" response function. As predicted by the monitoring hypothesis generated from a previous recognition study [Henson, R. N. A., Rugg, M. D., Shallice, T., Josephs, O., & Dolan, R. J. (1999a). Recollection and familiarity in recognition memory: An event-related fMRI study. Journal of Neuroscience, 19, 3962-3972], a right dorsolateral prefrontal region showed a greater response to correct low- versus correct high-confidence judgements. Several regions, including the precuneus, posterior cingulate, and left lateral parietal cortex, showed greater responses to correct old than correct new judgements. The anterior left and right prefrontal regions also showed an old-new difference, but for these regions the difference emerged relatively later in time. These results further support the proposal that different subregions of the prefrontal cortex subserve different functions during episodic retrieval. These functions are discussed in relation to a monitoring process, which operates when familiarity levels are close to response criterion and is associated with nonconfident judgements, and a recollective process, which is associated with the confident recognition of old words."
] |
Viperin suppresses hepatic and adipose lipogenesis in mice
|
Viperin is an interferon-inducible antiviral protein, responsible for antiviral response to a variety of viral infections. Here, we show that silencing viperin by antisense oligonucleotides (ASO) protects against diet-induced glucose intolerance, and yet exacerbates adipose tissue inflammation. In high-fat diet-fed mice, viperin ASO improves glucose homeostasis, reduces plasma triglyceride concentrations and ameliorates diet-induced hepatic steatosis. Peripheral delivery of viperin by adeno-associated virus elevates fasting plasma glucose and insulin concentrations and reduces insulin-stimulated glucose uptake in skeletal muscle. Viperin overexpression reduces epinephrine- stimulated lipolysis in white adipose tissue, whereas viperin ASO increases expression of lipolytic genes. Targeting viperin by antisense oligonucleotides promotes reciprocal regulation of hepatic and adipose lipogenesis by reducing hepatic lipid content and increasing triacylglycerol content in adipose tissue. These findings reveal viperin as an important target to improve glucose metabolism, and suggest that suppressing antiviral potential may improve the metabolic adaptability to high-fat diet.
|
[
"Obese adipose tissue is characterized by infiltration of macrophages. We and others recently showed that a specific subset of macrophages is recruited to obese adipose and muscle tissue. This subset expresses CD11c and produces high levels of proinflammatory cytokines that are linked to the development of obesity-associated insulin resistance. Here, we used a conditional cell ablation system, based on transgenic expression of the diphtheria toxin receptor under the control of the CD11c promoter, to study the effects of depletion of CD11c+ cells in obese mouse models. Our results show that CD11c+ cell depletion results in rapid normalization of insulin sensitivity. Furthermore, CD11c+ cell ablation leads to a marked decrease in inflammatory markers, both locally and systemically, as reflected by gene expression and protein levels. Together, these results indicate that these CD11c+ cells are a potential therapeutic target for treatment of obesity-related insulin resistance and type II diabetes.",
"A central paradox in type 2 diabetes is the apparent selective nature of hepatic insulin resistance--wherein insulin fails to suppress hepatic glucose production yet continues to stimulate lipogenesis, resulting in hyperglycemia, hyperlipidemia, and hepatic steatosis. Although efforts to explain this have focused on finding a branch point in insulin signaling where hepatic glucose and lipid metabolism diverge, we hypothesized that hepatic triglyceride synthesis could be driven by substrate, independent of changes in hepatic insulin signaling. We tested this hypothesis in rats by infusing [U-(13)C] palmitate to measure rates of fatty acid esterification into hepatic triglyceride while varying plasma fatty acid and insulin concentrations independently. These experiments were performed in normal rats, high fat-fed insulin-resistant rats, and insulin receptor 2'-O-methoxyethyl chimeric antisense oligonucleotide-treated rats. Rates of fatty acid esterification into hepatic triglyceride were found to be dependent on plasma fatty acid infusion rates, independent of changes in plasma insulin concentrations and independent of hepatocellular insulin signaling. Taken together, these results obviate a paradox of selective insulin resistance, because the major source of hepatic lipid synthesis, esterification of preformed fatty acids, is primarily dependent on substrate delivery and largely independent of hepatic insulin action.",
"Adipose tissue (AT) can accumulate macrophages and secrete several inflammatory mediators. Despite its pivotal role in the progression of chronic inflammatory processes such as atherosclerosis, the adaptive role of immunity in obesity remains poorly explored. Visceral AT of diet-induced obese C57BL/6 mice had higher numbers of both CD4(+) and CD8(+) T cells than lean controls, monitored by flow cytometry. When stimulated in vitro, T cells from obese AT produced more interferon (IFN)gamma than those from controls. AT from obese animals also had more cells expressing I-A(b), a mouse class II histocompatibility marker implicated in antigen presentation, as determined by immunostaining. Differentiated 3T3-L1 cells stimulated with recombinant IFNgamma or T-helper 1-derived supernatant produced several chemokines and their mRNAs. Obese IFNgamma-deficient animals had significantly reduced AT expression of mRNA-encoding inflammatory genes such as tumor necrosis factor-alpha and monocyte chemoattractant protein-1, decreased AT inflammatory cell accumulation, and better glucose tolerance than control animals consuming the same diet. Obese mice doubly deficient for IFNgamma receptor and apolipoprotein (Apo)E on a mixed 129SvEv/C57BL/6 (129/B6) genetic background, despite exhibiting similar AT mRNA levels of tumor necrosis factor-alpha and monocyte chemoattractant protein-1 as 129/B6-ApoE(-/-) controls, had decreased expression of important T cell-related genes, such as IFNgamma-inducible protein-10 and I-A(b), and lower plasma triglycerides and glucose. These results indicate a role for T cells and IFNgamma, a prototypical T-helper 1 cytokine, in regulation of the inflammatory response that accompanies obesity.",
"The liver's regulation of fatty acids (FAs) postprandially may contribute to risk of metabolic diseases. Measurements of steady-state metabolism were used to investigate sources of FAs used for very low-density lipoprotein (VLDL)-triacylglycerol (TG) synthesis during fasting and feeding in vivo. Subjects were duodenally fed a formula labeled with the stable isotope glyceryl tri-palmitate-d(31) and iv infused with [1,2,3,4-(13)C(4)]-palmitatic acid and [1-(13)C(1)]-acetate to quantitate the liver's use of FAs originating from adipose tissue and de novo lipogenesis. This study of healthy men (n = 12; body mass index, 24.4 +/- 2.7 kg/m(2)) was conducted at a General Clinical Research Center. Concentrations of metabolites during fasting and feeding, sources of FAs used for lipoprotein synthesis, rate of appearance of serum nonesterified FA (NEFA), and VLDL-TG were measured. During fasting, 77.2 +/- 14.0% of VLDL-TG was derived from adipose FA recycling and 4.0 +/- 3.6% from lipogenesis; with feeding, 43.6 +/- 18.6% came from adipose FAs (P < 0.001), 8.2 +/- 5.1% from lipogenesis (P < 0.001), 15.2 +/- 13.7% from uptake of chylomicron-remnant TG, and 10.3 +/- 6.9% from dietary FA spillover into the serum NEFA pool. Fed-state VLDL-TG from NEFA reesterification decreased in proportion to the reduction in adipose NEFA appearance. These data: 1) quantify the extent to which the healthy liver manages its use of different sources of FAs that flow to it, 2) demonstrate how the postprandial reduction in adipose-NEFA flux may be partially replaced by other sources, and 3) highlight the potential for dietary FA spillover to support the continued dominance of NEFA as a substrate for VLDL-TG synthesis.",
"Adipose tissue macrophages are important mediators of inflammation and insulin resistance in obesity. IFN-γ is a central regulator of macrophage function. The role of IFN-γ in regulating systemic inflammation and insulin resistance in obesity is unknown. We studied obese IFN-γ knockout mice to identify the role of IFN-γ in regulating inflammation and insulin sensitivity in obesity. IFN-γ-knockout C57Bl/6 mice and wild-type control litter mates were maintained on normal chow or a high fat diet for 13 weeks and then underwent insulin sensitivity testing then sacrifice and tissue collection. Flow cytometry, intracellular cytokine staining, and QRTPCR were used to define tissue lymphocyte phenotype and cytokine expression profiles. Adipocyte size was determined from whole adipose tissue explants examined under immunofluorescence microscopy. Diet-induced obesity induced systemic inflammation and insulin resistance, along with a pan-leukocyte adipose tissue infiltrate that includes macrophages, T-cells, and NK cells. Obese IFN-γ-knockout animals, compared with obese wild-type control animals, demonstrate modest improvements in insulin sensitivity, decreased adipocyte size, and an M2-shift in ATM phenotype and cytokine expression. These data suggest a role for IFN-γ in the regulation of inflammation and glucose homeostasis in obesity though multiple potential mechanisms, including effects on adipogenesis, cytokine expression, and macrophage phenotype.",
"Reductions in peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1alpha) levels have been associated with the skeletal muscle insulin resistance. However, in vivo, the therapeutic potential of PGC-1alpha has met with failure, as supra-physiological overexpression of PGC-1alpha induced insulin resistance, due to fatty acid translocase (FAT)-mediated lipid accumulation. Based on physiological and metabolic considerations, we hypothesised that a modest increase in PGC-1alpha levels would limit FAT upregulation and improve lipid metabolism and insulin sensitivity, although these effects may differ in lean and insulin-resistant muscle. Pgc-1alpha was transfected into lean and obese Zucker rat muscles. Two weeks later we examined mitochondrial biogenesis, intramuscular lipids (triacylglycerol, diacylglycerol, ceramide), GLUT4 and FAT levels, insulin-stimulated glucose transport and signalling protein phosphorylation (thymoma viral proto-oncogene 2 [Akt2], Akt substrate of 160 kDa [AS160]), and fatty acid oxidation in subsarcolemmal and intermyofibrillar mitochondria. Electrotransfection yielded physiologically relevant increases in Pgc-1alpha (also known as Ppargc1a) mRNA and protein ( approximately 25%) in lean and obese muscle. This induced mitochondrial biogenesis, and increased FAT and GLUT4 levels, insulin-stimulated glucose transport, and Akt2 and AS160 phosphorylation in lean and obese animals, while bioactive intramuscular lipids were only reduced in obese muscle. Concurrently, PGC-1alpha increased palmitate oxidation in subsarcolemmal, but not in intermyofibrillar mitochondria, in both groups. In obese compared with lean animals, the PGC-1alpha-induced improvement in insulin-stimulated glucose transport was smaller, but intramuscular lipid reduction was greater. Increases in PGC-1alpha levels, similar to those that can be induced by physiological stimuli, altered intramuscular lipids and improved fatty acid oxidation, insulin signalling and insulin-stimulated glucose transport, albeit to different extents in lean and insulin-resistant muscle. These positive effects are probably attributable to limiting the PGC-1alpha-induced increase in FAT, thereby preventing bioactive lipid accumulation as has occurred in transgenic PGC-1alpha animals.",
"Tumor necrosis factor-alpha (TNF-alpha) has been shown to have certain catabolic effects on fat cells and whole animals. An induction of TNF-alpha messenger RNA expression was observed in adipose tissue from four different rodent models of obesity and diabetes. TNF-alpha protein was also elevated locally and systemically. Neutralization of TNF-alpha in obese fa/fa rats caused a significant increase in the peripheral uptake of glucose in response to insulin. These results indicate a role for TNF-alpha in obesity and particularly in the insulin resistance and diabetes that often accompany obesity."
] |
Changes in oxidative-stress related markers by brain region in Alzheimer's disease and mild cognitive impairment brain tissue
|
Oxidative stress and decreased cellular responsiveness to oxidative stress are thought to influence brain aging and Alzheimer's disease, but the specific patterns of oxidative damage and the underlying mechanism leading to this damage are not definitively known. The objective of this study was to define the pattern of changes in oxidative-stress related markers by brain region in human Alzheimer's disease and mild cognitive impairment brain tissue. Observational case-control studies were identified from systematic queries of PubMed, ISI Web of Science and Scopus databases and studies were evaluated with appropriate quality measures. The data was used to construct a region-by-region meta-analysis of malondialdehyde, 4-hydroxynonenal, protein carbonylation, 8-hydroxyguanine levels and superoxide dismutase, glutathione peroxidase, glutathione reductase and catalase activities. We also evaluated ascorbic acid, tocopherol, uric acid and glutathione levels. The analysis was complicated in several cases by publication bias and/or outlier data. We found that malondialdehyde levels were slightly increased in the temporal and occipital lobes and hippocampus, but this analysis was significantly impacted by publication bias. 4-hydroxynonenal levels were unchanged in every brain region. There was no change in 8-hydroxyguanine level in any brain region and protein carbonylation levels were unchanged except for a slight increase in the occipital lobe. Superoxide dismutase, glutathione peroxidase and reductase and catalase activities were not decreased in any brain region. There was limited data reporting non-enzymatic antioxidant levels in Alzheimer's disease brain, although glutathione and tocopherol levels appear to be unchanged. Minimal quantitative data is available from brain tissue from patients with mild cognitive impairment. While there is modest evidence supporting minor regional changes in markers of oxidative damage, this analysis fails to identify a consistent pattern of pro-oxidative changes and accumulation of oxidative damage in bulk tissue analysis in the setting of Alzheimer's disease, as has been widely reported.
|
[
"Most human tissues used in research are of post mortem origin. This is the case for all brain samples, and due to the difficulty in obtaining a good number of samples, especially in the case of neurodegenerative diseases, male and female samples are often included in the same experimental group. However, the effects of post mortem interval (PMI) and gender differences in the endpoints being analyzed are not always fully understood, as is the case for DNA repair activities. To investigate these effects, in a controlled genetic background, base excision repair (BER) activities were measured in protein extracts obtained from Wistar rat brains from different genders and defined PMI up to 24 hours, using a novel fluorescent-based in vitro incision assay. Uracil and AP-site incision activity in nuclear and mitochondrial extracts were similar in all groups included in this study. Our results show that gender and PMI up to 24 hours have no influence in the activities of the BER proteins UDG and APE1 in rat brains. These findings demonstrate that these variables do not interfere on the BER activities included in these study, and provide a security window to work with UDG and APE1 proteins in samples of post mortem origin.",
"Metallothioneins (MTs) protect cells from oxidative damage by scavenging reactive oxygen species (ROS). Concurrent with protecting cells from ROS-mediated damage, MT transcription is induced by ROS. ROS activate transcription by affecting several signal transduction pathways, many of which have been implicated in regulating MT transcription. ROS-activated intracellular signaling is mediated by the stable lipid peroxide 4-hydroxynonenal (HNE). After determining the level of sensitivity of Hepa 1-6 cells to HNE, MT-1 mRNA expression was shown to be induced in a concentration and time-dependent manner after HNE exposure. Finally, using MT-based reporters, HNE was found to induce MT transcription via both antioxidant response and metal response elements. Thus, ROS may activate MT transcription by generating HNE that in turn affects signaling pathways that regulate MT transcription via the transcription factors AP-1 and MTF-1.",
"The measurement of gene expressions in brains with neurodegenerative diseases is a major area of brain research. The objective of our research was to determine whether quantitative real-time PCR could measure messenger RNA (mRNA) expression in brains with post-mortem intervals beyond 12h. In the present paper, we examined the quality of RNA from brain specimens of both Alzheimer's disease (AD) patients (n = 13) and non-demented normal control subjects (n = 6). To determine a unregulated endogenous reference gene in AD, we measured mRNA expressions of the commonly used reference genes beta-actin, 18S rRNA, and GAPDH. In addition, we determined whether post-mortem interval, brain weight, or age at death influences mRNA expression. Our real-time PCR analysis results indicate that mRNA expression can be detected in all brain specimens for beta-actin, 18S rRNA, GAPDH, and also synaptophysin, a known marker for AD. Further, using real-time PCR analysis, we found that beta-actin and 18S rRNA are differentially expressed in the brain specimens of both AD and control subjects, while GAPDH is similarly expressed in AD and control brain specimens. These findings suggest that GAPDH can be used as a endogenous reference gene in the study of AD brains. A comparative gene expression analysis also suggests that synaptophysin is down-regulated in AD brain specimens compared to control brain specimens.",
"Rising prevalence of neurodegenerative disorders with a steady increase in aged-population necessitates studies of the human brain to understand their pathophysiology. As animal models are not available, medical centers have established \"brain banks\" to provide autopsy brain samples for such research. Frozen tissues must be of optimal quality to permit molecular and protein studies. Post-mortem interval (PMI) is an important factor affecting tissue quality although its effects on brain physiology are unclear. We undertook this study to analyze the biochemical effects of PMI on protein stability in human brains collected at autopsy and stored at the brain bank of a tertiary care neurosciences institute in south India. Different neuroanatomical areas including frontal cortex (FC), cerebellum (CB), caudate nucleus (CD) and substantia nigra (SN) from autopsy human brains (n=9) with varying PMI (4-18 h) were analyzed for pH, protein insolubility, protein oxidation/ nitration and protein expression of glial fibrillary acidic protein (GFAP), synatophysin and neurofilament (NF). Histological changes at different PMI were also assessed. An increase in tissue pH was noted with increasing PMI. Although there was no significant alteration in solubility of proteins, SN showed increased protein oxidation/nitration events, GFAP and NF expression with increasing PMI. No major abnormalities in cell morphology or tissue integrity were noted. Immunohistochemistry with GFAP and NF did not show any significant increase in signal in FC at high PMI. In post-mortem human brains, although there were no gross structural changes at the tissue level with increasing PMI, biochemical events such as oxidative and nitrosative damage of cellular proteins, tissue pH could be considered as markers of tissue quality for biochemical research. Further, SN was found to be most susceptible to PMI related changes.",
"To verify the hypothesis that borderline diabetes may increase the risk of dementia and Alzheimer's disease, a community-based cohort of 1,173 dementia- and diabetes-free individuals aged >or=75 years was longitudinally examined three times to detect patients with dementia and Alzheimer's disease (Diagnostic and Statistical Manual of Mental Disorders, Revised Third Edition criteria). Borderline diabetes was defined as a random plasma glucose level of 7.8-11.0 mmol/l. Data were analyzed using Cox proportional hazards models. During the 9-year follow-up, 397 subjects developed dementia, including 307 Alzheimer's cases. At baseline, 47 subjects were identified with borderline diabetes. Borderline diabetes was associated with adjusted hazard ratios (95% CIs) of 1.67 (1.04-2.67) for dementia and 1.77 (1.06-2.97) for Alzheimer's disease; the significant associations were present after additional adjustment for future development of diabetes. Stratified analysis suggested a significant association between borderline diabetes and Alzheimer's disease only among noncarriers of APOE epsilon4 allele. There was an interaction between borderline diabetes and severe systolic hypertension on the risk of Alzheimer's disease (P = 0.04). We conclude that borderline diabetes is associated with increased risks of dementia and Alzheimer's disease; the risk effect is independent of the future development of diabetes. Borderline diabetes may interact with severe systolic hypertension to multiply the risk of Alzheimer's disease.",
"At autopsy, the time that has elapsed since the time of death is routinely documented and noted as the postmortem interval (PMI). The PMI of human tissue samples is a parameter often reported in research studies and comparable PMI is preferred when comparing different populations, i.e., disease versus control patients. In theory, a short PMI may alleviate non-experimental protein denaturation, enzyme activity, and other chemical changes such as the pH, which could affect protein and nucleic acid integrity. Previous studies have compared PMI en masse by looking at many different individual cases each with one unique PMI, which may be affected by individual variance. To overcome this obstacle, in this study human hippocampal segments from the same individuals were sampled at different time points after autopsy creating a series of PMIs for each case. Frozen and fixed tissue was then examined by Western blot, RT-PCR, and immunohistochemistry to evaluate the effect of extended PMI on proteins, nucleic acids, and tissue morphology. In our results, immunostaining profiles for most proteins remained unchanged even after PMI of over 50 h, yet by Western blot distinctive degradation patterns were observed in different protein species. Finally, RNA integrity was lower after extended PMI; however, RNA preservation was variable among cases suggesting antemortem factors may play a larger role than PMI in protein and nucleic acid integrity.",
"Post-mortem brain tissue provides a unique opportunity to uncover the genes or proteins involved in the pathophysiology of neuropsychiatric disorders. Protein phosphorylation is a common protein modification within intracellular signaling pathways that affects the distribution and function of protein, and has been hypothesized to be of major importance in both the pathophysiology and treatment of major neuropsychiatric disorders. Thus, we were interested in ascertaining the stability of the phosphorylated forms of proteins that are involved in cellular signaling. Antibodies against phospho-tyrosine, phospho-threonine, and phospho-PKA substrates were used to examine the PMI effects on the general amounts of proteins in their phosphorylated form. Phospho-specific antibodies for ERK, JNK, RSK, CREB, and ATF-2 were used to test the effects of PMI on specific proteins whose functioning are known to be regulated markedly by phosphorylation. We found that PMI rapidly decreased the levels of proteins in their phosphorylated states and also decreased the total levels of certain proteins. The PMI effects were observed in the samples stored at both 4 degrees C and room temperature, in both frontal cortex and hippocampus. Thus, it appears that measurements (such as two-dimensional gel electrophoresis and functional assays) that rely on the phosphorylation state of proteins would be extremely sensitive to PMI."
] |
The visceral role of misato in Drosophila and its implications for pathogenesis of visceral myopathy
|
Genetic mechanisms for the pathogenesis of visceral myopathy (VM) have been rarely demonstrated. Here we report the visceral role of misato (mst) in Drosophila and its implications for the pathogenesis of VM. Depletion of mst using three independent RNAi lines expressed by a pan-muscular driver elicited characteristic symptoms of VM, such as abnormal dilation of intestinal tracts, reduced gut motility, feeding defects, and decreased life span. By contrast, exaggerated expression of mst reduced intestine diameters, but increased intestinal motilities along with thickened muscle fibers, demonstrating a critical role of mst in the visceral muscle. Mst expression was detected in the adult intestine with its prominent localization to actin filaments and was required for maintenance of intestinal tubulin and actomyosin structures. Consistent with the subcellular localization of Mst, the intestinal defects induced by mst depletion were dramatically rescued by exogenous expression of an actin member. Upon ageing the intestinal defects were deteriorative with marked increase of apoptotic responses in the visceral muscle. Taken together, we propose the impairment of actomyosin structures induced by mst depletion in the visceral muscle as a pathogenic mechanism for VM.
|
[
"The conditional expression of hairpin constructs in Drosophila melanogaster has emerged in recent years as a method of choice in functional genomic studies. To date, upstream activating site-driven RNA interference constructs have been inserted into the genome randomly using P-element-mediated transformation, which can result in false negatives due to variable expression. To avoid this problem, we have developed a transgenic RNA interference vector based on the phiC31 site-specific integration method.",
"Although several neural pathways have been implicated in feeding behaviors in mammals [1-7], it remains unclear how the brain coordinates feeding motivations to maintain a constant body weight (BW). Here, we identified a neuropeptide pathway important for the satiety and BW control in Drosophila. Silencing of myoinhibitory peptide (MIP) neurons significantly increased BW through augmented food intake and fat storage. Likewise, the loss-of-function mutation of mip also increased feeding and BW. Suppressing the MIP pathway induced satiated flies to behave like starved ones, with elevated sensitivity toward food. Conversely, activating MIP neurons greatly decreased food intake and BW and markedly blunted the sensitivity of starved flies toward food. Upon terminating the activation protocol of MIP neurons, the decreased BW reverts rapidly to the normal level through a strong feeding rebound, indicating the switch-like role of MIP pathway in feeding. Surprisingly, the MIP-mediated BW decrease occurred independently of sex peptide receptor (SPR), the only known receptor for MIP, suggesting the presence of a yet-unknown MIP receptor. Together, our results reveal a novel anorexigenic pathway that controls satiety in Drosophila and provide a new avenue to study how the brain actively maintains a constant BW.",
"To facilitate the genetic analysis of muscle assembly and maintenance, we have developed a method for efficient RNA interference (RNAi) in Drosophila primary cells using double-stranded RNAs (dsRNAs). First, using molecular markers, we confirm and extend the observation that myogenesis in primary cultures derived from Drosophila embryonic cells follows the same developmental course as that seen in vivo. Second, we apply this approach to analyze 28 Drosophila homologs of human muscle disease genes and find that 19 of them, when disrupted, lead to abnormal muscle phenotypes in primary culture. Third, from an RNAi screen of 1140 genes chosen at random, we identify 49 involved in late muscle differentiation. We validate our approach with the in vivo analyses of three genes. We find that Fermitin 1 and Fermitin 2, which are involved in integrin-containing adhesion structures, act in a partially redundant manner to maintain muscle integrity. In addition, we characterize CG2165, which encodes a plasma membrane Ca2+-ATPase, and show that it plays an important role in maintaining muscle integrity. Finally, we discuss how Drosophila primary cells can be manipulated to develop cell-based assays to model human diseases for RNAi and small-molecule screens.",
"We describe a method for preparing highly enriched cultures of Drosophila myoblasts from a heterogeneous cell population derived from gastrulating embryos. Enriched cultures are prepared by plating this heterogeneous population of cells in medium from which much of the free calcium is chelated by ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA). Adhesion of myoblasts to tissue culture plastic is better than that of other cell types when plated in this medium. Data concerning cell identity, timing of S phase, and fusion kinetics document the degree of enrichment for myogenic cells and illustrate their synchronous differentiation in vitro.",
"Using primary embryonic Drosophila cell cultures, we have investigated the assembly of transcellular microtubule bundles in epidermal tendon cells. Muscles attach to the tendon cells of previously undescribed epidermal balls that form shortly after culture initiation. Basal capture of microtubule ends in cultured tendon cells is confined to discrete sites that occupy a relatively small proportion of the basal cell surface. These capturing sites are associated with hemiadherens junctions that link the ends of muscle cells to tendon cell bases. In vivo, muscle attachment and microtubule capture occur across the entire cell base. The cultured tendon cells reveal that the basal ends of their microtubules can be precisely targeted to small, pre-existing, structurally well-defined cortical capturing sites. However, a search and capture targeting procedure, such as that undertaken by kinetochore microtubules, cannot fully account for the precision of microtubule capture and positioning in tendon cells. We propose that cross-linkage of microtubules is also required to zip them into apicobasally oriented alignment, progressing from captured basal plus ends to apical minus ends. This involves repositioning of apical minus ends before they become anchored to an apical set of hemiadherens junctions. The proposal is consistent with our finding that hemiadherens junctions assemble at tendon cell bases before they do so at cell apices in both cultures and embryos. It is argued that control of microtubule positioning in the challenging spatial situations found in vitro involves the same procedures as those that operate in vivo.",
"The larval gut of Drosophila is coated with visceral muscles of mesodermal origin. In the midgut region this musculature comprises circular and longitudinal fibres. The complete visceral musculature is described to be removed during metamorphosis and to be replaced by a newly differentiated imaginal tissue resembling the morphology of the larval musculature. However, progenitors of this imaginal visceral musculature have never been detected prior to differentiation. Here I present results indicating that the longitudinal visceral musculature of the midgut completely persists through metamorphosis. Single cells expressing green fluorescent protein (GFP) as a marker were transplanted at the blastoderm stage. All clones contributing to the longitudinal visceral musculature detected in third instar larvae were recovered after metamorphosis in adult flies. Further evidence for the persistence of the larval visceral musculature was obtained from the P[Gal4] insertion line 5053A. It expresses GAL4 specifically in the longitudinal visceral muscles of the midgut of all developmental stages to the adult fly beginning at the end of embryogenesis. By using GFP as a reporter, it was possible to follow these cells through the entire metamorphosis. Although the muscles undergo dramatic morphological changes including the loss of their contractile system, no evidence for a replacement of the larval visceral musculature by imaginal precursor cells was detected."
] |
Association of serum folate level with the severity of white matter hyperintensity and presence of cerebral microbleeds
|
To investigate the association of serum folate level with the severity of white matter hyperintensity (WMH) and presence of cerebral microbleeds (CMB).
|
[
"Cerebral small vessel disease (SVD) is a common accompaniment of ageing. Features seen on neuroimaging include recent small subcortical infarcts, lacunes, white matter hyperintensities, perivascular spaces, microbleeds, and brain atrophy. SVD can present as a stroke or cognitive decline, or can have few or no symptoms. SVD frequently coexists with neurodegenerative disease, and can exacerbate cognitive deficits, physical disabilities, and other symptoms of neurodegeneration. Terminology and definitions for imaging the features of SVD vary widely, which is also true for protocols for image acquisition and image analysis. This lack of consistency hampers progress in identifying the contribution of SVD to the pathophysiology and clinical features of common neurodegenerative diseases. We are an international working group from the Centres of Excellence in Neurodegeneration. We completed a structured process to develop definitions and imaging standards for markers and consequences of SVD. We aimed to achieve the following: first, to provide a common advisory about terms and definitions for features visible on MRI; second, to suggest minimum standards for image acquisition and analysis; third, to agree on standards for scientific reporting of changes related to SVD on neuroimaging; and fourth, to review emerging imaging methods for detection and quantification of preclinical manifestations of SVD. Our findings and recommendations apply to research studies, and can be used in the clinical setting to standardise image interpretation, acquisition, and reporting. This Position Paper summarises the main outcomes of this international effort to provide the STandards for ReportIng Vascular changes on nEuroimaging (STRIVE).",
"Cerebral microbleeds (CMBs) are increasingly recognised neuroimaging findings in individuals with cerebrovascular disease and dementia, and in normal ageing. There has been substantial progress in the understanding of CMBs in recent years, particularly in the development of newer MRI methods for the detection of CMBs and the application of these techniques to population-based samples of elderly people. In this Review, we focus on these recent developments and their effects on two main questions: how CMBs are detected, and how CMBs should be interpreted. The number of CMBs detected depends on MRI characteristics, such as pulse sequence, sequence parameters, spatial resolution, magnetic field strength, and image post-processing, emphasising the importance of taking into account MRI technique in the interpretation of study results. Recent investigations with sensitive MRI techniques have indicated a high prevalence of CMBs in community-dwelling elderly people. We propose a procedural guide for identification of CMBs and suggest possible future approaches for elucidating the role of these common lesions as markers for, and contributors to, small-vessel brain disease.",
"We performed a systematic review and meta-analysis to assess whether the presence of cerebral microbleeds (CMBs) on pretreatment MRI of patients with acute ischemic stroke treated with IV thrombolysis is associated with increased risk of symptomatic intracerebral hemorrhage (ICH) and poor functional outcome. We searched PubMed for relevant studies and calculated pooled odds ratios (OR) for symptomatic ICH and poor (i.e., modified Rankin Scale score >2) 3- to 6-month functional outcome using random effects models with DerSimonian-Laird weights among individuals with vs without CMBs. Eight eligible studies including 2,601 stroke patients treated with IV thrombolysis were pooled in a meta-analysis. The cumulative CMBs prevalence was 24% (95% confidence interval [CI] 18%-30%). The pooled symptomatic ICH incidence was 5% (95% CI 4%-7%) among patients with CMBs and 3% (95% CI 2%-5%) in patients without CMBs. CMB presence was associated with higher risk of symptomatic ICH compared to patients without CMBs (OR 2.18; 95% CI 1.12-4.22; p = 0.021). Four studies (n = 1,665) reported data on 3- to 6-month poststroke functional outcome. The pooled incidence of poor functional outcome was 52% (95% CI 45%-59%) in patients with CMBs vs 41% (95% CI 35%-46%) in those without CMBs. Meta-analysis of these studies demonstrated OR for CMBs presence and adverse outcome to be 1.58 (95% CI 1.18-2.14; p = 0.002). CMBs are associated with greater symptomatic ICH risk and poor functional outcome in ischemic stroke patients undergoing thrombolytic therapy. In the absence of adjusted analyses and randomized evidence, this risk seems acceptable and should probably not discourage recanalization therapies in this patient population (Level B recommendation: nonrandomized Class IIa evidence).",
"To determine prevalence and severity of microbleeds (MBs) in a large cohort of patients attending a memory clinic. The authors consecutively included patients attending their memory clinic between January 2002 and April 2005. They analyzed prevalence and number of MBs according to demographic, diagnostic, and MRI data. The authors included 772 patients (53% men, age 66 +/- 11). One hundred twenty-seven patients (17%) exhibited at least one MB. The prevalence differed according to diagnostic groups (p < 0.0001): Sixty-five percent of patients with vascular dementia exhibited MBs vs 18% of Alzheimer disease patients, 20% of mild cognitive impairment patients, and 10% of patients with subjective complaints. The presence of MBs was associated with age, white matter hyperintensities, lacunar infarcts, and infarcts. The prevalence of microbleeds (MBs) in a large cohort of patients attending a memory clinic is higher than previously described in community samples and lower than reported in stroke patients. This finding of a relatively high proportion of MBs in Alzheimer disease and mild cognitive impairment provides further evidence for the involvement of vascular factors in neurodegenerative diseases such as Alzheimer disease.",
"Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) is a hereditary arteriopathy caused by mutations of the Notch3 gene. The risk factors for cerebral microhaemorrhages (CM), their relationship to other MRI lesions in the disease and their potential clinical impact have not been previously defined. Our purpose was to examine the frequency, number and location of microhaemorrhages in a multicentre cohort study, defining predisposing factors and associated radiographic markers in CADASIL patients. We collected clinical data from 147 consecutive patients enrolled in an ongoing prospective cohort study. Degree of neurological disability and cognitive impairment were assessed by standardized scales. T(1)-weighted, FLAIR and T2*-weighted gradient-echo (GE) MRI sequences were performed. Volume and location of lacunar infarcts and white matter hyperintensity (WMH) were assessed. Number and location of CM were recorded. CM were present in 35% patients, most commonly occurring in the thalamus, brainstem and basal ganglia. The location of CM qualitatively differed from areas of lacunar infarction and WMH. There was a significant association between the presence of CM and a history of hypertension (P = 0.005), systolic blood pressure (SBP) (P = 0.014), haemoglobin A1c (HbA1c) (P = 0.004) and the volume of lacunar infarcts (P = 0.010) and WMHs (P = 0.046). The number of CM was independently associated with SBP (P = 0.005), the diagnosis of hypertension (P = 0.0004), volume of WMH (P = 0.0005) and lacunar infarcts (P = 0.004). In contrast, no association was found between blood pressure or HbA1c and the load of WMH or lacunar infarcts. The presence of CM was independently associated with increased modified Rankin scores. CM are independently associated with blood pressure and HbA1c as well as with lacunar infarct and WMH volume in CADASIL. Both the vascular risk factors and regional distribution of CM appear distinct from those associated with other MRI markers, suggesting a distinct pathological process. These lesions have a potential clinical impact in CADASIL. These findings further suggest that modulation of blood pressure and glucose levels might influence the course of the disease.",
"Brain microbleeds (BMBs) are seen as small, homogeneous, round foci of low signal intensity on magnetic resonance imaging gradient echo (GRE) T2 sequences. BMBs might only be a biomarker for microangiopathy, or alternatively BMBs might provide useful diagnostic and prognostic information, potentially with therapeutic implications for the treatment of stroke. Because of the rapid expansion in recent BMB research, we systematically reviewed and critically appraised the published literature according to QUADAS, STARD and Cochrane principles. Our selection criteria were met by 54 studies of 53 case series involving 9073 participants, 4432 of whom were people with cerebrovascular diseases. There were significant biases in many of the studies: variation in MRI magnet strength, flip angle, slice gap and slice thickness; inconsistent definitions of BMB size (23% did not define size at all, and of those that did 44% chose a diameter of < or =5 mm); only 30% included participants who were representative of the disease under study; and only 53% mentioned that BMB evaluation was blinded to other factors of interest. By pooling data from similar studies, we found that the prevalence of BMBs was 5% [95% confidence interval (CI) 4-6] in healthy adults, 34% (95% CI 31-36) in people with ischaemic stroke, and 60% (95% CI 57-64) in people with non-traumatic intracerebral haemorrhage (ICH). In the studies where a distinction could be made, BMBs were more prevalent among recurrent strokes than first-ever strokes: they affected 23% (95% CI 18-29) with first-ever ischaemic stroke but 44% (95% CI 34-54) with recurrent ischaemic stroke, and 52% (95% CI 47-56) with first-ever ICH but 83% (95% CI 71-90) with recurrent ICH. By pooling data that could be extracted from similar studies, it appears that BMBs are associated with hypertension (OR 3.9, 95% CI 2.4-6.4) and diabetes mellitus (OR 2.2, 95% CI 1.2-4.2) in otherwise healthy adults, and that they are associated with hypertension (OR 2.3, 95% CI 1.7-3.0) in adults with cerebrovascular diseases. The association with hypertension was robust in sensitivity analyses. There is a pressing need for better designed studies to assess the diagnostic utility of BMBs, disentangle the many likely influences on their occurrence, and determine their prognostic utility and whether they should influence treatment. We conclude by proposing criteria for ideal study design and reporting.",
"Traumatic microbleeds (TMBs) can be regarded as a radiological marker of diffuse axonal injury (DAI). We sought to investigate the impact of the field strengths on the depiction of TMBs by T2*-weighted gradient echo magnetic resonance imaging (MRI). By the use of comparative MRI of 14 patients (age range, 22-62 years) on 1.5- and a 3 T (Tesla) systems at a median time interval of 61 months after traumatic brain injury (TBI), we found 239 (range 0.5-48.5, median 7.5) TMBs at 1.5 T, and 470 (range 2-118, median 18.5) TMBs at 3 T, respectively (p=0.001). However, in all but one patients MRI at 1.5 T also clearly showed TMBs. A significant negative correlation between the number of TMBs and the time interval TBI-MRI was observed, which was weaker for the imaging at 3 T (r(s)=-0.798; p=0.001; and r(s)=-0.649; p=0.012, respectively). In conclusion, T2*-weighted gradient-echo MRI at 3 T is superior as compared to MRI at 1.5 T for the detection of TMBs. Nevertheless, in clinical practice, MRI at 1.5 T seems to be sufficient for this purpose. MRI at 3 T may be appropriate if there is a strong clinical suspicion of DAI, despite unremarkable routine MRI, and possibly also if evidence of DAI is sought after a long interval from trauma."
] |
Immunotherapy for metastatic renal-cell carcinoma
|
The management of metastatic renal-cell carcinoma (mRCC) represents an important clinical challenge. Since being approved in the early 1990s, aspecific immunotherapy has been a mainstay of treatment for mRCC and the only therapy that has demonstrated long-term cures for mRCC. However, in recent times there have been landmark advances made in the field of specific immunotherapy for a number of malignancies, including kidney cancer. This review outlines the range of immunobased agents currently available for the treatment of mRCC.
|
[
"Although immune mechanisms can suppress tumour growth, tumours establish potent, overlapping mechanisms that mediate immune evasion. Emerging evidence suggests a link between angiogenesis and the tolerance of tumours to immune mechanisms. Hypoxia, a condition that is known to drive angiogenesis in tumours, results in the release of damage-associated pattern molecules, which can trigger the rejection of tumours by the immune system. Thus, the counter-activation of tolerance mechanisms at the site of tumour hypoxia would be a crucial condition for maintaining the immunological escape of tumours. However, a direct link between tumour hypoxia and tolerance through the recruitment of regulatory cells has not been established. We proposed that tumour hypoxia induces the expression of chemotactic factors that promote tolerance. Here we show that tumour hypoxia promotes the recruitment of regulatory T (T(reg)) cells through induction of expression of the chemokine CC-chemokine ligand 28 (CCL28), which, in turn, promotes tumour tolerance and angiogenesis. Thus, peripheral immune tolerance and angiogenesis programs are closely connected and cooperate to sustain tumour growth.",
"Although it was thought that apoptotic cells, when rapidly phagocytosed, underwent a silent death that did not trigger an immune response, in recent years a new concept of immunogenic cell death (ICD) has emerged. The immunogenic characteristics of ICD are mainly mediated by damage-associated molecular patterns (DAMPs), which include surface-exposed calreticulin (CRT), secreted ATP and released high mobility group protein B1 (HMGB1). Most DAMPs can be recognized by pattern recognition receptors (PRRs). In this Review, we discuss the role of endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) in regulating the immunogenicity of dying cancer cells and the effect of therapy-resistant cancer microevolution on ICD.",
"The Memorial Sloan-Kettering Cancer Center risk model classifies patients with metastatic renal cell carcinoma (RCC) by 5 pretreatment features as favorable, intermediate, and poor risk. The number of Memorial Sloan-Kettering Cancer Center patients in each risk group was examined by year of treatment to analyze stage migration. The distribution of risk groups was examined retrospectively in 789 Memorial Sloan-Kettering Cancer Center patients with metastatic RCC treated in a first-line therapy clinical trial from 1975 to 2007. Date of treatment onset was divided into 6 cohorts between 1975 and 2007 (1975-1980, 1981-1985, 1986-1990, 1991-1995, 1996-2001, and 2001-2007). The median age of the first-line metastatic RCC clinical trial patients was 59 years (range, 20-82 years). Most patients received cytokine therapy (55%), 37% received chemotherapy/other, and 8% received vascular endothelial growth factor-targeted therapies. Overall survival increased with each consecutive cohort year group (P < .001). Median survival was 0.43 years (95% confidence interval [CI], 0.27-0.68) in the 1973-1980 cohort and 1.5 years in the 2001-2007 cohort (95% CI, 1.15-2.11). Memorial Sloan-Kettering Cancer Center risk-group distribution shifted between 1975 and 2007 (P < .0001). The poor-risk group proportion became smaller (from 44% in 1975-1980 to 13% in 2001-2007), whereas the favorable-risk group increased (from 0% in 1975-1980 to 49% in 2001-2007). The intermediate-risk group remained stable at 50%. After adjusting for type of therapy, the shifts continue to be significant (P < .0001). The risk-group distribution for metastatic RCC patients in clinical trials shifted from 1975 to 2007. These shifts have direct implications for data analysis, interpretation of metastatic RCC trends, and drug development.",
"Mucosal tissues require constant immune surveillance to clear harmful pathogens while maintaining tolerance to self Ags. Regulatory T cells (Tregs) play a central role in this process and expression of alpha(E)beta(7) has been reported to define a subset of Tregs with tropism for inflamed tissues. However, the signals responsible for recruiting Tregs to epithelial surfaces are poorly understood. We have isolated a subset of CCR10-expressing CD25+CD4+Foxp3+ Tregs with potent anti-inflammatory properties from chronically inflamed human liver. The CCR10+ Tregs were detected around bile ducts that expressed increased levels of the CCR10 ligand CCL28. CCL28 was secreted by primary human cholangiocytes in vitro in response to LPS, IL-1beta, or bile acids. Exposure of CCR10+ Tregs to CCL28 in vitro stimulated migration and adhesion to mucosal addressin cell adhesion molecule-1 and VCAM-1. Liver-derived CCR10+ Tregs expressed low levels of CCR7 but high levels of CXCR3, a chemokine receptor associated with infiltration into inflamed tissue and contained a subset of alpha(E)beta7(+) cells. We propose that CXCR3 promotes the recruitment of Tregs to inflamed tissues and CCR10 allows them to respond to CCL28 secreted by epithelial cells resulting in the accumulation of CCR10+ Tregs at mucosal surfaces.",
"T-cell adhesion/costimulatory molecules and their cognate receptors on target cells play a major role in T-cell receptor (TCR)-mediated activities. Here, we compared the involvement of CD103 and LFA-1, and their respective ligands, in the maturation of the cytotoxic immune synapse (cIS) and in the activation of CTL effector functions. Our results indicate that cytotoxicity toward cancer cells and, to a lesser extent, cytokine production by specific CTL require, together with TCR engagement, the interaction of either CD103 with E-cadherin or LFA-1 with ICAM-1. Flow-based adhesion assay showed that engagement of CD103 or LFA-1, together with TCR, enhances the strength of the T-cell/target cell interaction. Moreover, electron microscopic analyses showed that integrin-dependent mature cIS (mcIS) displays a cohesive ultrastructure, with tight membrane contacts separated by extensive clefts. In contrast, immature cIS (icIS), which is unable to trigger target cell lysis, is loose, with multiple protrusions in the effector cell membrane. Experiments using confocal microscopy revealed polarized cytokine release and degranulation at the mcIS associated with target cell killing, whereas icIS is characterized by failure of IFN-γ and granzyme B relocalization. Thus, interactive forces between CTL and epithelial tumor cells, mainly regulated by integrin engagement, correlate with maturity and the ultrastructure of the cIS and influence CTL effector functions. These results provide new insights into molecular mechanisms regulating antitumor CTL responses and may lead to the development of more efficient cancer immunotherapy strategies.",
"The A2A adenosine receptor (A2AR) has been shown to be a critical and nonredundant negative regulator of immune cells in protecting normal tissues from inflammatory damage. We hypothesized that A2AR also protects cancerous tissues by inhibiting incoming antitumor T lymphocytes. Here we confirm this hypothesis by showing that genetic deletion of A2AR in the host resulted in rejection of established immunogenic tumors in approximately 60% of A2AR-deficient mice with no rejection observed in control WT mice. The use of antagonists, including caffeine, or targeting the A2 receptors by siRNA pretreatment of T cells improved the inhibition of tumor growth, destruction of metastases, and prevention of neovascularization by antitumor T cells. The data suggest that effects of A2AR are T cell autonomous. The inhibition of antitumor T cells via their A2AR in the adenosine-rich tumor microenvironment may explain the paradoxical coexistence of tumors and antitumor immune cells in some cancer patients (the \"Hellstrom paradox\"). We propose to target the hypoxia-->adenosine-->A2AR pathway as a cancer immunotherapy strategy to prevent the inhibition of antitumor T cells in the tumor microenvironment. The same strategy may prevent the premature termination of immune response and improve the vaccine-induced development of antitumor and antiviral T cells. The observations of autoimmunity during melanoma rejection in A2AR-deficient mice suggest that A2AR in T cells is also important in preventing autoimmunity. Thus, although using the hypoxia-->adenosine-->A2AR pathway inhibitors may improve antitumor immunity, the recruitment of this pathway by selective drugs is expected to attenuate the autoimmune tissue damage.",
"Nivolumab (a programmed death 1 [PD-1] checkpoint inhibitor) and ipilimumab (a cytotoxic T-lymphocyte-associated antigen 4 [CTLA-4] checkpoint inhibitor) have been shown to have complementary activity in metastatic melanoma. In this randomized, double-blind, phase 3 study, nivolumab alone or nivolumab plus ipilimumab was compared with ipilimumab alone in patients with metastatic melanoma. We assigned, in a 1:1:1 ratio, 945 previously untreated patients with unresectable stage III or IV melanoma to nivolumab alone, nivolumab plus ipilimumab, or ipilimumab alone. Progression-free survival and overall survival were coprimary end points. Results regarding progression-free survival are presented here. The median progression-free survival was 11.5 months (95% confidence interval [CI], 8.9 to 16.7) with nivolumab plus ipilimumab, as compared with 2.9 months (95% CI, 2.8 to 3.4) with ipilimumab (hazard ratio for death or disease progression, 0.42; 99.5% CI, 0.31 to 0.57; P<0.001), and 6.9 months (95% CI, 4.3 to 9.5) with nivolumab (hazard ratio for the comparison with ipilimumab, 0.57; 99.5% CI, 0.43 to 0.76; P<0.001). In patients with tumors positive for the PD-1 ligand (PD-L1), the median progression-free survival was 14.0 months in the nivolumab-plus-ipilimumab group and in the nivolumab group, but in patients with PD-L1-negative tumors, progression-free survival was longer with the combination therapy than with nivolumab alone (11.2 months [95% CI, 8.0 to not reached] vs. 5.3 months [95% CI, 2.8 to 7.1]). Treatment-related adverse events of grade 3 or 4 occurred in 16.3% of the patients in the nivolumab group, 55.0% of those in the nivolumab-plus-ipilimumab group, and 27.3% of those in the ipilimumab group. Among previously untreated patients with metastatic melanoma, nivolumab alone or combined with ipilimumab resulted in significantly longer progression-free survival than ipilimumab alone. In patients with PD-L1-negative tumors, the combination of PD-1 and CTLA-4 blockade was more effective than either agent alone. (Funded by Bristol-Myers Squibb; CheckMate 067 ClinicalTrials.gov number, NCT01844505.).",
"Mucosae-associated epithelial chemokine (MEC) is a novel chemokine whose mRNA is most abundant in salivary gland, with strong expression in other mucosal sites, including colon, trachea, and mammary gland. MEC is constitutively expressed by epithelial cells; MEC mRNA is detected in cultured bronchial and mammary gland epithelial cell lines and in epithelia isolated from salivary gland and colon using laser capture microdissection, but not in the endothelial, hemolymphoid, or fibroblastic cell lines tested. Although MEC is poorly expressed in skin, its closest homologue is the keratinocyte-expressed cutaneous T cell-attracting chemokine (CTACK; CCL27), and MEC supports chemotaxis of transfected lymphoid cells expressing CCR10, a known CTACK receptor. In contrast to CTACK, however, MEC also supports migration through CCR3. Consistent with this, MEC attracts eosinophils in addition to memory lymphocyte subsets. These results suggest an important role for MEC in the physiology of extracutaneous epithelial tissues, including diverse mucosal organs.",
"Myeloid suppressor cells with high arginase activity are found in tumors and spleen of mice with colon and lung cancer. These cells, described as macrophages or immature dendritic cells, deplete arginine and impair T cell proliferation and cytokine production. Although arginase activity has been described in cancer patients, it is thought to originate from tumor cells metabolizing arginine to ornithine needed to sustain rapid cell proliferation. The goal of this study was to determine whether myeloid suppressor cells producing high arginase existed in renal cell carcinoma patients. Peripheral blood mononuclear cells from 123 patients with metastatic renal cell carcinoma, prior to treatment, were found to have a significantly increased arginase activity. These patients had a markedly decreased cytokine production and expressed low levels of T cell receptor CD3zeta chain. Cell separation studies showed that the increased arginase activity was limited to a specific subset of CD11b+, CD14-, CD15+ cells with a polymorphonuclear granulocyte morphology and markers, instead of macrophages or dendritic cells described in mouse models. Furthermore, these patients had low levels of arginine and high levels of ornithine in plasma. Depletion of the CD11b+, CD14- myeloid suppressor cells reestablished T cell proliferation and CD3zeta chain expression. These results showed, for the first time, the existence of suppressor myeloid cells producing arginase in human cancer patients. In addition, it supports the concept that blocking arginase may be an important step in the success of immunotherapy.",
"Vascular endothelial growth factor (VEGF) is a major angiogenic factor. Osteosarcoma is characterised by hypervascularity and metastatic potential. We examined VEGF mRNA expression, VEGF isoform pattern and VEGF receptor (flt-1 and KDR) by RT-PCR analysis in 30 osteosarcomas. All 30 osteosarcomas expressed VEGF mRNA. 17 osteosarcomas (57%) expressed flt-1 mRNA, whilst 20 (67%) expressed KDR mRNA. 6/30 (20%) osteosarcomas were positive for VEGF121 only, 8 (27%) for VEGF121 + VEGF165, and 16 (53%) for VEGF121 + VEGF165 + VEGF189. Patients with osteosarcomas with VEGF165 (n = 24) had significantly poorer prognosis in comparison with those without VEGF165 (P = 0.022, Wilcoxon's test). The osteosarcomas with VEGF165 had significantly increased vascularity assessed on sections immunostained for CD34 (P < 0.001, Mann-Whitney U test). Although VEGF165 is a soluble isoform, it is also retained on the cellular surface. These results suggest that cell-retained VEGF isoforms (VEGF165, VEGF189) might be essential for neovascularisation in osteosarcoma, whilst the soluble VEGF121 isoform is not sufficient to stimulate neovascularisation in this type of neoplasm."
] |
Assessment of exercise tolerance in adolescents shortly after sport-related concussion
|
To evaluate (1) systematic assessment of exercise tolerance in adolescents shortly after sport-related concussion (SRC) and (2) the prognostic utility of such assessment.
|
[
"With heightened awareness of concussion, there is a need to assess and manage the concussed patient in a consistent manner. Unfortunately, concussion physical examination has not been standardized or supported by evidence. Important questions remain about the physical examination. Review of ClinicalKey, Cochrane, MEDLINE, and PubMed prior to July 2015 was performed using search terms, including concussion, mTBI, physical examination, mental status, cranial nerves, reflexes, cervical, vestibular, and oculomotor. The references of the pertinent articles were reviewed for other relevant sources. Clinical review. Level 3. The pertinent physical examination elements for concussion include evaluation of cranial nerves, manual muscle testing, and deep tendon reflexes; inspecting the head and neck for trauma or tenderness and cervical range of motion; Spurling maneuver; a static or dynamic balance assessment; screening ocular examination; and a mental status examination that includes orientation, immediate and delayed recall, concentration, mood, affect, insight, and judgment. Other examination elements to consider, based on signs, symptoms, or clinical suspicion, include testing of upper motor neurons, cervical strength and proprioception, coordination, pupillary reactivity, and visual acuity; examination of the jaw, temporomandibular joint, and thoracic spine; fundoscopic evaluation; orthostatic vital signs; assessment of dynamic visual acuity; and screening for depression, anxiety, substance abuse disorders, and preinjury psychiatric difficulties. Various elements of the physical examination, such as screening ocular examination, cervical musculoskeletal examination, static and/or dynamic balance assessment, and mental status examination, appear to have utility for evaluating concussion; however, data on validity are lacking.",
"Accommodative dysfunction is a common oculomotor sequelae of mild traumatic brain injury (mTBI). This study evaluated a range of dynamic (objective) and static (subjective) measures of accommodation in 12 nonstrabismic individuals with mTBI and near vision-related symptoms before and after oculomotor training (OMT) and placebo (P) training (6 wk, two sessions per week, 3 h of training each). Following OMT, the dynamics of accommodation improved markedly. Clinically, there was a significant increase in the maximum accommodative amplitude both monocularly and binocularly. In addition, the near vision symptoms reduced along with improved visual attention. None of the measures were found to change significantly following P training. These results provide evidence for a significant positive effect of the accommodatively based OMT on accommodative responsivity. Such improvement is suggestive of oculomotor learning, demonstrating considerable residual brain-visual system plasticity in the adult compromised brain.",
"To determine the prevalence of vision diagnoses after concussion in adolescents. Cross-sectional study from July 1, 2013 to February 28, 2014, of patients aged 11 to 17 years with concussion evaluated in a comprehensive concussion program. A total of 100 adolescents were examined, with a mean age of 14.5 years. Overall, 69% had one or more of the following vision diagnoses: accommodative disorders (51%), convergence insufficiency (49%), and saccadic dysfunction (29%). In all, 46% of patients had more than one vision diagnosis. A high prevalence of vision diagnoses (accommodative, binocular convergence, and saccadic eye movement disorders) was found in this sample of adolescents with concussion, with some manifesting more than one vision diagnosis. These data indicate that a comprehensive visual examination may be helpful in the evaluation of a subset of adolescents with concussion. Academic accommodations for students with concussion returning to the classroom setting should account for these vision diagnoses.",
"To compare symptoms in patients with physiologic postconcussion disorder (PCD) versus cervicogenic/vestibular PCD. We hypothesized that most symptoms would not be equivalent. In particular, we hypothesized that cognitive symptoms would be more often associated with physiologic PCD. Retrospective review of symptom reports from patients who completed a 22-item symptom questionnaire. University-based concussion clinic. Convenience sample of 128 patients who had symptoms after head injury for more than 3 weeks and who had provocative treadmill exercise testing. Subjects were classified as either physiologic PCD (abnormal treadmill performance and a normal cervical/vestibular physical examination) or cervicogenic/vestibular PCD (CGV, normal treadmill performance, and an abnormal cervical/vestibular physical examination). Self-reported symptoms. Univariate and multivariate methods, including t tests, tests of equivalence, a logistic regression model, k-nearest neighbor analysis, multidimensional scaling, and principle components analysis were used to see whether symptoms could distinguish PCD from CGV. None of the statistical methods used to analyze self-reported symptoms was able to adequately distinguish patients with PCD from patients with CGV. Symptoms after head injury, including cognitive symptoms, have traditionally been ascribed to brain injury, but they do not reliably discriminate between physiologic PCD and cervicogenic/vestibular PCD. Clinicians should consider specific testing of exercise tolerance and perform a physical examination of the cervical spine and the vestibular/ocular systems to determine the etiology of postconcussion symptoms. Symptoms after head injury, including cognitive symptoms, do not discriminate between concussion and cervical/vestibular injury."
] |
Human papilloma virus evasion of the host defence system
|
The DNA damage response (DDR) is a complex signalling network activated when DNA is altered by intrinsic or extrinsic agents. DDR plays important roles in genome stability and cell cycle regulation, as well as in tumour transformation. Viruses have evolved successful life cycle strategies in order to ensure a chronic persistence in the host, virtually avoiding systemic sequelae and death. This process promotes the periodic shedding of large amounts of infectious particles to maintain a virus reservoir in individual hosts, while allowing virus spreading within the community. To achieve such a successful lifestyle, the human papilloma virus (HPV) needs to escape the host defence systems. The key to understanding how this is achieved is in the virus replication process that provides by itself an evasion mechanism by inhibiting and delaying the host immune response against the viral infection. Numerous studies have demonstrated that HPV exploits both the ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia and rad3-related (ATR) DDR pathways to replicate its genome and maintain a persistent infection by downregulating the innate and cell-mediated immunity. This review outlines how HPV interacts with the ATM- and ATR-dependent DDR machinery during the viral life cycle to create an environment favourable to viral replication, and how the interaction with the signal transducers and activators of transcription (STAT) protein family and the deregulation of the Janus kinase (JAK)-STAT pathways may impact the expression of interferon-inducible genes and the innate immune responses.
|
[
"This study investigates the clinical course of low grade squamous intraepithelial lesions (LSIL), HPV status and HPV16-specific immune response in a large prospective study of 125 women with LSIL followed cytologically, virologically and histologically. Women with low-grade abnormal smears were recruited and followed-up for one year. Colposcopy, cervical biopsy for histology and brushings for HPV typing was performed at recruitment, 6 months (no biopsy) and upon completion of the study at one year. HPV16-specific T-cell responses were analysed by interferon-gamma ELISPOT at entry, 6 and 12 months. Infection with multiple HPV types was detected in 70% of all patients, HPV16 was found in 42% of the patients. LSIL lesions progressed to HSIL in 24%, persisted in 60% and regressed to normal in 16% of the patients. No difference was observed in the clearance rate of infections with single or multiple HPV types among the groups with a different histological outcome. HPV16-specific type 1 T-cell responses were detected in only half of the patients with an HPV16+ LSIL, and predominantly reactive to HPV16 E2 and E6. Interestingly, the presence of HPV16 E2-specific T-cell responses correlated with absence of progression of HPV16+ lesions (p = 0.005) while the detection of HPV16 E6 specific reactivity was associated with persistence (p = 0.05). This large prospective study showed that the majority of LSIL persisted or progressed within the first year.This was paralleled by immune failure as most of the patients with an HPV16+ LSIL failed to react to peptides of HPV16 E2, E6 or E7.",
"The mammalian target of rapamycin (mTOR) kinase acts as a cellular rheostat that integrates signals from a variety of cellular signal transduction pathways that sense growth factor and nutrient availability as well as intracellular energy status. It was previously reported that the human papillomavirus type 16 (HPV16) E6 oncoprotein may activate the S6 protein kinase (S6K) through binding and E6AP-mediated degradation of the mTOR inhibitor tuberous sclerosis complex 2 (TSC2) (Z. Lu, X. Hu, Y. Li, L. Zheng, Y. Zhou, H. Jiang, T. Ning, Z. Basang, C. Zhang, and Y. Ke, J. Biol. Chem. 279:35664-35670, 2004; L. Zheng, H. Ding, Z. Lu, Y. Li, Y. Pan, T. Ning, and Y. Ke, Genes Cells 13:285-294, 2008). Our results confirmed that HPV16 E6 expression causes an increase in mTORC1 activity through enhanced phosphorylation of mTOR and activation of downstream signaling pathways S6K and eukaryotic initiation factor binding protein 1 (4E-BP1). However, we did not detect a decrease in TSC2 levels in HPV16 E6-expressing cells. We discovered, however, that HPV16 E6 expression causes AKT activation through the upstream kinases PDK1 and mTORC2 under conditions of nutrient deprivation. We show that HPV16 E6 expression causes an increase in protein synthesis by enhancing translation initiation complex assembly at the 5' mRNA cap and an increase in cap-dependent translation. The increase in cap-dependent translation likely results from HPV16 E6-induced AKT/mTORC1 activation, as the assembly of the translation initiation complex and cap-dependent translation are rapamycin sensitive. Lastly, coexpression of the HPV16 E6 and E7 oncoproteins does not affect HPV16 E6-induced activation of mTORC1 and cap-dependent translation. HPV16 E6-mediated activation of mTORC1 signaling and cap-dependent translation may be a mechanism to promote viral replication under conditions of limited nutrient supply in differentiated, HPV oncoprotein-expressing proliferating cells.",
"Cervical cancer is similar to other human cancers in that it develops through a multistep process. However, infection with oncogenic human papillomaviruses (HPVs) is believed to be essential for the initiation of this disease. Although HPV may play a central role in the early stages of neoplasia, the accumulation of mutations in an assortment of genes precedes the development of malignant cervical carcinoma. The mechanisms by which abnormalities accumulate are various, but it is possible that viral proteins are involved. In particular, the viral E6 oncoprotein has been shown to interact with the cellular tumour suppressor protein p53, which is involved in DNA damage repair pathways. Hence, E6 may contribute to the genomic instability through this interaction with p53. We have tested this hypothesis by monitoring the effects of E6 upon DNA damage induced p53 transcriptional activity. This study shows that HPV-18 E6 inhibits p53 transcriptional activity following genotoxic stress with UV radiation. No effect was observed when a mutant E6 unable to direct the degradation of p53 was included in this assay. These results suggest that continued E6 expression may contribute to the accumulation of DNA damage associated with the progression of cervical cancer.",
"The study of human papillomavirus type 16 (HPV-16) replication has been impaired because of the lack of a cell culture system that stably maintains viral replication. Recently, cervical epithelial cell populations that stably maintain HPV-16 replicons at a copy number of approximately 1,000 per cell were derived from an HPV-16-infected patient (W12 cell clone 20863 [W12-E cells]). We used neutral/neutral and neutral/alkaline two-dimensional gel electrophoretic techniques to characterize HPV-16 DNA replication in these cells. When W12-E cells were maintained in an undifferentiated state mimicking the nonproductive stage of the life cycle, HPV-16 DNA was found to replicate primarily by theta structures in a bidirectional manner. The initiation site of HPV-16 DNA replication was mapped to approximately nucleotide 100, and the termination site was mapped to between nucleotides 3398 and 5990. To study the productive stage of HPV-16 DNA replication, W12-E cells were grown under culture conditions that promote differentiation of epithelial cell types. Under these conditions, where virus-like particles were detected, the mode of viral DNA replication changed from theta structure to what is apparently a rolling circle mode. Additionally, CIN 612-9E cells, which were derived from an HPV-31-infected patient and harbor HPV-31 extrachromosomally, exhibited the same switch in the mode of DNA replication upon induction of differentiation. These data argue that a fundamental switch in the mechanism of viral DNA replication occurs during the life cycle of the papillomavirus.",
"Human papillomavirus (HPV) infections are generally long lasting, and a host immune response to infection is hard to detect. Nevertheless immunocompromised subjects control HPV infection less well than those with intact immunity. Immune responses are best documented for the papillomavirus groups that cause evident human disease, particularly those responsible for anogenital cancers and genital warts. Humoral immunity to the viral capsid has been shown sufficient for protection against infection, while innate and adaptive cell mediated immunity appears important for eventual elimination of HPV infection. However, molecular and cellular mechanisms responsible for protection from and clearance of HPV infection are not completely established.",
"Chronic infection with human papillomavirus (HPV) can result in cervical cancer. To understand how HPV escapes immune eradication, we examined biophenotypes of immune cells in human normal cervix, cervical intraepithelial neoplasia (CIN), and cancer. Expression and cellular localization of Forkhead box protein-3 (FOXP3), indolamine 2,3-dioxygenase (IDO), interleukin (IL)-10, and interferon (IFN)-gamma were examined by immunofluorescence and immunohistochemistry. Mean cell densities of stromal FOXP3+ cells, IDO+ cells, IL-10+ cells, CD1a+ cells, and macrophages significantly increased from normal cervix to cancer, whereas densities of IFN-gamma+ and MMP-9+ cells increased from normal cervix to CIN but decreased in cancer. Flow cytometry confirmed significant elevation of cervical T cells expressing IFN-gamma and transforming growth factor-beta in CIN compared with normal cervix. Upon activation, a significantly increased proportion of cervical T cells expressed IFN-gamma in CIN than normal. A unique subset of morphologically immature stromal dendritic cells expressing IL-10 and IDO was more numerous in cancer than in normal cervix and CIN. The potentially suppressive immune milieu in the cervix may be permissive of HPV-associated cervical carcinogenesis.",
"The tumour suppressor p53 is activated following stress and initiates a heterogeneous response in a cell-, tissue- and stress-dependent manner. This heterogeneity is reflected in the different physiological outcomes that follow p53 activation. One mechanism that may contribute to this variability is the promoter selectivity of p53 target genes. p53 is at the hub of numerous signalling pathways that are triggered in response to particular stresses, all of which can leave their mark on p53 by way of post-translational modifications and interactions with cofactors. The precise combination of these marks, much like the bars in a barcode, dictates the behaviour of p53 in any given situation."
] |
Serum Heart-Type Fatty Acid Binding Protein Level and Cardiovascular Risk Factors in Elderly Diabetic Patients
|
Cardiovascular disease (CVD) is reported to be higher in elderly diabetics. Serum heart-type fatty acid binding protein (H-FABP) is a serum marker of myocardial ischemia. We aimed to investigate the association between serum H-FABP level and conventional cardiovascular risk factors, inflammatory markers and subclinical atherosclerosis in elderly diabetics without overt CVD.
|
[
"We aimed to assess the additive diagnostic value of measuring the serum levels of soluble human heart-type fatty acid binding protein (H-FABP) in the early diagnosis of acute myocardial infarction (AMI) in unselected patients with chest pain. A total of 97 consecutive patients with acute ischemic-type chest pain were prospectively enrolled and classified according to the American Heart Association/American College of Cardiology guidelines. The test characteristics of H-FABP and cardiac troponin T serum levels at admission revealed a greater sensitivity of H-FABP in the first 4 hours of symptoms (86% vs 42%, p <0.05). Combining H-FABP and cardiac troponin T also improved the sensitivity in the detection of AMI (97% vs 71%, p <0.05) but demonstrated a greater misclassification rate (25% vs 9%, p <0.05). The specificity of H-FABP was poor (65%, 95% confidence interval 58% to 71%). Receiver operating characteristics revealed a poor performance of H-FABP in patients with non-ST-elevation myocardial infarction. Classification tree analysis demonstrated that an H-FABP-related improvement in the early definite rule-out of AMI (reduction of false-negative rate from 11% to 3%) was at the expense of an increase in the false-positive rate to 5%. In conclusion, measurement of H-FABP, in addition to cardiac troponin T, serum levels within the first 4 hours of symptoms improves the sensitivity and negative predictive value for the detection of AMI at the cost of test accuracy and precision, especially in patients with non-ST-elevation myocardial infarction.",
"The long-term associations of established risk factors for coronary heart disease (CHD), for example cholesterol, are well known, but not for the less familiar hemostatic variables. To establish whether associations between hemostatic variables and CHD first identified nearly three decades ago have persisted long-term. The first Northwick Park Heart Study (NPHS-I) recruited 2167 white men and 941 white women, average age at entry 48 years, on whom measures of factor (F) VII activity (VIIc) and plasma fibrinogen were carried out, both at entry and at follow-up approximately 6 years later. During a median follow-up of 29 years, 231 male and 36 female CHD deaths were recorded from notifications by the Office for National Statistics. VIIc at recruitment was significantly related to CHD mortality, corrected rate ratio, RR, per 1 SD increase 1.56 (95% CI 1.29, 1.88) in men and RR 1.78 (95% CI 1.17, 2.72) in women. Recruitment fibrinogen was also strongly related to CHD mortality in men, RR 1.63 (95% CI 1.33, 1.99) but not in women, RR 0.75 (95% CI 0.40, 1.43). The associations persisted after controlling for confounders and were confirmed using 6-year follow-up measurements and in analyses omitting deaths within 10 years of recruitment. The hemostatic system contributes to CHD mortality, and its effect is stable over time. For VIIc, the effect was similar in men and women, while for fibrinogen it appeared to be present only in men.",
"To examine the association between unrecognised myocardial infarction (MI) as detected by electrocardiography and the long-term risk of heart failure. The Rotterdam Study is a prospective population-based cohort study of the general population of a suburb of the city of Rotterdam, The Netherlands. At baseline 2581 men and 3724 women aged > or =55 years were classified on the basis of electrocardiography, interview and clinical data into those with recognised MI, those with ECG-based unrecognised MI and those without MI. The participants were followed-up for incident heart failure, death or end of the study period on 12 October 2006. During a median follow-up time of 13.2 years, 823 cases of heart failure occurred, of which 403 in men. Independently of cardiovascular risk factors, recognised and unrecognised MIs yielded HRs of developing heart failure in men of 2.6 (95% CI 2.0 to 3.3) and 2.1 (95% CI 1.5 to 2.9), respectively. In women, recognised MI was associated with heart failure (HR=2.8; 95% CI 1.9 to 4.1), whereas unrecognised MI was not significantly related to the risk of heart failure (HR=1.1; 95% CI 0.7 to 1.7). Unrecognised MI detected by electrocardiography yields a long-term risk of heart failure equivalent to recognised MI in men, but is not significantly related to heart failure in women. In the light of the high incidence of both unrecognised MI and heart failure in the elderly, it may be worthwhile for both doctors and patients to improve responsiveness to typical and atypical symptoms of MI.",
"Epidemiological evidence suggests that fibrinogen and CRP are associated with coronary heart disease risk. High CRP in Indigenous Australians has been reported in previous studies including our 'Diabetes and Related diseases in Urban Indigenous population in Darwin region' (DRUID) Study. We studied levels of fibrinogen and its cross-sectional relationship with traditional and non-traditional cardiovascular risk factors in an urban Indigenous Australian cohort. Fibrinogen data were available from 287 males and 628 females (aged ≥ 15 years) from the DRUID study. Analysis was performed for associations with the following risk factors: diabetes, HbA1c, age, BMI, waist circumference, waist-hip ratio, total cholesterol, triglyceride, HDL cholesterol, C-reactive protein, homocysteine, blood pressure, heart rate, urine ACR, smoking status, alcohol abstinence. Fibrinogen generally increased with age in both genders; levels by age group were higher than those previously reported in other populations, including Native Americans. Fibrinogen was higher in those with than without diabetes (4.24 vs 3.56 g/L, p < 0.001). After adjusting for age and sex, the following were significantly associated with fibrinogen: BMI, waist, waist-hip ratio, systolic blood pressure, heart rate, fasting triglycerides, HDL cholesterol, HbA1c, CRP, ACR and alcohol abstinence. On multivariate regression (age and sex-adjusted) CRP and HbA1c were significant independent predictors of fibrinogen, explaining 27% of its variance; CRP alone explained 25% of fibrinogen variance. On factor analysis, both CRP and fibrinogen clustered with obesity in women (this factor explained 20% of variance); but in men, CRP clustered with obesity (factor explained 18% of variance) whilst fibrinogen clustered with HbA1c and urine ACR (factor explained 13% of variance). Fibrinogen is associated with traditional and non-traditional cardiovascular risk factors in this urban Indigenous cohort and may be a useful biomarker of CVD in this high-risk population. The apparent different associations of fibrinogen with cardiovascular disease risk markers in men and women should be explored further.",
"Pentraxin 3 (PTX3) is increased in circulating blood during the acute stage of acute coronary syndrome (ACS). Therefore, we compared diagnostic values of PTX3 for ACS with those of biomarkers for myocardial damage, such as troponin T (TnT) and heart-type fatty acid binding protein (H-FABP). Patients (n = 87) undergoing coronary angiography (CAG), consisting of 16 ACS and 71 non-ACS patients were enrolled. Non-ACS consists of 12 patients with normal CAG, 30 stable angina pectoris (SAP) patients controlled by medical treatment, and 29 SAP patients who required elective coronary revascularization (percutaneous coronary intervention or coronary artery bypass graft). Age, gender, or prevalence of diabetes, hypertension, or smoking was not significantly different between ACS and non-ACS groups. Serum total, high-density lipoprotein, or low-density lipoprotein cholesterol, or triglyceride levels were not significantly different between ACS and non-ACS. PTX3 levels were not significantly correlated with lipid profiles or different between those with and without conventional risk factors. Circulating PTX3, TnT, and H-FABP levels were significantly higher in ACS than non-ACS. In receiver-operating characteristic (ROC) curves, area under the curve (AUC) values for PTX3, TnT and H-FABP were 0.920, 0.674, and 0.690, respectively. ROC curves of PTX3 (AUC: 0.901), TnT (AUC: 0.731), and H-FABP (AUC: 0.633) for ST-elevation ACS were similar to those for whole ACS. In a TnT-negative subgroup, the AUC values of PTX3 and H-FABP for ACS were 0.981 and 0.489, respectively. PTX3 is a sensitive and specific biomarker for the diagnosis of ACS, and shows additional diagnostic values when measured in combination with TnT.",
"Hypothyroidism, whether overt or subclinical, has multiple effects on the cardiovascular system. Epicardial fat tissue (EFT) is closely related to cardiovascular disorders and atherosclerosis. Our study aimed to assess EFT thickness and carotid artery intima-media thickness (CIMT) in patients with Hashimoto's thyroiditis (HT) displaying overt and subclinical hypothyroidism (SCH). The study included 33 patients with SCH and 24 patients with overt hypothyroidism (OH) with HT as well as 32 healthy controls. EFT thickness, CIMT, thyroid hormone levels and lipid parameters were measured in all subjects. Correlation analysis and linear regression analysis were performed for EFT thickness. Mean EFT thickness was 2·89 ± 0·38, 3·53 ± 0·92 and 4·56 ± 1·61 mm in control, SCH and OH groups, respectively (P < 0·001). EFT thickness of OH patients was high compared with SCH and control subjects (P < 0·01 and <0·001, respectively). CIMT of OH patients was high compared with SCH and control subjects (P < 0·01 and <0·001, respectively). In addition, EFT was significantly thicker in SCH patients than in controls (P < 0·05). Correlation analysis showed that EFT thickness was significantly positively correlated with CIMT, age, body mass index, systolic blood pressure, thyroid-stimulating hormone, total and LDL cholesterol and triglyceride and negatively correlated with free T4. In the regression analysis, EFT thickness retained its independent and positive association with CIMT, patient group (particularly OH) and systolic blood pressure. Epicardial fat tissue thickness may be a useful indicator of early atherosclerosis in SCH and OH patients with HT.",
"To investigate the diagnostic and prognostic role of heart-type fatty acid-binding protein (hFABP) compared with copeptin and in addition to high-sensitivity cardiac troponin T (hs-cTnT) in patients with chest pain suspected of acute myocardial infarction (AMI). Diagnostic and prognostic performances of hFABP, copeptin and hs-cTnT were evaluated and compared. The final diagnosis was adjudicated by two independent cardiologists. This prospective observational multicentre study took place in four primary and one secondary hospital from April 2006 to September 2009. We enrolled 1247 consecutive patients with suspected AMI to the emergency department. For analysis, patients were included, if baseline levels for hs-cTnT and hFABP were available (n=1074), patients with ST-segment elevation myocardial infarction (STEMI) were excluded for the diagnostic analysis (n=43). Treatment was left to the discretion of the emergency physician. AMI and mortality. 4% of the patients had STEMI and 16% of the patients had non-STEMI. Patients with AMI had significantly higher levels of hFABP at presentation (p<0.001). Neither the combination with hFABP nor with copeptin increased the diagnostic accuracy of hs-cTnT at admission, quantified by the area under the receiver operating characteristic curve (AUC) (p>0.05). The negative predictive value regarding 90-day, 1-year and 2-year mortality was 100% (99-100), 99% (98-100) and 98% (96-99), respectively, for hFABP levels below the median (p<0.001). The accuracy of hFABP to predict 90-day mortality was moderate (AUC 0.83; 95% CI 0.77 to 0.90). hFABP and copeptin do not improve the diagnosis of patients with chest pain without ST-segment elevation, but may be useful for risk stratification beyond hs-TnT.",
"The negative impact of subclinical hypothyroidism (sHT) on cardiovascular risk, widely recognized in young adults (aged <55-60 y), is still debated in the elderly (>65 y), especially in the oldest olds (>80 y). We searched Medline for reports published with the following search terms: \"hypothyroidism,\" \"subclinical hypothyroidism,\" \"ageing,\" \"elderly,\" \"L-thyroxin,\" \"thyroid,\" \"guidelines,\" \"treatment,\" \"quality of life,\" \"cardiovascular risk,\" \"heart failure,\" \"coronary heart disease\" (CHD), \"atherosclerosis,\" and \"endothelial dysfunction.\" We limited our search to reports in English published after 1980, although we incorporated some reports published before 1980. We supplemented the search with records from personal files, textbooks, and relevant articles. Analyzed parameters included the epidemiology of thyroid failure, the effect of thyroid hormone on the aging process, cardiovascular function, and CHD risk factors. We also included the potential benefits of L-T4 therapy on the quality of life, cardiovascular events, and survival. TSH levels increase with age, even in older people without thyroid disease. Most longitudinal studies show an increased risk for CHD events and mortality in sHT participants. This increase is less evident in the elderly, mainly in cases of serum TSH values above 10 mIU/L. Lower mortality rate in a cohort of the oldest olds (>85 y) has been reported. sHT in older people should be not regarded as a unique condition, and moderately old patients (aged <70-75 y) could be considered clinically similar to the adult population, albeit with a higher optimal TSH target value. Conversely, the oldest old subjects should be carefully followed with a wait-and-see strategy, generally avoiding hormonal treatment. The decision to treat elderly people is still an unresolved clinical challenge--first, due to a lack of appropriately powered randomized controlled trials of L-T4 in sHT patients, examining cardiovascular hard endpoints in various classes of age; and second, because of the negative effects of possible overtreatment.",
"Hypothyroidism (HT) has an increased risk for cardiovascular mortality and morbidity due to increased atherosclerosis. Heart-type fatty acid binding protein (H-FABP) is abundant in the cytosol of cardiomyocytes, and transports fatty acids into these cells. Although H-FABP has been shown to increase in several atherosclerotic and inflammatory conditions, there is no literature data indicating an alteration in other atherosclerotic processes such as HT. A total of 39 patients with subclinical hypothyroidism (SCH), 26 patients with overt hypothyroidism (OH), and 29 healthy subjects were enrolled in this study. Carotid artery intima media thickness (CIMT) was measured by high resolution B mode ultrasonography. H-FABP levels, thyroid function test, and biochemical tests of all subjects were measured. The associations between H-FABP and thyroid test and CIMT were examined with correlation and regression analysis. OH patients had higher H-FABP levels (mean, 6.18 ± 3.08 ng/mL) than both the SCH (mean, 3.81 ± 2.16 ng/mL) and the controls (mean, 2.12 ± 1.27 ng/mL) (P < 0.01 and < 0.001, respectively). SCH patients had increased serum H-FABP levels compared with control subjects (P < 0.01). CIMT of both OH and SCH patients was also significantly greater compared with control subjects (both of p < 0.01). H-FABP was significantly and positively correlated with age, systolic blood pressure, thyroid stimulating hormone (TSH) levels, and CIMT, and negatively correlated with fT4 levels. The H-FABP levels retained an independent and positive association with systolic blood pressure, and a negative association with fT4 levels. Serum H-FABP levels progressively increased from the control group to the OH group. This suggests that H-FABP may be an indicator of low-level myocardial damage in HT, especially when used together with CIMT. Decreasing serum fT4 levels seem also to have an effect on H-FABP levels.",
"Heart type fatty acid binding protein (H-FABP) is a major cytoplasmic low-molecular weight protein and released into the circulation when the myocardium is injured. Previous studies have demonstrated that H-FABP is closely associated with acute coronary syndrome, hypertrophic and dilated cardiomyopathy, heart failure, stroke, obstructive sleep apnea syndrome, pulmonary embolism. The aim of this study was to investigate serum H-FABP value in the patients with metabolic syndrome (MetS). We measured serum H-FABP levels in 55 consecutive patients with MetS, and 73 age-matched control subjects by using a sandwich enzyme-linked immunosorbent assay. Serum H-FABP levels were significantly higher in patients with MetS than in control subjects 18.37 ± 13.0 and 7.9 ± 6.5 ng/ml, respectively, (P < 0.001). Serum H-FABP levels were significantly higher in patients with diabetic MetS than in without diabetic MetS, 24.0 ± 10.2 and 13.9 ± 12.6 ng/ml, respectively, (P: 0,003). There were statistically significant differences between patients without diabetic MetS and control subjects, 13.8 ± 12.6 and 7.9 ± 6.5 ng/ml, respectively, (P = 0.023). Patients with MetS have an increased risk of death from cardiovascular diseases. H-FABP seems to be a marker that will enable the detection of cardiac injury in the early asymptomatic period in patients with MetS."
] |
Flow cytometry-based xenograft assay predicts the rapid or delayed engraftment potential of noncharacterized acute myeloid leukemia samples
|
Xenograft assay allows functional analysis of leukemia-initiating cells of acute myeloid leukemia primary samples. However, 40% of samples derived from patients with better outcomes fail to engraft in immunodeficient mouse recipients when conventional protocols are followed. At diagnosis, the engraftment of intermediate-risk group samples cannot be anticipated. In this study, we decided to further explore the reasons for xenograft success and failure. No differences in extracellular phenotype, apoptosis, or cell cycle profile could distinguish samples that engraft (engrafter [E]) from samples that do not engraft (nonengrafter [NE]) in NSG mice. In addition, ex vivo long-term culture assay revealed, after 5 weeks, a lower content of leukemic-LTC-initiating cells in the NE samples associated with a lower expansion rate capacity. One-week co-cultures with mesenchymal or osteoblastic or endothelial cells did not influence the proliferation rate, suggesting that E and NE samples are genuinely rapidly or slowly expanding independent of external cue. Engraftment success for some NE samples was consistently observed in recipient mice analyzed 6 months later than the conventional 3-month period. Eventually we implemented a flow cytometry-based assay, which allowed us to predict, in 1 week, the fast or delayed engraftment potential of a noncharacterized acute myeloid leukemia sample. This approach will be especially useful in selecting intermediate-risk-group patient samples and restricting the experimental duration to a 3-month period and, eventually, in reducing the number of animals and the cost and effort of unnecessary xenograft failures.
|
[
"The nonobese diabetic/severe combined immunodeficient (NOD/SCID) assay is the current model for assessment of human normal and leukemic stem cells. We explored why 51% of 59 acute myeloid leukemia (AML) patients were unable to initiate leukemia in NOD/SCID mice. Increasing the cell dose, using more permissive recipients, and alternative tissue sources did not cause AML engraftment in most previously nonengrafting AML samples. Homing of AML cells to the marrow was the same between engrafters and nonengrafters. FLT3 internal tandem duplication (ITD) and nucleophosmin mutations occurred at a similar frequency in engrafters and nonengrafters. The only variable that was related to engraftment ability was the karyotypically defined risk stratification of individual AML cases. Of interest, follow-up of younger patients with intermediate-risk AML revealed a significant difference in overall survival between NOD/SCID engrafting and nonengrafting AMLs. Hence, the ability of AML to engraft in the NOD/SCID assay seems to be an inherent property of AML cells, independent of homing, conditioning, or cell frequency/source, which is directly related to prognosis. Our results suggest an important difference between leukemic initiating cells between engrafting and nonengrafting AML cases that correlates with treatment response.",
"Asymmetric cell division is an important mechanism for generating cellular diversity, however, techniques for measuring the distribution of fate-regulating molecules during mitosis have been hampered by a lack of objectivity, quantitation, and statistical robustness. Here we describe a novel imaging flow cytometric approach that is able to report a cells proliferative history and cell cycle position using dye dilution, pH3, and PI staining to then measure the spatial distribution of fluorescent signals during mitosis using CCD-derived imagery. Using Jurkat cells, resolution of the fluorescently labeled populations was comparable to traditional PMT based cytometers thus eliminating the need to sort cells with specific division histories for microscopy. Subdividing mitotic stages by morphology allowed us to determine the time spent in each cell cycle phase using mathematical modeling approaches. Furthermore high sample throughput allowed us to collect statistically relevant numbers of cells without the need to use blocking agents that artificially enrich for mitotic events. The fluorescent imagery was used to measure PKCζ protein and EEA-1+ endosome distribution during different mitotic phases in Jurkat cells. While telophase cells represented the favorable population for measuring asymmetry, asynchronously dividing cells spent approximately 43 seconds in this stage, explaining why they were present at such low frequencies. This necessitated the acquisition of large cell numbers. Interestingly we found that PKCζ was inherited asymmetrically in 2.5% of all telophasic events whereas endosome inheritance was significantly more symmetrical. Furthermore, molecular polarity at early mitotic phases was a poor indicator of asymmetry during telophase highlighting that, though rare, telophasic events represented the best candidates for asymmetry studies. In summary, this technique combines the spatial information afforded by fluorescence microscopy with the statistical wealth and objectivity of traditional flow cytometry, overcoming the key limitations of existing approaches for studying asymmetry during mitosis.",
"Repopulation of immunodeficient mice remains the primary method for functional assessment of human acute myeloid leukemia. Published data report engraftment in ~40-66% of cases, mostly of intermediate- or poor-risk subtypes. Here we report that extending follow-up beyond the standard analysis endpoints of 10 to 16 weeks after transplantation permitted leukemic engraftment from nearly every case of xenotransplanted acute myeloid leukemia (18/19, ~95%). Xenogeneic leukemic cells showed conserved immune pheno-types and genetic signatures when compared to corresponding pre-transplant cells and, furthermore, were able to induce leukemia in re-transplantation assays. Importantly, bone marrow biopsies taken at standardized time points failed to detect leukemic cells in 11/18 of cases that later showed robust engraftment (61%, termed \"long-latency engrafters\"), indicating that leukemic cells can persist over months at undetectable levels without losing disease-initiating properties. Cells from favorable-risk leukemia subtypes required longer to become detectable in NOD/SCID/IL2Rγnull mice (27.5±9.4 weeks) than did cells from intermediate-risk (21.9±9.4 weeks, P<0.01) or adverse-risk (17±7.6 weeks; P<0.0001) subtypes, explaining why the engraftment of the first was missed with previous protocols. Mechanistically, leukemic cells engrafting after a prolonged latency showed inferior homing to the bone marrow. Finally, we applied our model to favorable-risk acute myeloid leukemia with inv(16); here, we showed that CD34+ (but not CD34-) blasts induced robust, long-latency engraftment and expressed enhanced levels of stem cell genes. In conclusion, we provide a model that allows in vivo mouse studies with a wide range of molecular subtypes of acute myeloid leukemia subtypes which were previously considered not able to engraft, thus enabling novel insights into leukemogenesis.",
"A major limitation to clinical stem cell-mediated gene therapy protocols is the low levels of engraftment by transduced progenitors. We report that CXCR4 overexpression on human CD34+ progenitors using a lentiviral gene transfer technique helped navigate these cells to the murine bone marrow and spleen in response to stromal-derived factor 1 (SDF-1) signaling. Cells overexpressing CXCR4 exhibited significant increases in SDF-1-mediated chemotaxis and actin polymerization compared with control cells. A major advantage of CXCR4 overexpression was demonstrated by the ability of transduced CD34+ cells to respond to lower, physiologic levels of SDF-1 when compared to control cells, leading to improved SDF-1-induced migration and proliferation/survival, and finally resulting in significantly higher levels of in vivo repopulation of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice including primitive CD34+/CD38(-/low) cells. Importantly, no cellular transformation was observed following transduction with the CXCR4 vector. Unexpectedly, we documented lack of receptor internalization in response to high levels of SDF-1, which can also contribute to increased migration and proliferation by the transduced CD34+ cells. Our results suggest CXCR4 overexpression for improved definitive human stem cell motility, retention, and multilineage repopulation, which could be beneficial for in vivo navigation and expansion of hematopoietic progenitors."
] |
Targeted deletion/in cis complementation of the upstream homology sequence and downstream homology sequence in Borrelia hermsii.
|
In Borrelia hermsii, antigenic variation occurs as a result of a nonreciprocal gene conversion event that places one of ~60 silent variable major protein genes downstream of a single, transcriptionally active promoter. The upstream homology sequence (UHS) and downstream homology sequence (DHS) are two putative cis-acting DNA elements that have been predicted to serve as crossover points for homologous recombination. In this report, a targeted deletion/in cis complementation technique was used to directly evaluate the role for these elements in antigenic switching. The results demonstrate that deletion of the expression site results in an inability of the pathogen to relapse in immunocompetent mice, and that the utilized technique was successful in producing complemented mutants that are capable of antigenic switching. Additional complemented clones with mutations in the UHS and DHS of the expressed locus were then generated and evaluated for their ability to relapse in immunocompetent mice. Mutation of the UHS and inverted repeat sequence within the DHS rendered these mutants incapable of relapsing. Overall, the results establish the requirement of the inverted repeat of the DHS for antigenic switching, and support the importance of the UHS for B. hermsii persistence in the mammalian host.
|
[
"Borrelia parkeri is a relapsing fever agent that rarely causes human infection, unlike other North American species. B. parkeri strain HR1 was isolated from Ornithodoros parkeri ticks. The sequences of its linear chromosome and large plasmid were determined by next-generation sequencing. These confirmed its closer relatedness to Borrelia turicatae than to Borrelia hermsii.",
"The zoonotic pathogen Borrelia hermsii bears its multiple paralogous genes for variable antigens on several linear plasmids. Application of combined long-read and short-read next-generation sequencing provided complete sequences for antigen-encoding plasmids as well as other linear and circular plasmids and the linear chromosome of the genome.",
"Borrelia hermsii, an agent of relapsing fever, survives in mammals through antigenic variation. Change in serotype-specific variable outer membrane proteins (Vmps) occurs when a Vmp gene at an expression site is replaced with a previously silent gene for another Vmp. Silent and active genes are on separate linear plasmids. The upstream site for a nonreciprocal recombination between two linear plasmids is near the 5' ends of the expressed and silent genes. In the present study we sought the downstream recombination sites in two serotypes, 7 and 21. Restriction fragments containing plasmid telomeres were identified by susceptibility to digestion with BAL-31 and rapid reannealment following denaturation. Whereas both silent genes and a minority population of both expression-linked genes were several kilobases from the telomeres, the predominant population of both expressed genes had 3' ends near plasmid telomeres. Sequence analysis of the predominant expression plasmids revealed that the telomeric sequences were the same in serotypes 7 and 21. Identical sequence was also downstream of silent Vmp genes. Switching of Vmp genes appears to occur by recombination that involves both upstream and downstream sites. The expression plasmid's telomere is preserved in the recombination event.",
"The relapsing fever agent Borrelia hermsii undergoes multiphasic antigenic variation through gene conversion of a unique expression site on a linear plasmid by an archived variable antigen gene. To further characterize this mechanism we assessed the repertoire and organization of archived variable antigen genes by sequencing approximately 85% of plasmids bearing these genes. Most archived genes shared with the expressed gene a <or= 62 nucleotide (nt) region, the upstream homology sequence (UHS), that surrounded the start codon. The 59 archived variable antigen genes were arrayed in clusters with 13 repetitive, 214 nt long downstream homology sequence (DHS) elements distributed among them. A fourteenth DHS element was downstream of the expression locus. Informative nucleotide polymorphisms in UHS regions and DHS elements were applied to the analysis of the expression site of relapse serotypes from 60 infected mice in a prospective study. For most recombinations, the upstream crossover occurred in the UHS's second half, and the downstream crossover was in the DHS's second half. Usually the closest archival DHS element was used, but occasionally a more distant DHS was employed. The downstream extragenic crossover site in B. hermsii contrasts with the upstream [corrected] extragenic crossover site for antigenic variation in African trypanosomes."
] |
[Functional and molecular imaging in lung cancer].
|
Imaging in oncology is an essential tool for patient management but its potential is being profoundly underutilized. Each of the techniques used in the diagnostic process also conveys functional information that can be relevant in treatment decision-making. New imaging algorithms and techniques enhance our knowledge about the phenotype of the tumor and its potential response to different therapies. Functional imaging can be defined as the one that provides information beyond the purely morphological data, and include all the techniques that make it possible to measure specific physiological functions of the tumor, whereas molecular imaging would include techniques that allow us to measure metabolic changes. Functional and molecular techniques included in this document are based on multi-detector computed tomography (CT), 18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET), magnetic resonance imaging (MRI), and hybrid equipments, integrating PET with CT (PET/CT) or MRI (PET-MRI). Lung cancer is one of the most frequent and deadly tumors although survival is increasing thanks to advances in diagnostic methods and new treatments. This increased survival poises challenges in terms of proper follow-up and definitions of response and progression, as exemplified by immune therapy-related pseudoprogression. In this consensus document, the use of functional and molecular imaging techniques will be addressed to exploit their current potential and explore future applications in the diagnosis, evaluation of response and detection of recurrence of advanced NSCLC.
|
[
"The aim of preoperative computed tomographic (CT) assessment of patients with carcinoma of the bronchus is to stage the tumour accurately, and forewarn the surgeon of any possible local extrapulmonary extension of tumour in patients considered to have potentially resectable disease. The ability of CT scanning to differentiate between conventionally resectable lung cancer (TNM stages I and II), locally advanced but resectable lung cancer (TNM stage IIIa), and locally advanced but unresectable lung cancer (TNM stage IIIb) was determined in a group of patients accepted for surgery. Computed tomographic scans of 110 patients who underwent thoracotomy for intended resection of carcinoma of the bronchus, including 52 cases with stage III and 58 cases with stage I or II disease, were reviewed and the CT features and radiological interpretations correlated with the surgical and pathological findings. Thirteen CT scans were judged not to have been of diagnostic quality: of the remaining 97 cases 45 had stage III lung cancer, of whom 30 had successful resections, and 52 had stage I or stage II tumours. There was no difference in the frequencies of CT observations--including contiguity of tumour and mediastinum or chest wall, apparent mediastinal or chest wall invasion, proximity of tumour to the carina, mediastinal nodal enlargement, pulmonary collapse or consolidation and pleural effusion--in patients with stage I/II disease and patients with stage III disease. Similar results were found when the same observations were compared in all patients with resected disease and those with unresectable tumour. Sensitivity and specificity of CT was 27% and 96% respectively for tumour unresectability, 50% and 89% for mediastinal invasion, 14% and 99% for chest wall invasion, and 61% and 76% for mediastinal nodal metastases. Only 19 of 45 stage III tumours were correctly identified as being stage III and resectable or unresectable. In patients being considered for thoracotomy for resection of lung cancer, CT scanning used as the sole method of staging is of limited value for differentiating between stage I/II and stage III tumours. Patients should not be denied the opportunity for curative surgery on the basis of equivocal CT signs.",
"The aim of this study was to investigate whether apparent diffusion coefficients (ADCs) could be used as measures of cell density and necrotic fraction of tumors. Tumors of four human melanoma xenograft lines were subjected to diffusion-weighted magnetic resonance imaging (DWI). ADCs were calculated from the images and related to cell density and necrotic fraction, as determined from histological sections. A significant correlation was found between the ADC of the viable tissue and cell density, regardless of whether tumors of different lines or different regions within individual tumors were considered. Necrosis was found in two of the lines. A single region of massive necrosis that could be differentiated from the viable tissue in ADC maps was found in one line, whereas a number of smaller necrotic regions that could not be identified in ADC maps were found in the other line. Tumor ADC was significantly correlated with the necrotic fraction of the former, but not of the latter line. Our results suggest that ADCs can be used as measures of cell density and necrotic fraction of some but not of all tumors, depending on whether the individual necrotic regions are large enough to be differentiated from the viable tissue with the obtained spatial resolution of the DW images. Magn Reson Med 43:828-836, 2000.",
"To compare the results of whole-body magnetic resonance (MR) imaging with staging based on computed tomographic (CT), dedicated MR imaging, and nuclear scintigraphic results as standard of reference. Fifty-one patients with known malignant tumors were included in the study. Patients were placed on a rolling table platform capable of moving the patient rapidly through the isocenter of the magnet bore. The thorax and the abdomen were imaged by using fast breath-hold T2-weighted sequences in the transverse plane. After intravenous administration of a paramagnetic contrast agent, three-dimensional gradient-echo data sets were collected in five stations and covered the body from the skull to the knees. Location and size of cerebral, pulmonary, hepatic, and osseous metastases were documented by two experienced radiologists. Whole-body MR imaging findings were compared with results obtained at skeletal scintigraphy, CT, and dedicated MR imaging. The mean examination time for whole-body MR imaging was 14.5 minutes. All cerebral, pulmonary, and hepatic metastases greater than 6 mm in diameter could be identified with whole-body MR imaging. Small pulmonary metastases were missed with MR imaging, which did not change therapeutic strategies, but MR imaging depicted a single hepatic metastasis that was missed with CT. Skeletal scintigraphy depicted osseous metastases in 21 patients, whereas whole-body MR imaging revealed osseous metastases in 24 patients. The additional osseous metastases seen with MR imaging were confirmed at follow-up examinations but did not result in a change in therapy. Whole-body MR imaging performed on a per-patient basis revealed sensitivity and specificity values of 100%. Whole-body MR imaging for the evaluation of metastases compared well with the reference techniques for cerebral, pulmonary, and hepatic lesions. Whole-body MR imaging was more sensitive in the detection of hepatic and osseous metastases than were the reference techniques.",
"First, to investigate the diagnostic performance of fast T1-weighted sequences for lung nodule evaluation in oncologic magnetic resonance (MR)/positron emission tomography (PET). Second, to evaluate the influence of image acquisition in inspiration and expiration breath-hold on diagnostic performance. The study was approved by the local Institutional Review Board. PET/CT and MR/PET of 44 cancer patients were evaluated by 2 readers. PET/CT included lung computed tomography (CT) scans in inspiration and expiration (CTin, CTex). MR/PET included Dixon sequence for attenuation correction and fast T1-weighted volumetric interpolated breath-hold examination (VIBE) sequences (volume interpolated breath-hold examination acquired in inspiration [VIBEin], volume interpolated breath-hold examination acquired in expiration [VIBEex]). Diagnostic performance was analyzed for lesion-, lobe-, and size-dependence. Diagnostic confidence was evaluated (4-point Likert-scale; 1 = high). Jackknife alternative free-response receiver-operating characteristic (JAFROC) analysis was performed. Seventy-six pulmonary lesions were evaluated. Lesion-based detection rates were: CTex, 77.6%; VIBEin, 53.3%; VIBEex, 51.3%; and Dixon, 22.4%. Lobe-based detection rates were: CTex, 89.6%; VIBEin, 58.3%; VIBEex, 60.4%; and Dixon, 31.3%. In contrast to CT, inspiration versus expiration did not alter diagnostic performance in VIBE sequences. Diagnostic confidence was best for VIBEin and CTex and decreased in VIBEex and Dixon (1.2 ± 0.6; 1.2 ± 0.7; 1.5 ± 0.9; 1.7 ± 1.1, respectively). The JAFROC figure-of-merit of Dixon was significantly lower. All patients with malignant lesions were identified by CTex, VIBEin, and VIBEex, while 3 patients were false-negative in Dixon. Fast T1-weighted VIBE sequences allow for identification of patients with malignant pulmonary lesions. The Dixon sequence is not recommended for lung nodule evaluation in oncologic MR/PET patients. In contrast to CT, inspiration versus expiratory breath-hold in VIBE sequences was less crucial for lung nodule evaluation but was important for diagnostic confidence.",
"Therapeutic options in locally advanced non-small cell lung cancer (NSCLC) have expanded in the past decade to include a palate of targeted interventions such as high dose targeted thermal ablations, radiotherapy and growing platform of antibody and small molecule therapies and immunotherapies. Although these therapies have varied mechanisms of action, they often induce changes in tumour architecture and microenvironment such that response is not always accompanied by early reduction in tumour mass, and evaluation by criteria other than size is needed to report more effectively on response. Functional imaging techniques, which probe the tumour and its microenvironment through novel positron emission tomography and magnetic resonance imaging techniques, offer more detailed insights into and quantitation of tumour response than is available on anatomical imaging alone. Use of these biomarkers, or other rational combinations as readouts of pathological response in NSCLC have potential to provide more accurate predictors of treatment outcomes. In this article, the robustness of the more commonly available positron emission tomography and magnetic resonance imaging biomarker indices is examined and the evidence for their application in NSCLC is reviewed.",
"To evaluate the ability of dynamic contrast material-enhanced and diffusion-weighted (DW) magnetic resonance (MR) imaging to help detect early response to chemotherapy in patients with non-small cell lung cancer (NSCLC). This study was approved by the institutional review board, and written informed consent was obtained from all subjects. Twenty-eight patients with stage IIIB or IV NSCLC (17 women, 11 men; mean age, 64.8 years) who underwent chemotherapy were enrolled. All patients underwent MR imaging before and after the first course of chemotherapy. The time to peak enhancement, maximum enhancement ratio, and washout ratio were determined from the time-signal intensity curves of dynamic contrast-enhanced MR images. The apparent diffusion coefficient (ADC) of each lung carcinoma was calculated from DW MR images. The responses of these parameters to the first course of chemotherapy and the pretreatment ADC itself were compared with final tumor size reduction by using the Pearson correlation coefficient. Kaplan-Meier curves of progression-free survival and overall survival were generated, and comparisons between the group with a good response of the significant parameter (upper 50th percentile) and that with a poor response of the significant parameter (lower 50th percentile) were performed by using a two-sided log-rank test. Significant correlation was found only between early ADC change and final tumor size reduction rate (r(2) = 0.41, P = .00025). The median progression-free survival for the group with a good increase in ADC was 12.1 months, and that for the group with a stable or decreased ADC was 6.67 months (P = .021), while median overall survival was 22.4 and 12.3 months, respectively (P = .048). ADC seems to be a promising tool for monitoring the early response to or predicting prognosis after chemotherapy of NSCLC.",
"To prospectively investigate the relationship between the apparent diffusion coefficient (ADC) and cellularity in lung cancer. Sixty patients histopathologically confirmed with lung cancer (41 men, 19 women) underwent diffusion-weighted magnetic resonance imaging of the chest (with b values of 50 and 1000 s/mm2). The median mean ADC (ADC mean) value and median minimum ADC (ADC min) value within each primary tumour were calculated and compared with the median nucleo-cytoplasmic ratio (NCR), which was selected to represent the cellularity. The correlation between the NCR and ADC mean/ADC min was calculated with SPSS 18.0 software. The mean ADC mean values, ADC min values and median NCR were (1.07 ± 0.12) × 10(-3) mm2/s, (0.86 ± 0.14) × 10(-3) mm2/s, and (14.9 ± 2.6) %, respectively, in adenocarcinoma; (0.88 ± 0.10) × 10(-3) mm2/s, (0.73 ± 0.12) × 10(-3)) mm2/s, and (20.6 ± 4.4) %, respectively, in squamous cell carcinoma; and (0.89 ± 0.13) × 10(-3) mm2/s, (0.67 ± 0.13) × 10(-3) mm2/s, and (18.3 ± 3.5) %, respectively in small cell lung cancer. The NCR of squamous cell carcinoma and small cell lung cancer is greater than that of adenocarcinoma (P < 0.01 and P = 0.002, respectively). There was an inverse relationship between ADC mean/NCR and ADC min/NCR (r = -0.60, P = 0.001 and r = -0.47, P < 0.001, respectively). There is a significant inverse relationship between tumour cellularity and ADC in lung cancer. However, tumour cellularity most likely is not the sole determinant of the ADC."
] |
Role of skeletal muscle derived interleukin 6 in the coordination of metabolic responses during recovery from exercise
|
An acute bout of exercise imposes a major challenge on whole-body metabolism and metabolic adjustments are needed in multiple tissues during recovery to reestablish metabolic homeostasis. It is currently unresolved how this regulation is orchestrated between tissues. This study was undertaken to clarify the role of skeletal muscle derived interleukin 6 (IL-6) in the coordination of the metabolic responses during recovery from acute exercise. Skeletal muscle specific IL-6 knockout (IL-6 MKO) and littermate Control mice were rested or ran on a treadmill for 2h. Plasma, skeletal muscle, liver and adipose tissue were obtained after 6 and 10h of recovery. Non-exercised IL-6 MKO mice had higher plasma lactate and lower plasma non-esterified fatty acids than Controls. The activity of pyruvate dehydrogenase in the active form was, in skeletal muscle, higher in IL-6 MKO mice than Controls in non-exercised mice and 6h after exercise. IL-6 MKO mice had lower glucose transporter 4 protein content in inguinal adipose tissue (WAT) than Control in non-exercised mice and 10h after treadmill running. Epididymal WAT hormone sensitive lipase phosphorylation and inguinal WAT mitogen activated kinase P38 phosphorylation were higher in IL-6 MKO than Control mice 6h after exercise. These findings indicate that skeletal muscle IL-6 may play an important role in the regulation of substrate utilization in skeletal muscle, basal and exercise-induced adaptations in adipose tissue glucose uptake and lipolysis during recovery from exercise. Together this indicates that skeletal muscle IL-6 contributes to reestablishing metabolic homeostasis during recovery from exercise by regulating WAT and skeletal muscle metabolism.
|
[
"The mechanisms that mediate the tightly controlled production and clearance of glucose during muscular work are unclear, and it has been suggested that an unidentified \"work factor\" exists that influences the contraction-induced increase in endogenous glucose production (EGP). The cytokine interleukin (IL)-6 is released from skeletal muscle during contraction. Here we show that IL-6 contributes to the contraction-induced increase in EGP. Six men performed 2 h of bicycle exercise on three separate occasions, at a relatively high intensity (HI) or at a low intensity with (LO + IL-6) or without (LO) an infusion of recombinant human IL-6 that matched the circulating concentration of IL-6 seen in HI exercise. The stable isotope 6,6 (2)H(2) glucose was infused to calculate EGP (rate of glucose appearance [R(a)]), whole-body glucose disposal (rate of glucose disappearance [R(d)]), and metabolic clearance rate (MCR) of glucose. Glucose R(a), R(d), and MCR were higher (P < 0.05) at HI than at LO. Throughout exercise at LO + IL-6, glucose R(a) and R(d) were higher (P < 0.05) than LO, even though the exercise intensity was identical. In addition, MCR was higher (P < 0.05) at LO + IL-6 than at LO at 90 min. Insulin, glucagon, epinephrine, norepinephrine, cortisol, and growth hormone were identical when comparing LO + IL-6 with LO. These data suggest that IL-6 influences glucose homeostasis during exercise. Our results provide potential new insights into factors that mediate glucose production and disposal and implicates IL-6 in the so-called \"work factor.\"",
"Adipose tissue mitochondria have been implicated as key mediators of systemic metabolism. We have shown that IL-6 activates AMPK, a mediator of mitochondrial biogenesis, in adipose tissue; however, IL-6(-/-) mice fed a high fat diet have been reported to develop insulin resistance. These findings suggest that IL-6 may control adipose tissue mitochondrial content in vivo, and that reductions in adipose tissue mitochondria may be causally linked to the development of insulin resistance in IL-6(-/-) mice fed a high fat diet. On the other hand, IL-6 has been implicated as a negative regulator of insulin action. Given these discrepancies the purpose of the present investigation was to further evaluate the relationship between IL-6, adipose tissue mitochondrial content and whole body insulin action. In cultured epididymal mouse adipose tissue IL-6 (75 ng/ml) induced the expression of the transcriptional co-activators PGC-1α and PRC, reputed mediators of mitochondrial biogenesis. Similarly, IL-6 increased the expression of COXIV and CPT-1. These effects were absent in cultured subcutaneous adipose tissue and were associated with lower levels of GP130 and IL-6 receptor alpha protein content. Markers of mitochondrial content were intact in adipose tissue from chow fed IL-6(-/-) mice. When fed a high fat diet IL-6(-/-) mice were more glucose and insulin intolerant than controls fed the same diet; however this was not explained by decreases in adipose tissue mitochondrial content or respiration. Our findings demonstrate depot-specific differences in the ability of IL-6 to induce PGC-1α and mitochondrial enzymes and demonstrate that IL-6 is not necessary for the maintenance of adipose tissue mitochondrial content in vivo. Moreover, reductions in adipose tissue mitochondria do not explain the greater insulin resistance in IL-6(-/-) mice fed a high fat diet. These results question the role of adipose tissue mitochondrial dysfunction in the etiology of insulin resistance.",
"Endurance exercise training elicits an increase in mitochondrial density as well as GLUT-4 glucose transporter protein content in skeletal muscle. Corresponding increases in mRNA for respiratory enzymes and GLUT-4 indicate that pretranslational control mechanisms are involved in this adaptive process. To directly test whether transcription of the GLUT-4 gene is activated in response to exercise training, nuclei were isolated from red hindlimb skeletal muscle of rats after 1 wk of exercise training (8% grade, 32 m/min, 40 min, twice/day). Rats were killed either 30 min, 3 h, or 24 h after the last training session. GLUT-4 transcription, determined by nuclear run-on analysis, was unaltered after 30 min, increased by 1.8-fold after 3 h, but was no longer different from controls 24 h after exercise. A similar transient increase in GLUT-4 transcription was evident, but less pronounced (1.4-fold), in untrained rats after a single bout of exercise, suggesting that the postexercise induction in GLUT-4 gene transcription is enhanced by exercise training. GLUT-4 protein content was increased 1.7-fold after 1 wk of training in the absence of any corresponding change in GLUT-4 mRNA, providing evidence that the initial increase in GLUT-4 expression involves translational and/or posttranslational control mechanisms. These findings demonstrate that muscle GLUT-4 expression in response to exercise training is subject to both transcriptional and posttranscriptional regulation. We propose that the increase in GLUT-4 mRNA evident with extended periods of training may result from a shift to pretranslational control and is the cumulative effect of repeated postexercise transient increases in GLUT-4 gene transcription.",
"Reduced peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) expression and mitochondrial dysfunction in adipose tissue have been associated with obesity and insulin resistance. Whether this association is causally involved in the development of insulin resistance or is only a consequence of this condition has not been clearly determined. Here we studied the effects of adipose-specific deficiency of PGC-1α on systemic glucose homeostasis. Loss of PGC-1α in white fat resulted in reduced expression of the thermogenic and mitochondrial genes in mice housed at ambient temperature, whereas gene expression patterns in brown fat were not altered. When challenged with a high-fat diet, insulin resistance was observed in the mutant mice, characterized by reduced suppression of hepatic glucose output. Resistance to insulin was also associated with an increase in circulating lipids, along with a decrease in the expression of genes regulating lipid metabolism and fatty acid uptake in adipose tissues. Taken together, these data demonstrate a critical role for adipose PGC-1α in the regulation of glucose homeostasis and a potentially causal involvement in the development of insulin resistance."
] |
What is the name of this compound?
|
In the title compound, C
|
[
"In the title compound, C27H21FN2O4, the mean planes of the two indole ring systems (r.m.s. deviations = 0.0166 and 0.0086 Å) are approximately perpendic-ular to one another, making a dihedral angle of 87.8 (5)°; the fluorobenzene ring is twisted with respect to the mean planes of the two indole ring systems at 82.7 (5) and 85.5 (3)°. In the crystal, pairs of N-H⋯O hydrogen bonds link the mol-ecules into the inversion dimers, which are further linked by N-H⋯O hydrogen bonds into supra-molecular chains propagating along the b-axis direction. Weak C-H⋯π inter-actions are observed between neighbouring chains.",
"In the title compound, C27H21FN2O4, the mean planes of the indole ring systems (r.m.s. deviations = 0.0263 and 0.0160 Å) are approximately perpendic-ular to one another, making a dihedral angle of 84.0 (5)°; the fluoro-benzene ring is twisted with respect to the mean planes of the two indole ring systems at 89.5 (5) and 84.6 (3)°. In the crystal, pairs of N-H⋯O hydrogen bonds link the mol-ecules into inversion dimers, which are further linked by N-H⋯O hydrogen bonds into supra-molecular chains propagated along the b-axis direction. Weak C-H⋯π inter-actions are observed between neighbouring chains.",
"An account is given of the development of the SHELX system of computer programs from SHELX-76 to the present day. In addition to identifying useful innovations that have come into general use through their implementation in SHELX, a critical analysis is presented of the less-successful features, missed opportunities and desirable improvements for future releases of the software. An attempt is made to understand how a program originally designed for photographic intensity data, punched cards and computers over 10000 times slower than an average modern personal computer has managed to survive for so long. SHELXL is the most widely used program for small-molecule refinement and SHELXS and SHELXD are often employed for structure solution despite the availability of objectively superior programs. SHELXL also finds a niche for the refinement of macromolecules against high-resolution or twinned data; SHELXPRO acts as an interface for macromolecular applications. SHELXC, SHELXD and SHELXE are proving useful for the experimental phasing of macromolecules, especially because they are fast and robust and so are often employed in pipelines for high-throughput phasing. This paper could serve as a general literature citation when one or more of the open-source SHELX programs (and the Bruker AXS version SHELXTL) are employed in the course of a crystal-structure determination."
] |
Using Mechanistic Crosslinking Probes to Study Protein Conformations and Protein-Protein Interactions
|
Over the past decade, mechanistic crosslinking probes have been used to study protein-protein interactions in natural product biosynthetic pathways. This approach is highly interdisciplinary, combining elements of protein biochemistry, organic chemistry, and computational docking. The development of an experiment to engage undergraduate students in multidisciplinary research is described that leverages mechanistic crosslinking probes to study protein conformations and protein-protein interactions. This experiment provides students with a platform to learn chemoenzymatic synthesis, polyacrylamide gel electrophoresis, biochemical assays, and computational docking all while exploring a contemporary biochemical topic.
|
[
"Fatty acid biosynthesis is essential to life and represents one of the most conserved pathways in nature, preserving the same handful of chemical reactions across all species. Recent interest in the molecular details of the de novo fatty acid synthase (FAS) has been heightened by demand for renewable fuels and the emergence of multidrug-resistant bacterial strains. Central to FAS is the acyl carrier protein (ACP), a protein chaperone that shuttles the growing acyl chain between catalytic enzymes within the FAS. Human efforts to alter fatty acid biosynthesis for oil production, chemical feedstock, or antimicrobial purposes has been met with limited success, due in part to a lack of detailed molecular information behind the ACP-partner protein interactions inherent to the pathway. This review will focus on recently developed tools for the modification of ACP and analysis of protein-protein interactions, such as mechanism-based crosslinking, and the studies exploiting them. Discussion specific to each enzymatic domain will focus first on mechanism and known inhibitors, followed by available structures and known interactions with ACP. Although significant unknowns remain, new understandings of the intricacies of FAS point to future advances in manipulating this complex molecular factory.",
"Microalgae are a promising feedstock for renewable fuels, and algal metabolic engineering can lead to crop improvement, thus accelerating the development of commercially viable biodiesel production from algae biomass. We demonstrate that protein-protein interactions between the fatty acid acyl carrier protein (ACP) and thioesterase (TE) govern fatty acid hydrolysis within the algal chloroplast. Using green microalga Chlamydomonas reinhardtii (Cr) as a model, a structural simulation of docking CrACP to CrTE identifies a protein-protein recognition surface between the two domains. A virtual screen reveals plant TEs with similar in silico binding to CrACP. Employing an activity-based crosslinking probe designed to selectively trap transient protein-protein interactions between the TE and ACP, we demonstrate in vitro that CrTE must functionally interact with CrACP to release fatty acids, while TEs of vascular plants show no mechanistic crosslinking to CrACP. This is recapitulated in vivo, where overproduction of the endogenous CrTE increased levels of short-chain fatty acids and engineering plant TEs into the C. reinhardtii chloroplast did not alter the fatty acid profile. These findings highlight the critical role of protein-protein interactions in manipulating fatty acid biosynthesis for algae biofuel engineering as illuminated by activity-based probes.",
"beta-Keto acyl carrier protein reductase (BKR) catalyzes the pyridine-nucleotide-dependent reduction of a 3-oxoacyl form of acyl carrier protein (ACP), the first reductive step in de novo fatty acid biosynthesis and a reaction often performed in polyketide biosynthesis. The Brassica napus BKR enzyme is NADPH-dependent and forms part of a dissociable type II fatty acid synthetase (FAS). Significant sequence similarity is observed with enoyl acyl carrier protein reductase (ENR), the other reductase of FAS, and the short-chain alcohol dehydrogenase (SDR) family. The first crystal structure of BKR has been determined at 2.3 A resolution in a binary complex with an NADP(+) cofactor. The structure reveals a homotetramer in which each subunit has a classical dinucleotide-binding fold. A triad of Ser154, Tyr167 and Lys171 residues is found at the active site, characteristic of the SDR family. Overall BKR has a very similar structure to ENR with good superimposition of catalytically important groups. Modelling of the substrate into the active site of BKR indicates the need for conformational changes in the enzyme. A catalytic mechanism can be proposed involving the conserved triad. Helix alpha6 must shift its position to permit substrate binding to BKR and might act as a flexible lid on the active site. The similarities in fold, mechanism and substrate binding between BKR, which catalyzes a carbon-oxygen double-bond reduction, and ENR, the carbon-carbon double-bond oxidoreductase in FAS, suggest a close evolutionary link during the development of the fatty acid biosynthetic pathway.",
"The beta-ketoacyl-acyl carrier protein (ACP) synthases are key regulators of type II fatty acid synthesis and are the targets for two natural products, thiolactomycin (TLM) and cerulenin. The high resolution structures of the FabB-TLM and FabB-cerulenin binary complexes were determined. TLM mimics malonyl-ACP in the FabB active site. It forms strong hydrogen bond interactions with the two catalytic histidines, and the unsaturated alkyl side chain interaction with a small hydrophobic pocket is stabilized by pi stacking interactions. Cerulenin binding mimics the condensation transition state. The subtle differences between the FabB-cerulenin and FabF-cerulenin (Moche, M., Schneider, G., Edwards, P., Dehesh, K., and Lindqvist, Y. (1999) J. Biol. Chem. 244, 6031-6034) structures explain the differences in the sensitivity of the two enzymes to the antibiotic and may reflect the distinct substrate specificities that differentiate the two enzymes. The FabB[H333N] protein was prepared to convert the FabB His-His-Cys active site triad into the FabH His-Asn-Cys configuration to test the importance of the two His residues in TLM and cerulenin binding. FabB[H333N] was significantly more resistant to both antibiotics than FabB and had an affinity for TLM an order of magnitude less than the wild-type enzyme, illustrating that the two-histidine active site architecture is critical to protein-antibiotic interaction. These data provide a structural framework for understanding antibiotic sensitivity within this group of enzymes."
] |
Living donor liver transplantation versus deceased donor liver transplantation
|
There is no consensus regarding the difference in outcomes of HCV in patients who receive living donor liver transplantation (LDLT) or compared to deceased donor liver transplantation (DDLT). The aims of this study were to compare characteristics between LDLT and DDLT groups and to identify risk factors affecting patient survival.
|
[
"To compare long-term survival of living donor liver transplant (LDLT) at experienced transplant centers with outcomes of deceased donor liver transplant and identify key variables impacting patient and graft survival. The Adult-to-Adult Living Donor Liver Transplantation Cohort Study is a prospective multicenter National Institutes of Health study comparing outcomes of LDLT and deceased donor liver transplant and associated risks. Mortality and graft failure for 1427 liver recipients (963 LDLT) enrolled in the Adult-to-Adult Living Donor Liver Transplantation Cohort Study who received transplant between January 1, 1998, and January 31, 2014, at 12 North American centers with median follow-up 6.7 years were analyzed using Kaplan-Meier and multivariable Cox models. Survival probability at 10 years was 70% for LDLT and 64% for deceased donor liver transplant. Unadjusted survival was higher with LDLT (hazard ratio = 0.76, P = 0.02) but attenuated after adjustment (hazard ratio = 0.98, P = 0.90) as LDLT recipients had lower mean model for end-stage liver disease (15.5 vs 20.4) and fewer received transplant from intensive care unit, were inpatient, on dialysis, were ventilated, or with ascites. Posttransplant intensive care unit days were less for LDLT recipients. For all recipients, female sex and primary sclerosing cholangitis were associated with improved survival, whereas dialysis and older recipient/donor age were associated with worse survival. Higher model for end-stage liver disease score was associated with increased graft failure. Era of transplantation and type of donated lobe did not impact survival in LDLT. LDLT provides significant long-term transplant benefit, resulting in transplantation at a lower model for end-stage liver disease score, decreased death on waitlist, and excellent posttransplant outcomes. Recipient diagnosis, disease severity, renal failure, and ages of recipient and donor should be considered in decision making regarding timing of transplant and donor options.Clinical Trials ID: NCT00096733.",
"Infection with hepatitis C virus (HCV) is a common cause of chronic liver disease, and HCV-related cirrhosis and hepatocellular carcinoma are the leading causes for liver transplantation in the Western world. Recurrent infection of the transplanted liver allograft is universal in patients with detectable HCV viremia at the time of transplant and can cause a spectrum of disease, ranging from asymptomatic chronic infection to an aggressive fibrosing cholestatic hepatitis. Recurrent HCV is more aggressive in the post-transplant population and is a leading cause of allograft loss, morbidity, and mortality. Historically, treatment of recurrent HCV has been limited by low rates of treatment success and high side effect profiles. Over the past few years, promising new therapies have emerged for the treatment of HCV that have high rates of sustained virological response without the need for interferon based regimens. In addition to being highly effective, these treatments have higher rates of adherence and a lower side effect profile. The purpose of this review is to summarize current therapies in recurrent HCV infection, to review the recent advances in therapy, and to highlight areas of ongoing research.",
"Donor factors influence hepatitis C virus (HCV) disease severity in liver transplant (LT) recipients. Living donors, because they are typically young and have short cold ischemic times, may be advantageous for HCV-infected patients. Among HCV-infected patients in the Adult-to-Adult Living Donor Liver Transplantation Cohort Study (A2ALL) surviving >90 days and followed for a median 4.7 years, advanced fibrosis (Ishak stage ≥3) and graft loss were determined. The 5-year cumulative risk of advanced fibrosis was 44% and 37% in living donor LT (LDLT) and deceased donor LT (DDLT) patients (P = 0.16), respectively. Aspartate aminotransferase (AST) activity at LT (hazard ratio [HR] = 1.38 for doubling of AST, P = 0.005) and biliary strictures (HR = 2.68, P = 0.0001) were associated with advanced fibrosis, but LDLT was not (HR = 1.11, 95% confidence interval [CI] 0.73-1.69, P = 0.63). The 5-year unadjusted patient and graft survival probabilities were 79% and 78% in LDLT, and 77% and 75% in DDLT (P = 0.43 and 0.32), with 27% and 20% of LDLT and DDLT graft losses due to HCV (P = 0.45). Biliary strictures (HR = 2.25, P = 0.0006), creatinine at LT (HR = 1.74 for doubling of creatinine, P = 0.0004), and AST at LT (HR = 1.36 for doubling of AST, P = 0.004) were associated with graft loss, but LDLT was not (HR = 0.76, 95% CI: 0.49-1.18, P = 0.23). Donor type does not affect the probability of advanced fibrosis or patient and graft survival in HCV-infected recipients. Thus, while LDLT offers the advantage of shorter wait times, there is no apparent benefit for HCV disease progression. Biliary strictures have a negative effect on HCV fibrosis severity and graft survival, and a high AST at LT may be an important predictor of fibrosis risk post-LT.",
"Autoimmune hepatitis and cholestatic liver diseases have more favorable outcomes after liver transplantation as compared to viral hepatitis and alcoholic liver diseases. However, there are only few reports comparing outcomes of both living donor liver transplants (LDLT) and deceased donor liver transplants (DDLT) for these conditions. We aim to study the survival outcomes of patients undergoing LT for autoimmune and cholestatic diseases and to identify possible risk factors influencing survival. Survival outcomes for LDLT vs. DDLT are also to be compared for these diseases. A retrospective analysis of the UNOS database for patients transplanted between February 2002 until October 2006 for AIH, PSC, and PBC was performed. Survival outcomes for LDLT and DDLT patients were analyzed and factors influencing survival were identified. Among all recipients the estimated patient survival at 1, 3, and 5 years for LDLT was 95.5%, 93.6%,and 92.5% and for DDLT was 90.9%, 86.5%, and 84.9%, respectively (p = 0.002). The estimated graft survival at 1, 3, and 5 years for LDLT was 87.9%, 85.4%, and 84.3% and for DDLT 85.9%, 80.3%, and 78.6%, respectively (p = 0.123). On multivariate proportional hazard regression analysis after adjusting for age and MELD score, the effect of donor type was not found to be significant. The overall survival outcomes of LDLT were similar to DDLT in our patients with autoimmune and cholestatic liver diseases. It appears from our study that after adjusting for age and MELD score donor type does not significantly affect the outcome."
] |
Immunocytokines: a new class of bifunctional protein therapeutics
|
Immunocytokines are fusion proteins that combine the specific antigen binding capacities of an antibody or derivative thereof and the potent bioactivity of a cytokine partner. These novel biopharmaceuticals have been directed to various targets of oncological as well as non-oncological origin and a handful of promising constructs are currently advancing in the clinical trial pipeline. Several factors such as the choice of a disease specific antigen, the antibody format and the modulatory nature of the payload are crucial, not only for therapeutic efficacy and safety but also for the commercial success of such a product. In this review, we provide an overview of the basic principles and obstacles in immunocytokine design with a specific focus on single chain antibody fragment-based constructs that employ interleukins as the immunoactive component. Impact statement Selective activation of the immune system in a variety of malignancies represents an attractive approach when existing strategies have failed to provide adequate treatment options. Immunocytokines as a novel class of bifunctional protein therapeutics have emerged recently and generated promising results in preclinical and clinical studies. In order to harness their full potential, multiple different aspects have to be taken into consideration. Several key points of these fusion constructs are discussed here and should provide an outline for the development of novel products based on an overview of selected formats.
|
[
"The hu14.18-IL2 fusion protein consists of interleukin-2 molecularly linked to a humanized monoclonal antibody that recognizes the GD2 disialoganglioside expressed on neuroblastoma cells. This phase II study assessed the antitumor activity of hu14.18-IL2 in two strata of patients with recurrent or refractory neuroblastoma. Hu14.18-IL2 was given intravenously (12 mg/m(2)/daily) for 3 days every 4 weeks for patients with disease measurable by standard radiographic criteria (stratum 1) and for patients with disease evaluable only by [(123)I]metaiodobenzylguanidine (MIBG) scintigraphy and/or bone marrow (BM) histology (stratum 2). Response was established by independent radiology review as well as BM histology and immunocytology, and durability was assessed by repeat evaluation after more than 3 weeks. Thirty-nine patients were enrolled (36 evaluable). No responses were seen in stratum 1 (n = 13). Of 23 evaluable patients in stratum 2, five patients (21.7%) responded; all had a complete response (CR) of 9, 13, 20, 30, and 35+ months duration. Grade 3 and 4 nonhematologic toxicities included capillary leak, hypoxia, pain, rash, allergic reaction, elevated transaminases, and hyperbilirubinemia. Two patients required dopamine for hypotension, and one patient required ventilatory support for hypoxia. Most toxicities were reversible within a few days of completing a treatment course and were expected based on phase I results. Patients with disease evaluable only by MIBG and/or BM histology had a 21.7% CR rate to hu14.8-IL2, whereas patients with bulky disease did not respond. Hu14.18-IL2 warrants further testing in children with nonbulky high-risk neuroblastoma.",
"Pre-existing antibodies against a drug substance can significantly alter the pharmacokinetic profile of the drug in the circulation. Rapid clearance, mediated by complement or Fc receptors, occurs for crosslinked immune complexes, but not for complexes containing only one or two antibodies. With antibodies functioning as carrier proteins, monovalent antigens may enjoy a prolonged circulatory half-life, as observed in the case of digoxin, insulin, and various interleukins. While such an effect should be highly sensitive to fluctuations in antibody affinity and titer, it may present a means of extending the circulation of potent but rapidly cleared therapeutic agents. This mini-review attempts to delineate the causal relation between the factors influencing antibody binding and the circulatory life of a therapeutic agent, be it a small drug or a macromolecule.",
"We have developed a novel antibody-drug conjugate (ADC) that can selectively deliver the Lck inhibitor dasatinib to human T lymphocytes. This ADC is based on a humanized antibody that selectively binds with high affinity to CXCR4, an antigen that is selectively expressed on hematopoietic cells. The resulting dasatinib-antibody conjugate suppresses T-cell-receptor (TCR)-mediated T-cell activation and cytokine expression with low nM EC50 and has minimal effects on cell viability. This ADC may lead to a new class of selective immunosuppressive drugs with improved safety and extend the ADC strategy to the targeted delivery of kinase inhibitors for indications beyond oncology.",
"Cytotoxic effector cells interact with target cells through various mechanisms. CTLs use the antigen-specific T cell receptor, whereas Fc receptor-positive natural killer cells use this receptor to interact with antibody-coated target cells. We evaluated the tumor-binding and lymphocyte-activating capability of a recombinant fusion protein consisting of a tumor-selective human/mouse chimeric anti-ganglioside GD2 antibody (ch14.18) and recombinant human interleukin-2 (IL2) (ch14.18-IL2). This fusion protein bound specifically to GD2-positive melanoma and neuroblastoma tumor cell lines, and its IL2 component stimulated in vitro proliferation of an IL2-dependent cell line, as well as peripheral blood mononuclear cells, in healthy control individuals and in cancer patients receiving continuous infusion of IL2. The IL2 presented by the fusion protein, when bound to tumor cells, induced proliferation of IL2-responsive cells as well as a comparable amount of soluble IL2 did. This suggests that localization of IL2 at the site of contact between tumor and effector cells is an effective way of presenting this cytokine to IL2-responsive cells. The ch14.18-IL2 fusion protein also mediated antibody-dependent cellular cytotoxicity with Fc receptor-positive effector cells to an extent similar to ch14.18. These results, together with those of previous studies documenting antitumor efficacy against human tumor xenografts in SCID mice and GD2-positive murine tumors in immunocompetent syngeneic mice, suggest that the ch14.18-IL2 fusion protein should be tested in Phase I and II trials in patients with GD2-positive tumors.",
"Immunoglobulins (Ig) or antibodies are heavy plasma proteins, with sugar chains added to amino-acid residues by N-linked glycosylation and occasionally by O-linked glycosylation. The versatility of antibodies is demonstrated by the various functions that they mediate such as neutralization, agglutination, fixation with activation of complement and activation of effector cells. Naturally occurring antibodies protect the organism against harmful pathogens, viruses and infections. In addition, almost any organic chemical induces antibody production of antibodies that would bind specifically to the chemical. These antibodies are often produced from multiple B cell clones and referred to as polyclonal antibodies. In recent years, scientists have exploited the highly evolved machinery of the immune system to produce structurally and functionally complex molecules such as antibodies from a single B clone, heralding the era of monoclonal antibodies. Most of the antibodies currently in the clinic, target components of the immune system, are not curative and seek to alleviate symptoms rather than cure disease. Our group used a novel strategy to identify reparative human monoclonal antibodies distinct from conventional antibodies. In this chapter, we discuss the therapeutic relevance of both polyclonal and monoclonal antibodies in clinic.",
"The activation of the immune system for a selective removal of tumor cells represents an attractive strategy for the treatment of metastatic malignancies, which cannot be cured by existing methodologies. In this review, we examine the design and therapeutic potential of immunocytokines and bispecific antibodies, two classes of bifunctional products which can selectively activate the immune system at the tumor site. Certain protein engineering aspects, such as the choice of the antibody format, are common to both classes of therapeutic agents and can have a profound impact on tumor homing performance in vivo of individual products. However, immunocytokines and bispecific antibodies display different mechanisms of action. Future research activities will reveal whether an additive of even synergistic benefit can be obtained from the judicious combination of these two types of biopharmaceutical agents.",
"Clinical strategies which modulate the human anti-mouse antibody response (HAMA) in patients may have a profound influence on the idiotype network inducible by murine monoclonal antibodies (MoAb). Prior to myeloablative chemotherapy (ABMT), 9 patients with Stage IV neuroblastoma were imaged with 131I-3F8, a MoAb specific for the ganglioside GD2. Their serum HAMA, anti-idiotypic, anti-GD2, and anti-anti-idiotypic antibodies were assayed by enzyme-linked immunosorbent assay prior to, and at 3 and 6 months postimaging. HAMA and anti-idiotypic levels remained low, in contrast to the high levels in 10 patients imaged with 131I-3F8 without ABMT. Five of the 9 patients are long-term survivors; all had elevated anti-GD2 and anti-anti-idiotypic levels, significantly higher than those who died of disease. Although 131I-3F8 imaging prior to ABMT detected abnormal sites in 4 of 9 patients, 3 of the 4 patients have continued in remission for 24-63 months after ABMT, and all 3 mounted anti-GD2 and anti-anti-idiotypic antibody responses. We conclude that myeloablative therapy strongly suppressed the HAMA/anti-idiotypic response to murine MoAb and that the prognostic significance of host immune response to ganglioside GD2 MoAb deserves further investigation."
] |
OsMAR1 regulates the salt-stress response via the regulation of the OCPI2 protein in rice
|
Our results suggest that a rice E3 ligase, OsMAR1, physically interacts with a cytosolic protein OCPI2 and may play an important role under salinity stress. Salt is an important abiotic stressor that negatively affects plant growth phases and alters development. Herein, we found that a rice gene, OsMAR1 (Oryza sativa microtubule-associated RING finger protein 1), encoding the RING E3 ligase was highly expressed in response to high salinity, water deficit, and ABA treatment. Fluorescence signals of its recombinant proteins were clearly associated with the microtubules in rice protoplasts. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) showed that OsMAR1 interacted with a cytosolic protein OCPI2 (O. sativa chymotrypsin protease inhibitor 2) and led to its degradation via the 26S proteasome. Heterogeneous overexpression of OsMAR1 in Arabidopsis showed retarded root growth compared with that of control plants, and then led to hypersensitivity phenotypes under high salinity stress. Taken together, OsMAR1 negatively regulates the salt-stress response via the regulation of the OCPI2 protein in rice.
|
[
"Transcriptional regulation in response to hyperosmotic, high-salinity and oxidative stress, and abscisic acid (ABA) treatment in Arabidopsis suspension-cultured cell line T87 was investigated with a cDNA microarray containing 7000 independent full-length Arabidopsis cDNAs. The transcripts of 102, 11, 84 and 73 genes were increased more than 5-fold within 5h after treatment with 0.5M mannitol, 0.1M NaCl, 50 microM ABA and 10mM H2O2, respectively. On the other hand, the transcripts of 44, 57, 25 and 34 genes were down-regulated to less than one-third within 5h after treatment with 0.5M mannitol, 0.1M NaCl, 50 microM ABA and 10mM H2O2, respectively. Venn diagram analysis revealed 11 genes were induced significantly by mannitol, NaCl, and ABA, indicating crosstalk among these signaling pathways. Comparison of the genes induced by each stress revealed that 32%, 17% and 33% of mannitol-, NaCl- and ABA-inducible genes were also induced by H2O2, indicating the crosstalk between the signaling pathways for osmotic stress and oxidative stress. Although the expression profiles revealed that the T87 cells had most of the regulatory systems seen in Arabidopsis seedlings, the T87 cells did not have one of ABA-dependent signaling pathways.",
"Many studies have been conducted to understand plant stress responses to salinity because irrigation-dependent salt accumulation compromises crop productivity and also to understand the mechanism through which some plants thrive under saline conditions. As mechanistic understanding has increased during the last decades, discovery-oriented approaches have begun to identify genetic determinants of salt tolerance. In addition to osmolytes, osmoprotectants, radical detoxification, ion transport systems, and changes in hormone levels and hormone-guided communications, the Salt Overly Sensitive (SOS) pathway has emerged to be a major defense mechanism. However, the mechanism by which the components of the SOS pathway are integrated to ultimately orchestrate plant-wide tolerance to salinity stress remains unclear. A higher-level control mechanism has recently emerged as a result of recognizing the involvement of GIGANTEA (GI), a protein involved in maintaining the plant circadian clock and control switch in flowering. The loss of GI function confers high tolerance to salt stress via its interaction with the components of the SOS pathway. The mechanism underlying this observation indicates the association between GI and the SOS pathway and thus, given the key influence of the circadian clock and the pathway on photoperiodic flowering, the association between GI and SOS can regulate growth and stress tolerance. In this review, we will analyze the components of the SOS pathways, with emphasis on the integration of components recognized as hallmarks of a halophytic lifestyle.",
"It is well known that salinization (high-pH) has been considered as a major environmental threat to agricultural systems. The aim of this study was to investigate the differences between salt stress and alkali stress in metabolic profiles and nutrient accumulation of wheat; these parameters were also evaluated to determine the physiological adaptive mechanisms by which wheat tolerates alkali stress. The harmful effect of alkali stress on the growth and photosynthesis of wheat were stronger than those of salt stress. High-pH of alkali stress induced the most of phosphate and metal ions to precipitate; as a result, the availability of nutrients significantly declined. Under alkali stress, Ca sharply increased in roots, however, it decreased under salt stress. In addition, we detected the 75 metabolites that were different among the treatments according to GC-MS analysis, including organic acids, amino acids, sugars/polyols and others. The metabolic data showed salt stress and alkali stress caused different metabolic shifts; alkali stress has a stronger injurious effect on the distribution and accumulation of metabolites than salt stress. These outcomes correspond to specific detrimental effects of a highly pH environment. Ca had a significant positive correlation with alkali tolerates, and increasing Ca concentration can immediately trigger SOS Na exclusion system and reduce the Na injury. Salt stress caused metabolic shifts toward gluconeogenesis with increased sugars to avoid osmotic stress; energy in roots and active synthesis in leaves were needed by wheat to develop salt tolerance. Alkali stress (at high pH) significantly inhibited photosynthetic rate; thus, sugar production was reduced, N metabolism was limited, amino acid production was reduced, and glycolysis was inhibited.",
"As sessile organisms, plants must cope with abiotic stress such as soil salinity, drought, and extreme temperatures. Core stress-signaling pathways involve protein kinases related to the yeast SNF1 and mammalian AMPK, suggesting that stress signaling in plants evolved from energy sensing. Stress signaling regulates proteins critical for ion and water transport and for metabolic and gene-expression reprogramming to bring about ionic and water homeostasis and cellular stability under stress conditions. Understanding stress signaling and responses will increase our ability to improve stress resistance in crops to achieve agricultural sustainability and food security for a growing world population.",
"Growth and carbon (C) fluxes are severely altered in plants exposed to soil water deficit. Correspondingly, it has been suggested that plants under water deficit suffer from C shortage. In this study, we test this hypothesis in Arabidopsis (Arabidopsis thaliana) by providing an overview of the responses of growth, C balance, metabolites, enzymes of the central metabolism, and a set of sugar-responsive genes to a sustained soil water deficit. The results show that under drought, rosette relative expansion rate is decreased more than photosynthesis, leading to a more positive C balance, while root growth is promoted. Several soluble metabolites accumulate in response to soil water deficit, with K(+) and organic acids as the main contributors to osmotic adjustment. Osmotic adjustment costs only a small percentage of the daily photosynthetic C fixation. All C metabolites measured (not only starch and sugars but also organic acids and amino acids) show a diurnal turnover that often increased under water deficit, suggesting that these metabolites are readily available for being metabolized in situ or exported to roots. On the basis of 30 enzyme activities, no in-depth reprogramming of C metabolism was observed. Water deficit induces a shift of the expression level of a set of sugar-responsive genes that is indicative of increased, rather than decreased, C availability. These results converge to show that the differential impact of soil water deficit on photosynthesis and rosette expansion results in an increased availability of C for the roots, an increased turnover of C metabolites, and a low-cost C-based osmotic adjustment, and these responses are performed without major reformatting of the primary metabolism machinery.",
"Drought stress from soil or air limits plant growth and development, leading to a reduction in crop productivity. Several E3 ligases positively or negatively regulate the drought stress response. In the present study, we show that the pepper (Capsicum annuum) Drought Induced RING type E3 ligase 1, CaDIR1, regulates the drought stress response via abscisic acid (ABA)-mediated signaling. CaDIR1 contains a C3HC4-type RING finger domain in the N-terminal region; this domain functions during protein degradation via attachment of ubiquitins to the substrate target proteins. The expression levels of the CaDIR1 gene were suppressed and induced by ABA and drought treatments, respectively. We conducted loss-of-function and gain-of function genetic studies to examine the in vivo function of CaDIR1 in response to ABA and drought stress. CaDIR1-silenced pepper plants displayed a drought-tolerant phenotype characterized by a low level of transpirational water loss via increased stomatal closure and elevated leaf temperatures. CaDIR1-overexpressing (OX) Arabidopsis plants exhibited an ABA-hypersensitive phenotype during the germination stage, but an ABA-hyposensitive phenotype-characterized by decreased stomatal closure and reduced leaf temperatures-at the adult stage. Moreover, adult CaDIR1-OX plants exhibited a drought-sensitive phenotype characterized by high levels of transpirational water loss. Our results indicate that CaDIR1 functions as a negative regulator of the drought stress response via ABA-mediated signaling. Our findings provide a valuable insight into the plant defense mechanism that operates during drought stress.",
"Thermotolerance is very important for plant survival when plants are subjected to lethally high temperature. However, thus far little is known about the functions of RING E3 ligase in response to heat shock in plants. This study found that one rice gene encoding the RING finger protein was specifically induced by heat and cold stress treatments but not by salinity or dehydration and named it OsHCI1 (Oryza sativa heat and cold induced 1). Subcellular localization results showed that OsHCI1 was mainly associated with the Golgi apparatus and moved rapidly and extensively along the cytoskeleton. In contrast, OsHCI1 may have accumulated in the nucleus under high temperatures. OsHCI1 physically interacted with nuclear substrate proteins including a basic helix-loop-helix transcription factor. Transient co-overexpression of OsHCI1 and each of three nuclear proteins showed that their fluorescent signals moved into the cytoplasm as punctuate formations. Heterogeneous overexpression of OsHCI1 in Arabidopsis highly increased survival rate through acquired thermotolerance. It is proposed that OsHCI1 mediates nuclear-cytoplasmic trafficking of nuclear substrate proteins via monoubiquitination and drives an inactivation device for the nuclear proteins under heat shock.",
"The TRIM family of proteins is distinguished by its tripartite motif (TRIM). Typically, TRIM proteins contain a RING finger domain, one or two B-box domains, a coiled-coil domain and the more variable C-terminal domains. TRIM16 does not have a RING domain but does harbour two B-box domains. Here we showed that TRIM16 homodimerized through its coiled-coil domain and heterodimerized with other TRIM family members; TRIM24, Promyelocytic leukaemia (PML) protein and Midline-1 (MID1). Although, TRIM16 has no classic RING domain, three-dimensional modelling of TRIM16 suggested that its B-box domains adopts RING-like folds leading to the hypothesis that TRIM16 acts as an ubiquitin ligase. Consistent with this hypothesis, we demonstrated that TRIM16, devoid of a classical RING domain had auto-polyubiquitination activity and acted as an E3 ubiquitin ligase in vivo and in vitro assays. Thus via its unique structure, TRIM16 possesses both heterodimerization function with other TRIM proteins and also has E3 ubiquitin ligase activity.",
"To investigate the importance of different processes to heat stress tolerance, 45 Arabidopsis (Arabidopsis thaliana) mutants and one transgenic line were tested for basal and acquired thermotolerance at different stages of growth. Plants tested were defective in signaling pathways (abscisic acid, salicylic acid, ethylene, and oxidative burst signaling) and in reactive oxygen metabolism (ascorbic acid or glutathione production, catalase) or had previously been found to have temperature-related phenotypes (e.g. fatty acid desaturase mutants, uvh6). Mutants were assessed for thermotolerance defects in seed germination, hypocotyl elongation, root growth, and seedling survival. To assess oxidative damage and alterations in the heat shock response, thiobarbituric acid reactive substances, heat shock protein 101, and small heat shock protein levels were determined. Fifteen mutants showed significant phenotypes. Abscisic acid (ABA) signaling mutants (abi1 and abi2) and the UV-sensitive mutant, uvh6, showed the strongest defects in acquired thermotolerance of root growth and seedling survival. Mutations in nicotinamide adenine dinucleotide phosphate oxidase homolog genes (atrbohB and D), ABA biosynthesis mutants (aba1, aba2, and aba3), and NahG transgenic lines (salicylic acid deficient) showed weaker defects. Ethylene signaling mutants (ein2 and etr1) and reactive oxygen metabolism mutants (vtc1, vtc2, npq1, and cad2) were more defective in basal than acquired thermotolerance, especially under high light. All mutants accumulated wild-type levels of heat shock protein 101 and small heat shock proteins. These data indicate that, separate from heat shock protein induction, ABA, active oxygen species, and salicylic acid pathways are involved in acquired thermotolerance and that UVH6 plays a significant role in temperature responses in addition to its role in UV stress.",
"The time-resolved response of Arabidopsis thaliana towards changing light and/or temperature at the transcriptome and metabolome level is presented. Plants grown at 21°C with a light intensity of 150 μE m⁻² sec⁻¹ were either kept at this condition or transferred into seven different environments (4°C, darkness; 21°C, darkness; 32°C, darkness; 4°C, 85 μE m⁻² sec⁻¹; 21 °C, 75 μE m⁻² sec⁻¹; 21°C, 300 μE m⁻² sec⁻¹ ; 32°C, 150 μE m⁻² sec⁻¹). Samples were taken before (0 min) and at 22 time points after transfer resulting in (8×) 22 time points covering both a linear and a logarithmic time series totaling 177 states. Hierarchical cluster analysis shows that individual conditions (defined by temperature and light) diverge into distinct trajectories at condition-dependent times and that the metabolome follows different kinetics from the transcriptome. The metabolic responses are initially relatively faster when compared with the transcriptional responses. Gene Ontology over-representation analysis identifies a common response for all changed conditions at the transcriptome level during the early response phase (5-60 min). Metabolic networks reconstructed via metabolite-metabolite correlations reveal extensive environment-specific rewiring. Detailed analysis identifies conditional connections between amino acids and intermediates of the tricarboxylic acid cycle. Parallel analysis of transcriptional changes strongly support a model where in the absence of photosynthesis at normal/high temperatures protein degradation occurs rapidly and subsequent amino acid catabolism serves as the main cellular energy supply. These results thus demonstrate the engagement of the electron transfer flavoprotein system under short-term environmental perturbations."
] |
Diffusion-weighted magnetic resonance imaging of prostate cancer
|
Diffusion-weighted magnetic resonance imaging (DWI) enables non-invasive, quantitative staging of prostate cancer via measurement of the apparent diffusion coefficient (ADC) of water within tissues. In cancer, more advanced disease is often characterized by higher cellular density (cellularity), which is generally accepted to correspond to a lower measured ADC. A quantitative relationship between tissue structure and
|
[
"Using a mouse prostate reconstitution (MPR) model system, strain-specific responses to the ras and myc oncogenes were investigated. When ras + myc were introduced into both the mesenchymal and epithelial compartments of the urogenital sinus, poorly differentiated prostate cancer was produced at a high frequency (> 90%) in inbred C57BL/6 mice. In contrast, under similar conditions, inbred BALB/c MPRs formed benign prostatic hyperplasia that converted to cancer at a low frequency (< 10%). Restricting the oncogenes to the mesenchymal or epithelial compartments revealed that oncogene activities were more pronounced in the mesenchyme of C57BL/6 mice and resulted in elevated transforming growth factor-beta 1 expression along with a severe desmoplastic reaction. Heterologous MPRs composed of BALB/c mesenchyme and C57BL/6 epithelium or vice versa demonstrated that intrinsic properties of BALB/c mesenchyme can arrest the progression of ras + myc-initiated C57BL/6 epithelium from benign hyperplasia to malignant carcinoma.",
"The mouse prostate is an attractive model for studying the relationship between epithelial-mesenchymal interactions and the mechanism of androgen action because of the volume of information on tissue interactions in the development of the prostate of this species and the existence of a mutant mouse lacking functional androgen receptors (Tfm mouse). In this paper the major proteins of the mouse dorsolateral prostate (DLP) have been described, and antibodies to these proteins have been characterized. The two most abundant secreted proteins were of 110,000-115,000 (Mj1) and 55,000-62,000 (Mj2) mol wt. They were glycosylated, androgen dependent, and appeared to exist in an oligomeric complex. Antibodies raised against mouse DLP secretion reacted mainly with Mj1, Mj2, and a minor protein of 140,000 mol wt (Mn1). The antibodies were of a high titer and recognized these three mouse DLP proteins by Western blotting, immunoprecipitation, and immunocytochemical techniques. Mj1 and Mj2 were antigenically similar to proteins in the mouse coagulating gland and in the rat DLP, but were not found in other organs. Immunocytochemical staining of the DLP from intact mice revealed many ducts that were lined by a tall columnar epithelium whose cells stained intensely. However, ducts that were distended with luminal secretion had a low columnar epithelium that rarely showed intracellular staining. These marker proteins and the antibodies to them will be useful for detecting androgen-dependent functional activity in tissue recombinant studies with a variety of experimental tissues.",
"Progress toward understanding the biology of prostate cancer has been slow due to the few animal research models available to study the spectrum of this uniquely human disease. To develop an animal model for prostate cancer, several lines of transgenic mice were generated by using the prostate-specific rat probasin promoter to derive expression of the simian virus 40 large tumor antigen-coding region. Mice expressing high levels of the transgene display progressive forms of prostatic disease that histologically resemble human prostate cancer, ranging from mild intraepithelial hyperplasia to large multinodular malignant neoplasia. Prostate tumors have been detected specifically in the prostate as early as 10 weeks of age. Immunohistochemical analysis of tumor tissue has demonstrated that dorsolateral prostate-specific secretory proteins were confined to well-differentiated ductal epithelial cells adjacent to, or within, the poorly differentiated tumor mass. Prostate tumors in the mice also display elevated levels of nuclear p53 and a decreased heterogeneous pattern of androgen-receptor expression, as observed in advanced human prostate cancer. The establishment of breeding lines of transgenic mice that reproducibly develop prostate cancer provides an animal model system to study the molecular basis of transformation of normal prostatic cells and the factors influencing the progression to metastatic prostate cancer.",
"Regulatory sequences derived from the rat whey acidic protein gene have been used to preferentially overexpress a murine 172Arg-->Leu mutant p53 in the mammary gland of transgenic mice. Several different lines of mice expressing the 172Arg-->Leu mutant p53 displayed an impaired ability to lactate, and the mice expressing the highest levels of mutant p53 were unable to nurse their young. This failure was related to the inhibition of normal lobuloalveolar development that occurred during late pregnancy and a marked decrease in milk protein gene expression at early lactation. Interestingly, immunohistochemical analysis revealed that the mutant p53 was localized predominantly in the cytoplasm of alveolar cells. Ductal development was not overtly impaired in these mice. Expression of the 172Arg-->Leu mutant p53 resulted in radiation-induced apoptosis, and transactivation or repression of the expression of a number of genes, including mdm-2 and proliferating cell nuclear antigen, known properties of wild-type p53. The availability of lines of mice preferentially expressing specific p53 mutants in the mammary gland should facilitate evaluation of the roles of other factors, such as hormones, oncogenes and chemical carcinogens, in the etiology of breast cancer."
] |
Prevalence of factors associated with hypertension in adolescents in the city of Goiânia City, Central Brazil
|
Adolescents are a population with unique lifestyle challenges, including physical inactivity, inadequate nutrition, and obesity, all of which increase the risk of developing hypertension (HTN). The objective of this study has been to estimate the prevalence of factors associated with hypertension in adolescents in the city of Goiânia City, Central Brazil.
|
[
"Hypertension is one of the major risk factors of cardiovascular diseases, but despite a century of clinical and basic research, the discrete etiology of this disease is still not fully understood. The same is true for obesity, which is recognized as a major global epidemic health problem nowadays. Obesity is associated with an increasing prevalence of the metabolic syndrome, a cluster of risk factors including hypertension, abdominal obesity, dyslipidemia, and hyperglycemia. Epidemiological studies have shown that excess weight gain predicts future development of hypertension, and the relationship between BMI and blood pressure (BP) appears to be almost linear in different populations. There is no doubt that obesity-related hypertension is a multifactorial and polygenic trait, and multiple potential pathogenetic mechanisms probably contribute to the development of higher BP in obese humans. These include hyperinsulinemia, activation of the renin-angiotensin-aldosterone system, sympathetic nervous system stimulation, abnormal levels of certain adipokines such as leptin, or cytokines acting at the vascular endothelial level. Moreover, some genetic and epigenetic mechanisms are also in play. Although the full manifestation of both hypertension and obesity occurs predominantly in adulthood, their roots can be traced back to early ontogeny. The detailed knowledge of alterations occurring in the organism of experimental animals during particular critical periods (developmental windows) could help to solve this phenomenon in humans and might facilitate the age-specific prevention of human obesity-related hypertension. In addition, better understanding of particular pathophysiological mechanisms might be useful in so-called personalized medicine.",
"The prevalence of hypertension in childhood is increasing, and investigation of its distribution is important for planning timely interventions. This study assessed the prevalence of high blood pressure (HBP) and associated factors in students between 9 and 11 years of age enrolled in public and private schools in Maceió, Brazil. A cross-sectional study was performed in a probabilistic sample of students (10.3 ± 0.5 years). The students were selected from a systematic sampling of 80 schools (40 public and 40 private). To maintain similar proportions of students existing in public and private schools in Maceió, 21 and 14 students were randomly selected from each public and private school, respectively. The prevalence ratio (PR) was estimated using Poisson regression. A total of 1,338 students were evaluated (800 from public schools and 538 from private schools). No differences were observed between school types in terms of student age and gender (p > 0.05). The prevalence of obesity (19.9% vs. 9.0%; PR = 2.2; 95% CI = 1.67-2.92) and hypertension (21.2% vs. 11.4%; PR = 1.86; 95% CI = 1.45-2.40) were higher in private schools. The association between high blood pressure and type of school (public or private) remained statistically significant even after adjustment for obesity (PR = 1.53; 95% CI = 1.19-1.97). (a) students from private schools have higher socioeconomic status, BMI, and HBP prevalence compared to those of public school; (b) among the evaluated students, the prevalence of obesity only partially explained the higher prevalence of high blood pressure among students from private schools. Other factors related to lifestyle of children from private schools may explain the higher prevalence of HBP. This results show the need to implement measures to promote healthy lifestyles in the school environment, since children with HBP are more likely to become hypertensive adults. Therefore, early detection and intervention in children with HBP is an important action for the prevention of hypertension in adulthood.",
"To evaluate the sensitivity and specificity of anthropometric measurements of body fat in a sample of Brazilian adolescents for the prediction of hypertension. The arterial blood pressure was measured on two visits in a sample of 456 students aged 12 to 17 years, from public and private schools of the Fonseca neighborhood, in Niterói, between 2003 and 2004. A subject was defined as hypertensive if he/she had systolic and diastolic pressures above the 95th percentile for sex, age and height. A questionnaire was applied and Body Mass Index (BMI) and waist circumference (WC) measurements were made. A statistically significant correlation was observed between hypertension and the cutoff points considered unfavorable, both for BMI and WC. The greatest association was with the cutoff point proposed for the Brazilian population. As to the BMI sensitivity used for American Black or White populations or for the Brazilian population, we found 52.4% to 57.1% and 52.4%, respectively. And BMI specificity was 69.3%, 70.0% and 80.88%, respectively. The sensitivity found in our sample, relative to the cutoff points for WC proposed for all American ethnic groups, was also low (45.0%) and specificity was a little higher (77.6% and 74.5%, respectively). Existing American WC measurements showed low sensitivity and specificity for hypertension in our population. As to BMI, the available cutoff points also showed a low level of sensitivity. There is a need to establish body fat cutoff points that can provide a better prediction of cardiovascular risk.",
"The prevalence of hypertension among children and adolescents is estimated to range between 1% and 13%. Excess weight and central obesity are related to blood pressure levels in adults, and may be important in the early pathogenesis of SH when present in childhood. To study the association between anthropometric variables and blood pressure levels in schoolchildren from the 5th and 8th grades, and to identify which parameter was more strongly correlated with blood pressure levels. Contemporary cross-sectional study with probabilistic population-based cluster sampling of schoolchildren enrolled from the 5th to the 8th grades in public elementary schools of Porto Alegre. Data on familial risk factors and anthropometry were collected. Statistical analysis included correlations and cluster-adjusted confidence intervals. The mean age of participants was 12.57 (± 1.64) years, and 55.2% of them were females. Abnormal blood pressure levels were found in 11.3% of the sample and borderline values, in 16.2%. Among the anthropometric variables analyzed, hip circumference was the one with the strongest correlation with increased blood pressure (r = 0.462, p < 0.001), followed by waist circumference (r = 0.404, p < 0.001) and abdominal skinfold (r = 0.291, p < 0.001). We observed an association of waist circumference and skinfolds with increased blood pressure levels in the schoolchildren of the sample. Therefore, it is of the utmost importance that early measurements of blood pressure, and waist and hip circumferences become a routine in health services in order to prevent this condition.",
"OBJECTIVE To estimate the prevalences of tobacco use, tobacco experimentation, and frequent smoking among Brazilian adolescents. METHODS We evaluated participants of the cross-sectional, nation-wide, school-based Study of Cardiovascular Risks in Adolescents (ERICA), which included 12- to 17-year-old adolescents from municipalities of over 100 thousand inhabitants. The study sample had a clustered, stratified design and was representative of the whole country, its geographical regions, and all 27 state capitals. The information was obtained with self-administered questionnaires. Tobacco experimentation was defined as having tried cigarettes at least once in life. Adolescents who had smoked on at least one day over the previous 30 days were considered current cigarette smokers. Having smoked cigarettes for at least seven consecutive days was an indicator for regular consumption of tobacco. Considering the complex sampling design, prevalences and 95% confidence intervals were estimated according to sociodemographic and socio-environmental characteristics. RESULTS We evaluated 74,589 adolescents. Among these, 18.5% (95%CI 17.7-19.4) had smoked at least once in life, 5.7% (95%CI 5.3-6.2) smoked at the time of the research, and 2.5% (95%CI 2.2-2.8) smoked often. Adolescents aged 15 to 17 years had higher prevalences for all indicators than those aged 12 to 14 years. The prevalences did not differ significantly between sexes. The highest prevalences were found in the South region and the lowest ones, in the Northeast region. Regardless of sex, the prevalences were found to be higher for adolescents who had had paid jobs, who lived with only one parent, and who reported having been in contact with smokers either inside or outside their homes. Female public school adolescents were found to smoke more than the ones from private schools. CONCLUSIONS Tobacco use among adolescents is still a challenge. Intending to reduce the prevalence of tobacco use among young people, especially the ones under socioeconomic vulnerability conditions, Brazil must consolidate and increase effective public health care measures.",
"OBJECTIVE To estimate the prevalence of arterial hypertension and obesity and the population attributable fraction of hypertension that is due to obesity in Brazilian adolescents. METHODS Data from participants in the Brazilian Study of Cardiovascular Risks in Adolescents (ERICA), which was the first national school-based, cross-section study performed in Brazil were evaluated. The sample was divided into 32 geographical strata and clusters from 32 schools and classes, with regional and national representation. Obesity was classified using the body mass index according to age and sex. Arterial hypertension was defined when the average systolic or diastolic blood pressure was greater than or equal to the 95th percentile of the reference curve. Prevalences and 95% confidence intervals (95%CI) of arterial hypertension and obesity, both on a national basis and in the macro-regions of Brazil, were estimated by sex and age group, as were the fractions of hypertension attributable to obesity in the population. RESULTS We evaluated 73,399 students, 55.4% female, with an average age of 14.7 years (SD = 1.6). The prevalence of hypertension was 9.6% (95%CI 9.0-10.3); with the lowest being in the North, 8.4% (95%CI 7.7-9.2) and Northeast regions, 8.4% (95%CI 7.6-9.2), and the highest being in the South, 12.5% (95%CI 11.0-14.2). The prevalence of obesity was 8.4% (95%CI 7.9-8.9), which was lower in the North region and higher in the South region. The prevalences of arterial hypertension and obesity were higher in males. Obese adolescents presented a higher prevalence of hypertension, 28.4% (95%CI 25.5-31.2), than overweight adolescents, 15.4% (95%CI 17.0-13.8), or eutrophic adolescents, 6.3% (95%CI 5.6-7.0). The fraction of hypertension attributable to obesity was 17.8%. CONCLUSIONS ERICA was the first nationally representative Brazilian study providing prevalence estimates of hypertension in adolescents. Regional and sex differences were observed. The study indicates that the control of obesity would lower the prevalence of hypertension among Brazilian adolescents by 1/5.",
"Limited evidence exists on the accuracy of oscillometric devices for blood pressure measurement in children. This study validated the Omron 705 IT monitor (Omron Healthcare Europe BV, Hoofddorp, The Netherlands) in normotensive children and adolescents. Simultaneous blood pressure measurements were taken by two observers (connected mercury sphygmomanometers) four times, sequentially with three measurements by using the tested device. Absolute device-observer blood pressure differences were classified into three zones (within 5, 10 and 15 mmHg) and assessed using the European Society of Hypertension International Protocol criteria. The number of readings with a difference within 5 mmHg was calculated for each individual. The American Association for the Advancement of Medical Instrumentation criterion (mean difference+ or -SD <5+ or -8 mmHg) was also applied. A total of 197 study participants (591 readings) were included (99 boys, mean age+ or -SD 10.6+ or -2.4 years (range 6-16 years). The device produced 378 (64%), 532 (90%) and 579 (98%) measurements within 5, 10 and 15 mmHg, respectively, for systolic blood pressure, and 415 (70%), 525 (89%) and 577 (98%) measurements, respectively, for diastolic blood pressure. In all, 136 study participants (69%) had at least two systolic blood pressure differences within 5 mmHg and 16 (8%) had no differences within 5 mmHg (for diastolic blood pressure 147 (75%) and nine (5%) participants, respectively). Mean systolic blood pressure difference was 4.0+ or -4.8 mmHg and diastolic blood pressure -2.1+ or -5.9 mmHg and was <5+ or -8 mmHg in quartiles of participants divided by age, height, body mass index, arm circumference, blood pressure or pulse rate. The Omron 705 IT appears to be an accurate device for blood pressure measurement in normotensive children and adolescents."
] |
Sleep quality, circadian phase, and mood: A self-report questionnaire for sleep quality, circadian phase, and mood
|
Sleep quality, circadian phase, and mood are highly interdependent processes. Remarkably, there is currently no self-report questionnaire that measures all three of these clinically significant functions: The aim of this project was to address this deficit. In Study 1, 720 participants completed a set of potential items was generated from existing questionnaires in each of the three domains and refined to follow a single presentation format. Study 2 used an independent sample (
|
[
"The National Institutes of Health (NIH) Patient-Reported Outcomes Measurement Information System (PROMIS) Roadmap initiative (www.nihpromis.org) is a 5-year cooperative group program of research designed to develop, validate, and standardize item banks to measure patient-reported outcomes (PROs) relevant across common medical conditions. In this article, we will summarize the organization and scientific activity of the PROMIS network during its first 2 years. The network consists of 6 primary research sites (PRSs), a statistical coordinating center (SCC), and NIH research scientists. Governed by a steering committee, the network is organized into functional subcommittees and working groups. In the first year, we created an item library and activated 3 interacting protocols: Domain Mapping, Archival Data Analysis, and Qualitative Item Review (QIR). In the second year, we developed and initiated testing of item banks covering 5 broad domains of self-reported health. The domain mapping process is built on the World Health Organization (WHO) framework of physical, mental, and social health. From this framework, pain, fatigue, emotional distress, physical functioning, social role participation, and global health perceptions were selected for the first wave of testing. Item response theory (IRT)-based analysis of 11 large datasets supplemented and informed item-level qualitative review of nearly 7000 items from available PRO measures in the item library. Items were selected for rewriting or creation with further detailed review before the first round of testing in the general population and target patient populations. The NIH PROMIS network derived a consensus-based framework for self-reported health, systematically reviewed available instruments and datasets that address the initial PROMIS domains. Qualitative item research led to the first wave of network testing which began in the second year.",
"Insomnia is a prevalent disabling chronic disorder. The aim of this paper is fourfold: (a) to review evidence suggesting that dysfunctional forms of cognitive control, such as thought suppression, worry, rumination, and imagery control, are associated with sleep disturbance; (b) to review a new budding field of scientific investigation - the role of dysfunctional affect control in sleep disturbance, such as problems with down-regulating negative and positive affective states; (c) to review evidence that sleep disturbance can impair next-day affect control; and (d) to outline, on the basis of the reviewed evidence, how the repetitive-thought literature and the affective science literature can be combined to further understanding of, and intervention for, insomnia.",
"Item response theory (IRT) is increasingly being applied to health-related quality of life instrument development and refinement. This article discusses results obtained using categorical confirmatory factor analysis (CCFA) to check IRT model assumptions and the application of IRT in item analysis and scale evaluation. To demonstrate the value of CCFA and IRT in examining a health-related quality of life measure in children and adolescents. This illustration uses data from 10,241 children and their parents on items from the 4 subscales of the PedsQL 4.0 Generic Core Scales. CCFA was applied to confirm domain dimensionality and identify possible locally dependent items. IRT was used to assess the strength of the relationship between the items and the constructs of interest and the information available across the latent construct. CCFA showed generally strong support for 1-factor models for each domain; however, several items exhibited evidence of local dependence. IRT revealed that the items generally exhibit favorable characteristics and are related to the same construct within a given domain. We discuss the lessons that can be learned by comparing alternate forms of the same scale, and we assess the potential impact of local dependence on the item parameter estimates. This article describes CCFA methods for checking IRT model assumptions and provides suggestions for using these methods in practice. It offers insight into ways information gained through IRT can be applied to evaluate items and aid in scale construction.",
"Measurement of headache impact is important in clinical trials, case detection, and the clinical monitoring of patients. Computerized adaptive testing (CAT) of headache impact has potential advantages over traditional fixed-length tests in terms of precision, relevance, real-time quality control and flexibility. To develop an item pool that can be used for a computerized adaptive test of headache impact. We analyzed responses to four well-known tests of headache impact from a population-based sample of recent headache sufferers (n = 1016). We used confirmatory factor analysis for categorical data and analyses based on item response theory (IRT). In factor analyses, we found very high correlations between the factors hypothesized by the original test constructers, both within and between the original questionnaires. These results suggest that a single score of headache impact is sufficient. We established a pool of 47 items which fitted the generalized partial credit IRT model. By simulating a computerized adaptive health test we showed that an adaptive test of only five items had a very high concordance with the score based on all items and that different worst-case item selection scenarios did not lead to bias. We have established a headache impact item pool that can be used in CAT of headache impact.",
"To evaluate the feasibility of computerized adaptive testing (CAT) and the reliability and validity of CAT-based estimates of headache impact scores in comparison with 'static' surveys. Responses to the 54-item Headache Impact Test (HIT) were re-analyzed for recent headache sufferers (n = 1016) who completed telephone interviews during the National Survey of Headache Impact (NSHI). Item response theory (IRT) calibrations and the computerized dynamic health assessment (DYNHA) software were used to simulate CAT assessments by selecting the most informative items for each person and estimating impact scores according to pre-set precision standards (CAT-HIT). Results were compared with IRT estimates based on all items (total-HIT), computerized 6-item dynamic estimates (CAT-HIT-6), and a developmental version of a 'static' 6-item form (HIT-6-D). Analyses focused on: respondent burden (survey length and administration time), score distributions ('ceiling' and 'floor' effects), reliability and standard errors, and clinical validity (diagnosis, level of severity). A random sample (n = 245) was re-assessed to test responsiveness. A second study (n = 1103) compared actual CAT surveys and an improved 'static' HIT-6 among current headache sufferers sampled on the Internet. Respondents completed measures from the first study and the generic SF-8 Health Survey; some (n = 540) were re-tested on the Internet after 2 weeks. In the first study, simulated CAT-HIT and total-HIT scores were highly correlated (r = 0.92) without 'ceiling' or 'floor' effects and with a substantial reduction (90.8%) in respondent burden. Six of the 54 items accounted for the great majority of item administrations (3603/5028, 77.6%). CAT-HIT reliability estimates were very high (0.975-0.992) in the range where 95% of respondents scored, and relative validity (RV) coefficients were high for diagnosis (RV = 0.87) and severity (RV = 0.89); patient-level classifications were accurate 91.3% for a diagnosis of migraine. For all three criteria of change, CAT-HIT scores were more responsive than all other measures. In the second study, estimates of respondent burden, item usage, reliability and clinical validity were replicated. The test-retest reliability of CAT-HIT was 0.79 and alternate forms coefficients ranged from 0.85 to 0.91. All correlations with the generic SF-8 were negative. CAT-based administrations of headache impact items achieved very large reductions in respondent burden without compromising validity for purposes of patient screening or monitoring changes in headache impact over time. IRT models and CAT-based dynamic health assessments warrant testing among patients with other conditions."
] |
Curcumin attenuates carboplatin-induced myelosuppression by activating the DNA repair pathway in bone marrow cells
|
Carboplatin, a second-generation platinum agent, has been used as a cancer therapy for decades and exhibits strong anti-tumor activity. However, the wide application of carboplatin is largely limited due to its side effects, especially myelosuppression. Here, we combined carboplatin with curcumin, a natural product that improves tumor-induced anemia, for the treatment of fibrosarcoma to improve the side effects of carboplatin. We first examined the synergistic and attenuated effects of the two agents in a T241-bearing mouse model. The combination therapy caused no obvious synergistic effect, but curcumin significantly improved the survival rate of carboplatin-treated mice. Histologic analysis of the kidney and bone marrow revealed that curcumin improved carboplatin-induced myelosuppression but did not affect the kidney. To determine the mechanism involved, we introduced a probe derived from curcumin to identify its targets in bone marrow cells and the results provided us a clue that curcumin might affect the DNA repair pathway. Western blot analysis revealed that curcumin up-regulated BRCA1, BRCA2 and ERCC1 expression in bone marrow. In conclusion, curcumin attenuates carboplatin-induced myelosuppression by activating the DNA repair pathway in bone marrow cells.
|
[
"Methylglyoxal (MG) is a reactive dicarbonyl compound endogenously produced mainly from glycolytic intermediates. Elevated MG levels in diabetes patients are believed to contribute to diabetic complications. MG is cytotoxic through induction of apoptosis. Curcumin, the yellow pigment of Curcuma longa, is known to have antioxidant and anti-inflammatory properties. In the present study, we investigated the effect of curcumin on MG-induced apoptotic events in human hepatoma G2 cells. We report that curcumin prevented MG-induced cell death and apoptotic biochemical changes such as mitochondrial release of cytochrome c, caspase-3 activation, and cleavage of PARP (poly [ADP-ribose] polymerase). Using the cell permeable dye 2',7'-dichlorofluorescein diacetate (DCF-DA) as an indicator of reactive oxygen species (ROS) generation, we found that curcumin abolished MG-stimulated intracellular oxidative stress. The results demonstrate that curcumin significantly attenuates MG-induced ROS formation, and suggest that ROS triggers cytochrome c release, caspase activation, and subsequent apoptotic biochemical changes.",
"Although prostaglandin research began about 50 years ago, many of the most important advances in understanding the biochemistry, physiology and pharmacology have taken place within the past five to ten years. There is great potential for the extension of this research to the clinical practice of medicine. At this time, the most common interaction that clinicians have with the prostaglandin field is in administering nonsteroidal anti-inflammatory drugs, which function by inhibiting prostaglandins. The uses of these drugs include treating not only inflammation, but also dysmenorrhea, some renal disease, thrombotic diseases and some metabolic disorders. Prostaglandin analogs, with their potent effects on uterine contraction, are in common use in obstetrics. Other analogs, with gastric and duodenal cytoprotective effects are useful in treating peptic ulcer disease. Future benefits from prostaglandin and leukotriene research may include new therapy for inflammatory and hypersensitivity diseases such as asthma, inflammatory bowel diseases and dermatitis.",
"Serum gastrin has been reported to increase after therapy with some agents effective in the treatment of duodenal ulcer (DU). Misoprostol, a prostaglandin E1 analog, is effective in the treatment of DU, with healing rates similar to those achieved with vigorous antacid therapy or H2-receptor antagonists. Misoprostol reduces gastric acid secretion and also possesses cytoprotective properties. This multicenter study examined serum gastrin levels before and after treatment of DU patients with misoprostol. Sera for gastrin measurement were obtained from DU patients after an 8-hr fast, and 15 and 30 min after a standard mixed meal. DU patients were studied before and after two to four weeks of treatment with placebo or misoprostol: in one study, misoprostol 100 micrograms qid (without antacid) was compared with placebo; in the other study, misoprostol at 50- or 200-micrograms qid dosages (with limited antacid, Amphojel up to 54 meq/day) was compared with placebo. In addition, the serum gastrin values obtained in healthy subjects were compared with those from DU patients. Fasting and postprandial serum gastrin concentrations were essentially similar for DU patients and healthy subjects. There were no significant differences, either in fasting serum gastrin or in integrated gastrin responses, in DU patients after treatment with placebo or misoprostol at 100 micrograms (P = 0.32), 50 micrograms, or 200 micrograms doses (P = 0.85). It is concluded that misoprostol, when administered four times daily for two to four weeks at dosages required for the acceleration of DU healing, does not affect serum gastrin levels.",
"Misoprostol is an analogue of prostaglandin E1 and is the first synthetic prostaglandin analogue to be made available for the treatment of peptic ulcer disease. It inhibits gastric acid secretion in man, and there is also some evidence that it limits the extent of gastrointestinal damage induced by ulcerogenic agents in animals and healthy volunteers at doses lower than those required to inhibit acid secretion. This 'cytoprotective' activity has been explained by several mechanisms, but its contribution to the clinical efficacy of misoprostol in healing established ulcers is doubtful since the drug does not appear to be effective in healing peptic ulcers at non-antisecretory dosages. In clinical trials, ulcer healing has been reported in 60 to 85% of patients with duodenal ulcers and 32 to 54% with gastric ulcers receiving misoprostol 200 micrograms 4 times daily for 4 weeks--the recommended dosage. In comparative studies, the percentage of patients with healed ulcers after misoprostol (800 micrograms daily) was not significantly different from that with cimetidine (1200 mg daily), although there was greater pain relief with cimetidine. No study has yet been published concerning the use of misoprostol as maintenance therapy for the prevention of ulcer recurrence, and no long term tolerability data are available. However, in acute ulcer healing studies (2 to 12 weeks in duration) misoprostol has been well tolerated. Diarrhoea was the most commonly reported symptom, and this was only rarely of sufficient severity to interfere with treatment. No evidence of histopathological changes in the gastric mucosa induced by misoprostol have been reported in man. Evidence of uterine stimulant effects in women receiving misoprostol during the first trimester of pregnancy has resulted in the drug being contraindicated during pregnancy. Thus, misoprostol is a new type of antiulcer drug, providing an alternative approach to the therapy of peptic ulcer disease. It has been shown to be effective and well tolerated in the healing of both gastric and duodenal ulcers. Future studies need to identify the specific types of patients likely to obtain most benefit from treatment, in order to define more clearly the place of misoprostol in the treatment of these indications, as well as addressing the possibility of ulcer prevention with lower doses of misoprostol.",
"Platinating agents, including cisplatin, carboplatin, and oxaliplatin, have been used clinically for nearly 30years as part of the treatment of many types of cancers, including head and neck, testicular, ovarian, cervical, lung, colorectal and relapsed lymphoma. The cytotoxic lesion of platinating agents is thought to be the platinum intrastrand crosslink that forms on DNA, although treatment activates a number of signal transduction pathways. Treatment with these agents is characterized by resistance, both acquired and intrinsic. This resistance can be caused by a number of cellular adaptations, including reduced uptake, inactivation by glutathione and other anti-oxidants, and increased levels of DNA repair or DNA tolerance. Here we investigate the pathways that treatment with platinating agents activate, the mechanisms of resistance, potential candidate genes involved in the development of resistance, and associated clinical toxicities. Although the purpose of this review is to provide an overview of cisplatin, carboplatin, and oxaliplatin, we have focused primarily on preclinical data that has clinical relevance generated over the past five years.",
"Germline mutations in BRCA1 confer a high risk of breast and ovarian tumors. The role of BRCA1 in tumor suppression is not yet understood, but both transcription and repair functions have been ascribed. Evidence that BRCA1 is involved in DNA repair stems from its association with RAD51, a homolog of the yeast protein involved in the repair of DNA double-strand breaks (DSBs) by homologous recombination. We report here that Brca1-deficient mouse embryonic stem cells have impaired repair of chromosomal DSBs by homologous recombination. The relative frequencies of homologous and nonhomologous DNA integration and DSB repair were also altered. The results demonstrate a caretaker role for BRCA1 in preserving genomic integrity by promoting homologous recombination and limiting mutagenic nonhomologous repair processes.",
"The rational treatment of gastric ulcer (GU) requires both an understanding of the various causative factors responsible for what is best considered a spectrum of disorders, as well as a familiarity with the newer antisecretory and cytoprotective therapies that are available. Gastric ulcers that fulfill the criteria to be peptic ulcers (ie, occur in the antrum in the presence of excess luminal acid, with or without a coexisting duodenal ulcer) are best treated with a histamine H2-receptor antagonist. For GUs that develop in the setting of normal or reduced acid output, or those that occur as a result of direct (ie, drug-induced or bile acid-related) mucosal injury, use of a cytoprotective agent (eg, sucralfate) is the treatment of choice. Any GU that fails to heal within 12 to 15 weeks should be carefully examined to exclude the presence of a malignant neoplasm and should be considered for surgical resection."
] |
what is alps
|
Autoimmune lymphoproliferative syndrome (ALPS) is an incurable disease mainly caused by the defect of Fas-mediated apoptosis and characterized by nonmalignant autoimmune lymphoproliferation. Stabilized
|
[
"IL-2, a cytokine with pleiotropic effects, is critical for immune cell activation and peripheral tolerance. Although the therapeutic potential of IL-2 has been previously suggested in autoimmune diseases, the mechanisms whereby IL-2 mitigates autoimmunity and prevents organ damage remain unclear. Using an inducible recombinant adeno-associated virus vector, we investigated the effect of low systemic levels of IL-2 in lupus-prone MRL/Fas(lpr/lpr) (MRL/lpr) mice. Treatment of mice after the onset of disease with IL-2-recombinant adeno-associated virus resulted in reduced mononuclear cell infiltration and pathology of various tissues, including skin, lungs, and kidneys. In parallel, we noted a significant decrease of IL-17-producing CD3(+)CD4(-)CD8(-) double-negative T cells and an increase in CD4(+)CD25(+)Foxp3(+) immunoregulatory T cells (Treg) in the periphery. We also show that IL-2 can drive double-negative (DN) T cell death through an indirect mechanism. Notably, targeted delivery of IL-2 to CD122(+) cytotoxic lymphocytes effectively reduced the number of DN T cells and lymphadenopathy, whereas selective expansion of Treg by IL-2 had no effect on DN T cells. Collectively, our data suggest that administration of IL-2 to lupus-prone mice protects against end-organ damage and suppresses inflammation by dually limiting IL-17-producing DN T cells and expanding Treg.",
"Podocytes maintain the structure and function of the glomerular filtration barrier. However, podocytes have recently been implicated in the innate immune response, and their function as non-haematopoietic antigen-presenting cells was highlighted. We have shown previously that excessive expression of the chemokine CXCL13 is a distinctive early event for nephritis in a murine model of systemic lupus erythematosus (SLE). Furthermore, we found that CXCL13 is elevated significantly in the serum of patients with SLE-nephritis. In this study, we were able to show for the first time that (i) CXCL13 is expressed locally in glomeruli in a model for SLE-nephritis in mice and that (ii) incubation of human podocytes with CXCL13 induces receptor stimulation of CXCR5 with activation of signalling pathways, resulting in (iii) secretion of proinflammatory cytokines and chemokines in culture supernatant. This cytokine/chemokine cocktail can lead to (iv) a neutrophil respiratory burst in isolated human granulocytes. Taken together, our results provide further evidence that CXCL13 is involved in the pathogenesis of glomerulonephritis and that podocytes can play an active role in local proinflammatory immune responses. Thus, CXCL13 could be a direct target for the therapy of glomerulonephritis in general and for SLE-nephritis in particular.",
"Different studies over the last decade have linked the B cell-attracting chemokine CXC ligand 13 (CXCL13) to the autoimmune disease systemic lupus erythematosus (SLE). A pathogenetic role of this chemokine for disease manifestation in SLE was described initially in mouse models for SLE. Mechanisms of CXCL13 actions were also identified in SLE patients. Moreover, various clinical studies have identified CXCL13 serum levels as a useful biomarker in patients with SLE of different ethnicities for disease activity. In addition, CXCL13 seems to be a promising marker for the diagnosis of lupus nephritis, one of the most severe complications of SLE. However, its exact place within the mechanisms that lead to SLE remains to be defined. Further research is needed to resolve more details of the pathomechanism and the signalling pathway of CXCL13 in SLE. Blocking CXCL13 or the signal pathways of CXCL13 is seen as a promising therapeutic approach for SLE and will be addressed in the near future. This review summarizes all papers that linked CXCL13 to SLE and highlights its importance in the pathogenesis and diagnosis of SLE.",
"Lupus nephritis (LN) is a major cause of morbidity and mortality in the patients with systemic lupus erythematosus (SLE), an autoimmune disease with damage to multiple organs. Leukocyte recruitment into the inflamed kidney is a critical step to promote LN progression, and the chemokine/chemokine receptor system is necessary for leukocyte recruitment. In this review, we summarize recent studies on the roles of chemokines and chemokine receptors in the development of LN and discuss the potential and hurdles of developing novel, chemokine-based drugs to treat LN.",
"Intrarenal B cell infiltrates resembling secondary lymphoid tissue have been found in several forms of inflammatory kidney disease. Their role in renal inflammation is not well defined, perhaps because B cell clusters have been regarded as a single entity while being quite heterogeneous. Therefore we characterized intrarenal lymphoid clusters of 32 patients diagnosed with lupus nephritis and 16 with ANCA associated nephritis. We identified four increasingly organized levels of intrarenal aggregates from scattered B cells to highly compartmentalized B cell clusters with central follicular dendritic cell networks. Most B cells displayed a mature non-antibody producing phenotype with antigen presenting ability. In regions of B cell infiltration, expression of the lymphoid chemokine BCA-1 was found in cells of a dendritic-like morphology and most B cells expressed the corresponding receptor CXCR5. Biopsies containing B cells had significantly higher levels of BCA-1 mRNA expression compared to those without, suggesting a role of BCA-1 and CXCR5 for B cell infiltration into the kidney. Our study proposes a new classification of B cell clusters in lupus and ANCA associated nephritis which might help to study the function of intrarenal B cell clusters in a more differentiated manner.",
"The recruitment of immune cells to sites of tissue inflammation is orchestrated by chemokine/chemokine receptor networks. Among these, the CXCL13/CXCR5 axis is thought to be involved critically in systemic lupus erythematosus (SLE) and lupus nephritis pathogenesis. Beyond B cell abnormalities, another hallmark of SLE disease is the occurrence of aberrant T cell responses. In particular, double-negative (DN) T cells are expanded in the peripheral blood of patients with SLE and in lupus-prone mice. DN T cells induce immunoglobulin production, secrete proinflammatory cytokines and infiltrate inflamed tissue, including kidneys. We aimed to investigate how CXCR5 deficiency changes immune cell trafficking in murine lupus. We therefore crossed CXCR5(-/-) mice with B6/lpr mice, a well-established murine lupus model. B cell numbers and B cellular immune responses were diminished in CXCR5-deficient B6/lpr mice. In addition, we observed reduced accumulation of DN T cells in spleen and lymph nodes, paralleled by reduced splenomegaly and lymphadenopathy. In-vivo migration assays revealed reduced migration of CXCR5-deficient DN T cells into lymph nodes, and ex-vivo-activated CXCR5-deficient DN T cells failed to infiltrate kidneys of recipients. Moreover, DN T cells and B cells of CXCR5-deficient B6/lpr mice failed to migrate towards CXCL13 in vitro. We propose that CXCR5 is involved critically in B cell trafficking and germinal cell (GC) formation in murine lupus and in guiding pathogenic DN T cells into lymphoid organs and kidneys, and we therefore describe new pathomechanisms for the CXCL13/CXCR5 axis in SLE."
] |
Changes in Stimulant/Nitrite Use and Risky Sexual Behavior Among Seroconverting MSM
|
The use of stimulant drugs alone or in combination with amyl nitrites (stimulant/nitrites) has been associated with higher rates of risky sexual behavior and predictive of HIV infection among men who have sex with men. However, the temporal pattern of stimulant/nitrite use pre- and post-seroconversion has not been well established. This study assessed changes in stimulant/nitrite use and risky sexual behavior among seroconverting MSM over time. Data were collected in the Baltimore-Washington, DC; Pittsburgh; Chicago; and Los Angeles sites of the Multicenter AIDS Cohort Study (MACS), a longitudinal study of the natural history of HIV infection among MSM. We used propensity scores to select 1044 MSM from 7087 MACS participants composed of 348 seroconverting, 348 seronegative, and 348 seroprevalent participants matched on demographics, recruitment cohort, and study visits. We centered up to four-years of semi-annual data around the seroconversion visit of the seroconverting case within each matched group of participants. Mixed effects regressions estimated the effects of serostatus, recruitment cohort, and time on self-reported stimulant/nitrite use, numbers of male intercourse partners, and numbers of unprotected receptive anal intercourse (URAI) partners. Covariates included demographics, binge drinking, and marijuana use. Seroconverters had the highest odds of stimulant/inhaled nitrite use (AOR 10.3, CI 4.8-22.0), incident rates of intercourse (IRR 1.6, CI 1.3-2.1), and URAI partners (IRR 5.1, CI 3.5-7.3). All participants decreased drug use and sexual risk behavior over time. However, the decreases were largest for seroconverters who nevertheless maintained the highest rates of stimulant/nitrite use and sexual risk. Cohort-related effects were associated with sharp reductions in stimulant/nitrite use and URAI in the early 1990s that rebounded considerably within the first decade of the 2000s. Although all participants decreased risky sexual behavior and stimulant and/or nitrite use over time, seroconverters had the largest decreases. There was no evidence for abrupt or substantial increases in drug use or risky sex post-seroconversion. However, there was substantial variation at the individual level, with the factors underlying this variation not well understood and worth further study. Moreover, stimulant/nitrite use and risky sexual behavior appear to have been strongly influenced by contextual historical and socio-cultural effects. The manner in which contextual factors influence individual behavior is also not well understood and also warrants further study.
|
[
"Illicit drug use before or during sex - known as sexualized drug use (colloquially 'chemsex' or 'party and play') - has evolved as novel psychoactive substances have entered the market in many parts of the world. Here, we review key conceptual issues in associations between illicit drug use and sexual risk-behaviour in MSM. Although many studies have confirmed that MSM use drugs with greater prevalence than the general population, evidence is of variable quality and a sampling frame is difficult to establish. Moreover, psychosocial hypotheses linking drug use and sexual risk, including cognitive escape and sensation seeking, are unsatisfactory and generally ignore strategic use of drugs for sexual aims. Person-level associations between drug use history and both sexual risk behaviour and HIV infection tend to be consistent around the world, but evidence comparing encounters within subjects is generally unclear and out of date. There is a need for interventions for harm reduction targeted at MSM that account specifically for the social and cultural contexts of sexualized drug use. Expanded attention to surveillance of emerging drug use trends can help clinicians in sexual health and infectious diseases best anticipate the needs of their service users.",
"The Multicenter AIDS Cohort Study was designed to elucidate the natural history of the infection causing acquired immunodeficiency syndrome (AIDS), identify risk factors for occurrence and clinical expression of the infection, and establish a repository of biologic specimens for future study. A variety of recruitment techniques, including special assurance of confidentiality, were used to enroll participants. Nearly 5,000 homosexual men volunteered for semiannual interview, physical examination, and laboratory testing in four metropolitan areas. A significant majority of these men in each center (69-83%) reported having 50 or more lifetime sexual partners, and over 80% had engaged in receptive anal intercourse with at least some of their partners in the previous two years. By the time of the participants' initial evaluation (April 1984-April 1985), infection with the human immunodeficiency virus (HIV) had occurred in higher proportions of men in Los Angeles (51%) and Chicago (43%) than in Baltimore/Washington, DC (31%) and Pittsburgh (21%), presumably as a result of the higher number of partners and proportion with whom these men had engaged in high-risk practices (e.g., receptive anal intercourse). Follow-up evaluations are underway in this comprehensive longitudinal investigation of HIV infection.",
"Sexual risk behaviors of young gay and bisexual men must be understood within the context of other health concerns (e.g., anxiety, substance abuse), population specific factors (i.e., the coming-out process and gay-related stress), childhood sexual abuse, and other theoretical factors (e.g., safer-sex intentions). The current report proposes and longitudinally examines a model of risk factors for subsequent sexual risk behaviors among young gay and bisexual men in New York City. As hypothesized, more negative attitudes toward homosexuality, more substance abuse symptoms, and poorer intentions for safer sex were directly associated with a greater likelihood of unprotected anal sex over the following year. Furthermore, lower self-esteem, more anxious symptoms, and childhood sexual abuse were related to more unprotected anal sex indirectly through more sexual partners, sexual encounters, and substance abuse symptoms. These findings suggest that interventions targeting sexual risk behaviors of young gay and bisexual men may be more effective if they also address mental health concerns and aspects of the coming-out process.",
"This study examined patterns of drug use among gay men and other men who have sex with men (MSM) to identify sub-categories of men whose drug use and sexual behavior place them at especially high risk for HIV. A latent class analysis of a sample of MSM yielded a four-class model with two distinct high drug use sub-groups: one whose drug use concentrated on \"sex-drugs\" (SDU); and a distinct polydrug use class that showed higher probabilities of using all other drugs assessed. Comparative follow-up analyses indicated the SDU group was also more likely to engage in particular potentially high-risk sexual behaviors, be older, and to be HIV positive. Implications of distinguishing between patterns of drug use for HIV-risk prevention efforts with MSM are discussed.",
"Studied internal consistencies of the 1961 and 1978 versions of the Beck Depression Inventory in two different samples of psychiatric patients. The alpha coefficient for the 598 inpatients and outpatients who were administered the 1961 version was .88, and the alpha coefficient for the 248 outpatients who were self-administered the 1978 version was .86. The patterns of corrected item-total correlations were also similar, and it was concluded that the internal consistencies of both versions were comparable.",
"The persistence of disparities in STI/HIV risk among a new generation of emerging adult gay, bisexual, and other men who have sex with men (YMSM) warrant holistic frameworks and new methodologies for investigating the behaviors related to STI/HIV in this group. In order to better understand the continued existence of these disparities in STI/HIV risk among YMSM, the present study evaluated the presence and persistence of syndemic conditions among YMSM by examining the co-occurrence of alcohol and drug use, unprotected sexual behavior, and mental health burden over time. Four waves of data, collected over the first 18 months of a 7 wave, 36-month prospective cohort study of YMSM (n=600) were used to examine the extent to which measurement models of drug use, unprotected sexual behavior, and mental health burden remained consistent across time using latent class modeling. Health challenges persisted across time as these YMSM emerged into young adulthood and the measurement models for the latent constructs of drug use and unprotected sexual behavior were essentially consistent across time whereas models for mental health burden varied over time. In addition to confirming the the robustness of our measurement models which capture a more holistic understandings of the health conditions of drug use, unprotected sex, and mental health burden, these findings underscore the ongoing health challenges YMSM face as they mature into young adulthood. These ongoing health challenges, which have been understood as forming a syndemic, persist over time, and add further evidence to support ongoing and vigilant comprehensive health programming for sexual minority men that move beyond a sole focus on HIV.",
"The social identity of HIV/AIDS in the U.S. has been shaped, for the most part, by two factors, the prevailing configuration of social relations across class, racial, gender, and sexual orientation, on the one hand, and the prevailing array of public health, especially epidemiological, categories of disease transmission, on the other. Focusing on the AIDS epidemic among inner city people of color, this paper challenges the distortions wrought in our understanding from both of these factors and instead develops an alternative perspective for AIDS research among medical anthropologists and health social scientists generally."
] |
Proteases and synaptic plasticity
|
Long-term synaptic plasticity in the hippocampus is thought to underlie the formation of certain forms of memory, including spatial memory. The early phase of long-term synaptic potentiation and synaptic depression depends on post-translational modifications of synaptic proteins, while protein synthesis is also required for the late-phase of both forms of synaptic plasticity (L-LTP and L-LTD). Numerous pieces of evidence show a role for different types of proteases in synaptic plasticity, further increasing the diversity of mechanisms involved in the regulation of the intracellular and extracellular protein content. The cleavage of extracellular proteins is coupled to changes in postsynaptic intracellular mechanisms, and additional alterations in this compartment result from the protease-mediated targeting of intracellular proteins. Both mechanisms contribute to initiate signaling cascades that drive downstream pathways coupled to synaptic plasticity. In this review we summarize the evidence pointing to a role for extracellular and intracellular proteases, with distinct specificities, in synaptic plasticity. Where in the cells the proteases are located, and how they are regulated is also discussed. The combined actions of proteases and translation mechanisms contribute to a tight control of the synaptic proteome relevant for long-term synaptic potentiation and synaptic depression in the hippocampus. Additional studies are required to elucidate the mechanisms whereby these changes in the synaptic proteome are related with plasticity phenomena.
|
[
"Brain-derived neurotrophic factor (BDNF) is a neurotrophic factor involved in neuronal development and synaptic plasticity. Although the physiological effects of BDNF have been examined in detail, target proteins which mediate its actions remain largely unknown. Here, we report that BDNF stimulates the expression of tissue-type plasminogen activator (tPA) in primary cultures of cortical neurons in a time- and concentration-dependent manner. Among the other members of the neurotrophin family, neurotrophin-4 (NT-4) and to a lesser extent neurotrophin-3 (NT-3) also increased tPA mRNA expression, while nerve growth factor (NGF) was devoid of any effect. Induction of tPA expression by BDNF is accompanied by an increase in the proteolytic activity of tPA associated with cortical neurons and a release of tPA into the extracellular space. Release of tPA induced by BDNF depends on extracellular Ca2+ since it is markedly reduced in the presence of ethylene glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). Up-regulation of tPA expression by BDNF is followed by the induction of plasminogen activator inhibitor 2 (PAI-2), an inhibitor of tPA. Together these results suggest that activation of tPA by BDNF may contribute to structural changes associated with neuronal development or synaptic plasticity.",
"Neuropsin mRNA expression was analyzed and mapped in the mouse brains after kindling epileptogenesis by using in situ hybridization histochemistry. Dynamic increases of the neuropsin mRNA were observed in the layer II of prelimbic, somatosensory, auditory, perirhinal, entorhinal, and piriform cortices in an activity-dependent manner, though no neuropsin gene was expressed in these areas in control mice. In addition to the confirmation of our previous studies showing increases of mRNA in the hippocampus and amygdaloid complex, there were also remarkable increases of the neuropsin mRNA in the limbic areas, such as the accessory olfactory nucleus, the medial and lateral septal nucleus, the nucleus of diagonal band, the substantia innominata and the zona incerta. The dynamic activity-dependent changes of the gene expression and the site-specificity of neuropsin localization are suggesting that this molecule is implicated in cortical- and limbic-specific neuronal reorganization.",
"Adult cortical neurons can produce tissue-type plasminogen activator (tPA), an extracellular protease that plays a critical role in fibrinolysis and tissue remodelling processes. There is growing evidence that extracellular proteolysis may be involved in synaptic plasticity, axonal remodelling and neurotoxicity in the adult central nervous system. Here we show that transgenic mice overexpressing tPA in post-natal neurons have increased and prolonged hippocampal long-term potentiation (LTP), and improved performance in spatial orientation learning tasks. Extracellular proteolysis catalysed by tPA may facilitate synaptic micro-remodelling, and thereby play a role in activity-dependent neuronal plasticity and learning.",
"Motor learning is thought to involve modulation of synaptic inputs in the cerebellar cortex, including granule neuron/Purkinje neuron contacts. During a complex motor task requiring mice to walk across irregularly spaced pegs, cerebellar granule neurons show a rapid and transient induction of mRNA for the extracellular protease tissue plasminogen activator (tPA). This induction of tPA mRNA is cerebellar specific, is not seen in the cerebella of exercised or stressed animals, and is distinct from simple performance phenomena. Knock-out mice lacking the tPA gene show a significant reduction in both rate and extent of learning. Furthermore, blocking tPA activity during training dramatically impaired motor learning. Thus, tPA plays an important role in motor learning, in which tPA may facilitate remodeling of the active synaptic zone.",
"Proteolysis by the ubiquitin-proteasome pathway appears to have a complex role in synaptic plasticity, but its various functions remain to be elucidated. Using late phase long-term potentiation (L-LTP) in the hippocampus of the mouse as a model for long-term synaptic plasticity, we previously showed that inhibition of the proteasome enhances induction but blocks maintenance of L-LTP. In this study, we investigated the possible mechanisms by which proteasome inhibition has opposite effects on L-LTP induction and maintenance. Our results show that inhibiting phosphatidyl inositol-3 kinase or blocking the interaction between eukaryotic initiation factors 4E (eIF4E) and 4G (eIF4G) reduces the enhancement of L-LTP induction brought about by proteasome inhibition suggesting interplay between proteolysis and the signaling pathway mediated by mammalian target of rapamycin (mTOR). Also, proteasome inhibition leads to accumulation of translational activators in the mTOR pathway such as eIF4E and eukaryotic elongation factor 1A (eEF1A) early during L-LTP causing increased induction. Furthermore, inhibition of the proteasome causes a buildup of translational repressors, such as polyadenylate-binding protein interacting protein 2 (Paip2) and eukaryotic initiation factor 4E-binding protein 2 (4E-BP2), during late stages of L-LTP contributing to the blockade of L-LTP maintenance. Thus, the proteasome plays a critical role in regulating protein synthesis during L-LTP by tightly controlling translation. Our results provide novel mechanistic insights into the interplay between protein degradation and protein synthesis in long-term synaptic plasticity.",
"Expression of N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) in the CA1 region of the hippocampus can be divided into an early (1-2 h), protein synthesis-independent phase and a late (>4 h), protein synthesis-dependent phase. In this study we have addressed whether the de novo protein synthesis required for the expression of late-LTP can be sustained solely from the translation of mRNAs located in the dendrites of CA1 pyramidal neurones. Our results show that late-LTP, lasting at least 5 h, can be maintained in hippocampal slices where the dendrites located in stratum radiatum have been isolated from their cell bodies by a microsurgical cut. The magnitude of the potentiation of the slope of field EPSPs in these 'isolated' slices was similar to that recorded in 'intact' slices. Incubation of the slices with the mRNA translation inhibitor cycloheximide or the mammalian target of rapamycin (mTOR) inhibitor rapamycin blocked late-LTP in both 'intact' and 'isolated' slice preparations. In contrast, incubation of slices with the transcription inhibitor, actinomycin D, resulted in a reduction of sustained potentiation, at 4 h, in 'intact' slices while in 'isolated' slices the magnitude of potentiation was similar to that seen in untreated slices. These results indicate that late-LTP can be induced and maintained in 'isolated' dendritic preparations via translation of pre-existing mRNAs.",
"A hippocampal pyramidal neuron receives more than 10(4) excitatory glutamatergic synapses. Many of these synapses contain the molecular machinery for messenger RNA translation, suggesting that the protein complement (and thus function) of each synapse can be regulated on the basis of activity. Here, local postsynaptic protein synthesis, triggered by synaptic activation of metabotropic glutamate receptors, was found to modify synaptic transmission within minutes.",
"More than 20 matrix metalloproteinases (MMPs) and four of their endogenous tissue inhibitors (TIMPs) act together to control tightly temporally restricted, focal proteolysis of extracellular matrix. In the neurons of the adult brain several components of the TIMP/MMP system are expressed and are responsive to changes in neuronal activity. Furthermore, functional studies, especially involving blocking of MMP activities, along with the identification of MMP substrates in the brain strongly suggest that this enzymatic system plays an important physiological role in adult brain neurons, possibly being pivotal for neuronal plasticity.",
"Synaptic plasticity requires an activity-dependent, rapid, and long-lasting modification of synaptic character, including morphology and coupling strength. Here we show that a serine protease, neuropsin, directly and specifically modifies the synaptic adhesion molecule L1, which was localized to the presynaptic site of the asymmetric synapse in the mouse hippocampus. Increased neural activity triggered the rapid, transient activation of the precursor form of neuropsin in an NMDA receptor-dependent manner. The activated neuropsin immediately cleaved L1 and released a neuropsin-specific extracellular 180 kDa fragment. This neuropsin-specific L1-cleaving system is involved in NMDA receptor-dependent synaptic plasticity, such as the Schaffer collateral long-term potentiation.",
"Neurofascin belongs to the L1 subgroup of the immunoglobulin superfamily of cell adhesion molecules and is implicated in axonal growth and fasciculation. We used yeast two-hybrid screening to identify proteins that interact with neurofascin intracellularly and therefore might link it to trafficking, spatial targeting, or signaling pathways. Here, we demonstrate that rat syntenin-1, previously published as syntenin, mda-9, or TACIP18 in human, is a neurofascin-binding protein that exhibits a wide-spread tissue expression pattern with a relative maximum in brain. Syntenin-1 was found not to interact with other vertebrate members of the L1 subgroup such as L1 itself or NrCAM. We confirmed the specificity of the neurofascin-syntenin-1 interaction by ligand-overlay assay, surface plasmon resonance analysis, and colocalization of both proteins in heterologous cells. The COOH terminus of neurofascin was mapped to interact with the second PDZ domain of syntenin-1. Furthermore, we isolated syntenin-2 that may be expressed in two isoforms. Despite their high sequence similarity to syntenin-1, syntenin-2alpha, which interacts with neurexin I, and syntenin-2beta do not bind to neurofascin or several other transmembrane proteins that are binding partners of syntenin-1. Finally, we report that syntenin-1 and -2 both form homodimers and can interact with each other."
] |
what is a monoclonal antibody against influenza A
|
Broadly reactive human monoclonal antibodies against the HA stem of influenza A virus are being developed as therapeutic agents as well as to understand the epitopes that are essential for a universal influenza virus vaccine.
|
[
"Neutralizing antibodies against influenza viruses have traditionally been thought to provide protection exclusively through their variable region; the contributions of mechanisms conferred by the Fc domain remain controversial. We investigated the in vivo contributions of Fc interactions with their cognate receptors for a collection of neutralizing anti-influenza antibodies. Whereas five broadly neutralizing monoclonal antibodies (bNAbs) targeting the conserved stalk region of hemagglutinin (HA) required interactions between the antibody Fc and Fc receptors for IgG (FcγRs) to confer protection from lethal H1N1 challenge, three strain-specific monoclonal Abs (mAbs) against the variable head domain of HA were equally protective in the presence or absence of FcγR interactions. Although all antibodies blocked infection, only anti-stalk bNAbs were capable of mediating cytotoxicity of infected cells, which accounts for their FcγR dependence. Immune complexes generated with anti-HA stalk mAb efficiently interacted with FcγRs, but anti-HA head immune complexes did not. These results suggest that FcγR binding capacity by anti-HA antibodies was dependent on the interaction of the cognate Fab with antigen. We exploited these disparate mechanisms of mAb-mediated protection to reengineer an anti-stalk bNAb to selectively enhance FcγR engagement to augment its protective activity. These findings reveal a previously uncharacterized property of bNAbs and guide an approach toward enhancing mAb-mediated antiviral therapeutics.",
"Recent advances enabling the cloning of human immunoglobulin G genes have proven effective for discovering monoclonal antibodies with therapeutic potential. However, these antibody-discovery methods are often arduous and identify only a few candidates from numerous antibody-secreting plasma cells or plasmablasts. We describe an in vivo enrichment technique that identifies broadly neutralizing human antibodies with high frequency. For this technique, human peripheral blood mononuclear cells from vaccinated donors are activated and enriched in an antigen-specific manner for the production of numerous antigen-specific plasmablasts. Using this technology, we identified four broadly neutralizing influenza A antibodies by screening only 840 human antibodies. Two of these antibodies neutralize every influenza A human isolate tested and perform better than the current anti-influenza A therapeutic, oseltamivir, in treating severe influenza infection in mice and ferrets. Furthermore, these antibodies elicit robust in vivo synergism when combined with oseltamivir, thus highlighting treatment strategies that could benefit influenza-infected patients.",
"The 2009 pandemic H1N1 influenza pandemic demonstrated the global health threat of reassortant influenza strains. Herein, we report a detailed analysis of plasmablast and monoclonal antibody responses induced by pandemic H1N1 infection in humans. Unlike antibodies elicited by annual influenza vaccinations, most neutralizing antibodies induced by pandemic H1N1 infection were broadly cross-reactive against epitopes in the hemagglutinin (HA) stalk and head domain of multiple influenza strains. The antibodies were from cells that had undergone extensive affinity maturation. Based on these observations, we postulate that the plasmablasts producing these broadly neutralizing antibodies were predominantly derived from activated memory B cells specific for epitopes conserved in several influenza strains. Consequently, most neutralizing antibodies were broadly reactive against divergent H1N1 and H5N1 influenza strains. This suggests that a pan-influenza vaccine may be possible, given the right immunogen. Antibodies generated potently protected and rescued mice from lethal challenge with pandemic H1N1 or antigenically distinct influenza strains, making them excellent therapeutic candidates.",
"Influenza is a major concern for intensivists in all communities in the U.S. While there is considerable concern whether or not the country will be ready for a pandemic influenza, even seasonal influenza poses a major challenge to hospitals. The objective of this review is to summarize current knowledge of influenza with emphasis on the issues that intensivist will encounter. Intensive care unit in a 450-bed, tertiary care, teaching hospital. Source data were obtained from a PubMed search of the medical literature. PubMed \"related articles\" search strategies were likewise employed frequently. Seasonal influenza causes more than 200,000 hospitalizations and 41,000 deaths in the U.S. every year, and is the seventh leading cause of death in the U.S. Despite this impact there is a shortcoming in knowledge of influenza among many health care workers, and a paucity of clinical data and studies to guide therapy. Intensivists need to recognize the importance of seasonal influenza as a cause of severe morbidity and mortality. This review summarizes current knowledge of the diagnosis, complications, therapy, and infection control measures associated with influenza."
] |
Antimicrobial activity of Aspergillus aculeatus strain KKU-CT2
|
The bioactive compounds of the fungus Aspergillus aculeatus strain KKU-CT2, have been studied. The crude extracts from this fungus showed good antimicrobial activity against human pathogens, including Gram-positive and Gram-negative bacteria and yeast-like fungi. Its chemical components were isolated and purified by chromatographic methods. The structures of the secondary metabolites were elucidated by spectroscopic methods (IR,
|
[
"Plants of the genera Ferula and Ferulago are known for their complex content in bioactive secondary metabolites such as coumarins, phenylpropanoids, and sesquiterpenes. We used the ground parts of Ferula communis subsp. communis, Ferula glauca subsp. glauca and Ferulago campestris as natural sources for the isolation of four coumarins (CU-1 to CU-4), two phenylpropanoids (PE-1 and PE-2), one polyacetylene (PA-1) and 16 daucane esters (DE-1 to DE-16). The cytotoxic activity of the isolated compounds was evaluated against a panel of seven human tumor cell lines. Fourteen of the daucane derivatives showed antiproliferative activity at least against one of the human tumor cell lines tested, four compounds (DE-5, DE-8, DE-11, and DE-16) were active against all the tested cell lines. Among them DE-11 was the most cytotoxic compound against HeLa (4.4 ± 0.7 μM), A549 (2.8 ± 1.4 μM), HL-60 (2.6 ± 0.4 μM), K562 (26.5 ± 6.0 μM) RS 4;11 (1.7 ± 0.3 μM) and SEM (2.4 ± 0.1 μM) cell lines, while DE-8 was the most active against Jurkat (3.3 ± 0.8 μM). Preliminary structure-activity relationship suggests that the most active compounds in the daucane series present the trans fusion of the penta- and hepta-atomic cycles, and lipophylic ester groups linked to position 6. Isomeric derivatives such as DE-8 and DE-9 or DE-3, DE-4, and DE-5 exhibited significant differences in their IC(50) supporting that the β orientation for the ester group in the position 2 enhances the cytotoxic activity. Furthermore, the pro-apoptotic effect of the most active compounds evaluated in Jurkat cell line showed that these compounds are able to induce apoptosis in a time and concentration-dependent manner. Our findings suggest the potential role of daucane derivatives as models for the development of proapoptotic compounds.",
"Microorganisms and in particular actinomycetes and microfungi are known to produce a vast number of bioactive secondary metabolites. For industrially important fungal genera such as Penicillium and Aspergillus the production of these compounds has been demonstrated to be very consistent at the species level. This means that direct metabolite profiling techniques such as direct injection mass spectrometry or NMR can easily be used for chemotyping/metabolomics of strains from both culture collections and natural samples using modern informatics tools. In this review we discuss chemotyping/metabolomics as part of intelligent screening and highlight how it can be used for identification and classification of filamentous fungi and for the discovery of novel compounds when used in combination with modern methods for dereplication. In our opinion such approaches will be important for future effective drug discovery strategies, especially for dereplication of culture collections in order to avoid redundancy in the selection of species. This will maximize the chemical diversity of the microbial natural product libraries that can be generated from fungal collections.",
"Six sesquiterpenoids 1-6, including two new ones, an ent-daucane-type sesquiterpenoid, asperaculane A (1), and a nordaucane one, asperaculane B (2), and four known nordaucane derivatives, aculenes A-D 3-6, together with the known secalonic acid D (7), were isolated from a fermentation culture of the fungus Aspergillus aculeatus. Their structures and absolute configurations were established by analyses of their spectroscopic data, including 1D and 2D-NMR spectra, HR-ESIMS, electronic circular dichroism (ECD) data, and quantum chemical calculations. These metabolites were evaluated for in vitro cytotoxic activity against two cell lines, human cancer cell lines (HeLa) and one normal hamster cell line (CHO).",
"Semisynthetic analogues of the natural product 1-O-acetylbritannilactone (ABL), a sesquiterpene isolated from the medicinal plant Inula britannica, have been prepared and exhibited significant in vitro cytotoxic activities against four cell lines including three human cancer cell lines (HCT116, HEp-2 and HeLa) and one normal hamster cell line (CHO). Structure-activity relationships indicate that esterification of 6-OH (enhanced lipophilicity) and α-methylene-γ-lactone functionalities play important roles in conferring cytotoxicity. Among the tested compounds, 14 bearing a lauroyl group (12C) at the 6-OH position displayed most potent in vitro cytotoxic activity, with IC50 values between 2.91 and 6.78 μM, comparable to the positive control etoposide (VP-16, IC50 values between 2.13 and 4.79 μM). Moreover, the compound 14 triggered remarkable apoptosis at a low concentration, and induced cell cycle arrest in G2/M phase in HCT116 cells. The biological assays conducted with normal cells (CHO) revealed that all the synthetic compounds are no selective against cancer cell lines tested."
] |
Endoplasmic reticulum-Golgi trafficking enhances transport kinetics in breast cancer metastasis
|
In order to better understand the process of breast cancer metastasis, we have generated a mammary epithelial progression series of increasingly aggressive cell lines that metastasize to lung. Here we demonstrate that upregulation of an endoplasmic reticulum (ER) to Golgi trafficking gene signature in metastatic cells enhances transport kinetics, which promotes malignant progression. We observe increased ER-Golgi trafficking, an altered secretome and sensitivity to the retrograde transport inhibitor brefeldin A (BFA) in cells that metastasize to lung. CREB3 was identified as a transcriptional regulator of upregulated ER-Golgi trafficking genes ARF4, COPB1, and USO1, and silencing of these genes attenuated the metastatic phenotype in vitro and lung colonization in vivo. Furthermore, high trafficking gene expression significantly correlated with increased risk of distant metastasis and reduced relapse-free and overall survival in breast cancer patients, suggesting that modulation of ER-Golgi trafficking plays an important role in metastatic progression.
|
[
"Two hours of transient focal brain ischemia causes acute neuronal death in the striatal core region and a somewhat more delayed type of neuronal death in neocortex. The objective of the current study was to investigate protein aggregation and neuronal death after focal brain ischemia in rats. Brain ischemia was induced by 2 hours of middle cerebral artery occlusion. Protein aggregation was analyzed by electron microscopy, laser-scanning confocal microscopy, and Western blotting. Two hours of focal brain ischemia induced protein aggregation in ischemic neocortical neurons at 1 hour of reperfusion, and protein aggregation persisted until neuronal death at 24 hours of reperfusion. Protein aggregates were found in the neuronal soma, dendrites, and axons, and they were associated with intracellular membranous structures during the postischemic phase. High-resolution confocal microscopy showed that clumped protein aggregates surrounding nuclei and along dendrites were formed after brain ischemia. On Western blots, ubiquitinated proteins (ubi-proteins) were dramatically increased in neocortical tissues in the postischemic phase. The ubi-proteins were Triton-insoluble, indicating that they might be irreversibly aggregated. The formation of ubi-protein aggregates after ischemia correlated well with the observed decrease in free ubiquitin and neuronal death. The authors concluded that proteins are severely damaged and aggregated in neurons after focal ischemia. The authors propose that protein damage or aggregation may contribute to ischemic neuronal death.",
"Endoplasmic reticulum (ER) stress transducers IRE1, PERK and ATF6 are well known to transduce signals from the ER to the cytoplasm and nucleus when unfolded proteins are accumulated in the ER. Here, we identified OASIS as a novel ER stress transducer. OASIS is a basic leucine zipper (bZIP) transcription factor of the CREB/ATF family with a transmembrane domain that allows it to associate with the ER. The molecule is cleaved at the membrane in response to ER stress, and its cleaved amino-terminal cytoplasmic domain, which contains the bZIP domain, translocates into the nucleus where it activates the transcription of target genes that are mediated by ER stress-responsive and cyclic AMP-responsive elements. Intriguingly, OASIS was induced at the transcriptional level during ER stress in astrocytes of the central nervous system, but not in other cell types examined. Furthermore, overexpression of OASIS resulted in induction of BiP and suppression of ER-stress-induced cell death, whereas knockdown partially reduced BiP levels and led to ER stress in susceptible astrocytes. Our results reveal pivotal roles for OASIS in modulating the unfolded protein response in astrocytes, and the possibility that cell type-specific UPR signalling also exists in other cells.",
"Luman (or LZIP, CREB3) is a transcription factor with an endoplasmic reticulum (ER)-transmembrane domain. Due to its structural similarities with ATF6, it is thought that Luman might also be involved in cellular stress responses. Here we report that Luman can bind and activate transcription from the consensus unfolded protein response element (UPRE). Mutations that disrupted the binding of Luman to the UPREs impaired its ability to activate transcription from these sites. Overexpression of Luman stimulated transcription of EDEM, a downstream effector of the mammalian unfolded protein response involved in ER-associated degradation (ERAD). Unlike ATF6, however, Luman was not activated by proteolytic cleavage in response to endoplasmic reticulum stressors such as tunicamycin and thapsigargin. These results suggest that the activation of ERAD by Luman is likely through a pathway different from the common ER stress response, and that additional factor(s) are required for the activation of this Luman-mediated pathway.",
"We previously reported that the spermatid-specific transcription factor Tisp40 functions through UPRE and CRE. To investigate Tisp40 function in vivo, we generated TISP40(-/-) mice. TISP40(-/-) mice were born at expected ratios, were healthy, and mutant males bred normally. However, the ER stress-response protein Grp78/BiP accumulated in the TISP40(-/-) testis and RAMP4 (Ribosome-associated membrane protein 4) mRNA level was up-regulated. Disruption of TISP40 caused ER stress and activation of caspase 12 but not caspase 9, leading to apoptosis of meiotic/postmeiotic germ cells. On the other hand, DAPI staining and electron microscopy revealed that epididymal sperm nuclei were abnormally relaxed in the TISP40(-/-) testis, a phenotype that was independent of the expression and maturation of transition proteins and protamines but due to abnormally retained histones. Histones localized to the cytoplasm as well as to the nucleus and were also retained in epididymal sperm. Histones H2A and H4 were dramatically up-regulated and the acetylation of H2A, H2B and H4 was also enhanced in the TISP40(-/-) testis. Taken together, we conclude that Tisp40 plays an important role in the unfolded protein response of the testis and in regulating the maturation of sperm head nuclei.",
"The Golgi stress response is a homeostatic mechanism that controls the capacity of the Golgi apparatus in accordance with cellular demands. When the capacity of the Golgi apparatus becomes insufficient (Golgi stress), transcription levels of Golgi-related genes encoding glycosylation enzymes, a Golgi structural protein, and components of vesicular transport are upregulated through a common cis-acting enhancer-the Golgi apparatus stress response element (GASE). Here, we identified the transcription factor MLX as a GASE-binding protein. MLX resides in the cytoplasm and does not bind to GASE in normal growth conditions, whereas MLX translocates into the nucleus and specifically binds to GASE in response to Golgi stress. Suppression of MLX expression increased transcriptional induction of target genes of the Golgi stress response, whereas overexpression of MLX reduced GASE-binding of TFE3 as well as transcriptional induction from GASE, suggesting that MLX is a transcriptional repressor of the mammalian Golgi stress response.",
"The endoplasmic reticulum (ER) responds to the accumulation of unfolded proteins in its lumen (ER stress) by activating intracellular signal transduction pathways - cumulatively called the unfolded protein response (UPR). Together, at least three mechanistically distinct arms of the UPR regulate the expression of numerous genes that function within the secretory pathway but also affect broad aspects of cell fate and the metabolism of proteins, amino acids and lipids. The arms of the UPR are integrated to provide a response that remodels the secretory apparatus and aligns cellular physiology to the demands imposed by ER stress.",
"The expression of liver-specific genes is regulated by unequivocally allocated transcription factors via proper responsible elements within their promoters. We identified a novel transcription factor, CREB-H, and found that its expression was restricted in the liver among 16 human tissues tested. A region of CREB-H exhibited significant homology to the basic leucine zipper (b-Zip) domain of members of the CREB/ATF family: mammalian LZIP and Drosophila BBF-2 that binds to box-B, a Drosophila enhancer modulating the fat-body-specific gene expression. CREB-H contained a hydrophobic region representing a putative transmembrane domain, like LZIP. Constructing a variety of CREB-H fusion proteins with the GAL4 DNA-binding domain disclosed that CREB-H functioned as a transcriptional activator and its N-terminal 149 amino acids accounted for the activation ability. Gel mobility sift assays revealed that CREB-H did not bind to the C/EBP, AP-1 and NF-kappaB elements but specifically bound to CRE and the box-B element. Luciferase reporter assays demonstrated that like BBF-2, CREB-H activated transcription via the box-B element and that a deletion of the putative transmembrane domain increased the activation of reporter expression significantly. Furthermore, a fusion protein of GFP and full-length CREB-H was localized in reticular structures surrounding the nucleus, whereas a fusion protein of GFP and a deletion mutant lacking the putative transmembrane domain was mainly in the nucleus. These findings suggest that CREB-H plays an important role in transcriptional regulation of genes specifically expressed in the liver, and that the putative transmembrane domain may be associated with modulation of its function as the transcriptional activator.",
"The protein kinase PERK couples protein folding in the endoplasmic reticulum (ER) to polypeptide biosynthesis by phosphorylating the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha), attenuating translation initiation in response to ER stress. PERK is highly expressed in mouse pancreas, an organ active in protein secretion. Under physiological conditions, PERK was partially activated, accounting for much of the phosphorylated eIF2alpha in the pancreas. The exocrine and endocrine pancreas developed normally in Perk-/- mice. Postnatally, ER distention and activation of the ER stress transducer IRE1alpha accompanied increased cell death and led to progressive diabetes mellitus and exocrine pancreatic insufficiency. These findings suggest a special role for translational control in protecting secretory cells from ER stress.",
"Endoplasmic reticulum (ER) stress transducers IRE1 (inositol requiring 1), PERK (PKR-like endoplasmic reticulum kinase), and ATF6 (activating transcription factor 6) are well known to transduce signals from the ER to the cytoplasm and nucleus when unfolded proteins accumulate in the ER. Recently, we identified OASIS (old astrocyte specifically induced substance) as a novel ER stress transducer expressed in astrocytes. We report here that BBF2H7 (BBF2 human homolog on chromosome 7), an ER-resident transmembrane protein with the bZIP domain in the cytoplasmic portion and structurally homologous to OASIS, is cleaved at the membrane in response to ER stress. The cleaved fragments of BBF2H7 translocate into the nucleus and can bind directly to cyclic AMP-responsive element sites to activate transcription of target genes. Interestingly, although BBF2H7 protein is not expressed under normal conditions, it is markedly induced at the translational level during ER stress, suggesting that BBF2H7 might contribute to only the late phase of unfolded protein response signaling. In a mouse model of focal brain ischemia, BBF2H7 protein is prominently induced in neurons in the peri-infarction region. Furthermore, in a neuroblastoma cell line, BBF2H7 overexpression suppresses ER stress-induced cell death, while small interfering RNA knockdown of BBF2H7 promotes ER stress-induced cell death. Taken together, our results suggest that BBF2H7 is a novel ER stress transducer and could play important roles in preventing accumulation of unfolded proteins in damaged neurons.",
"Stress-induced eukaryotic translation initiation factor 2 (eIF2) alpha phosphorylation paradoxically increases translation of the metazoan activating transcription factor 4 (ATF4), activating the integrated stress response (ISR), a pro-survival gene expression program. Previous studies implicated the 5' end of the ATF4 mRNA, with its two conserved upstream ORFs (uORFs), in this translational regulation. Here, we report on mutation analysis of the ATF4 mRNA which revealed that scanning ribosomes initiate translation efficiently at both uORFs and ribosomes that had translated uORF1 efficiently reinitiate translation at downstream AUGs. In unstressed cells, low levels of eIF2alpha phosphorylation favor early capacitation of such reinitiating ribosomes directing them to the inhibitory uORF2, which precludes subsequent translation of ATF4 and represses the ISR. In stressed cells high levels of eIF2alpha phosphorylation delays ribosome capacitation and favors reinitiation at ATF4 over the inhibitory uORF2. These features are common to regulated translation of GCN4 in yeast. The metazoan ISR thus resembles the yeast general control response both in its target genes and its mechanistic details."
] |
Endoplasmic reticulum calcium entry in mushroom spines
|
Mushroom spines form strong synaptic contacts and are essential for memory storage. We have previously demonstrated that neuronal store-operated calcium entry (nSOC) in hippocampal neurons is regulated by STIM2 protein. This pathway plays a key role in stability of mushroom spines and is compromised in different mice models of Alzheimer's disease (AD). Actin was thought to be the sole cytoskeleton compartment presented in dendritic spines, however, recent studies demonstrated that dynamic microtubules with EB3 capped plus-ends transiently enter spines. We showed that STIM2 forms an endoplasmic reticulum (ER) Ca
|
[
"Soluble oligomers of amyloid beta (Abeta) play a role in the memory impairment characteristic of Alzheimer's disease. Acting as pathogenic ligands, Abeta oligomers bind to particular synapses and perturb their function, morphology, and maintenance. Events that occur shortly after oligomer binding have been investigated here in live hippocampal neurons by single particle tracking of quantum dot-labeled oligomers and synaptic proteins. Membrane-attached oligomers initially move freely, but their diffusion is hindered markedly upon accumulation at synapses. Concomitantly, individual metabotropic glutamate receptors (mGluR5) manifest strikingly reduced lateral diffusion as they become aberrantly clustered. This clustering of mGluR5 elevates intracellular calcium and causes synapse deterioration, responses prevented by an mGluR5 antagonist. As expected, clustering by artificial crosslinking also promotes synaptotoxicity. These results reveal a mechanism whereby Abeta oligomers induce the abnormal accumulation and overstabilization of a glutamate receptor, thus providing a mechanistic and molecular basis for Abeta oligomer-induced early synaptic failure.",
"Recent evidence suggests that high molecular weight soluble oligomeric Abeta (oAbeta) assemblies (also known as Abeta-derived diffusible ligands, or ADDLs) may represent a primary neurotoxic basis for cognitive failure in Alzheimer disease (AD). To date, most in vivo studies of oAbeta/ADDLs have involved injection of assemblies purified from the cerebrospinal fluid of human subjects with AD or from the conditioned media of Abeta-secreting cells into experimental animals. We sought to study the bioactivities of endogenously formed oAbeta/ADDLs generated in situ from the physiological processing of human amyloid precursor protein (APP) and presenitin1 (PS1) transgenes. We produced and histologically characterized single transgenic mice overexpressing APP(E693Q) or APP(E693Q) X PS1DeltaE9 bigenic mice. APP(E693Q) mice were studied in the Morris water maze (MWM) task at 6 and 12 months of age. Following the second MWM evaluation, mice were sacrificed, and brains were assayed for Abetatotal, Abeta40, Abeta42, and oAbeta/ADDLs by enzyme-linked immunosorbent assay (ELISA) and were also histologically examined. Based on results from the oAbeta/ADDL ELISA, we assigned individual APP(E693Q) mice to either an undetectable oAbeta/ADDLs group or a readily detectable oAbeta/ADDLs group. A days to criterion (DTC) analysis was used to determine delays in acquisition of the MWM task. Both single transgenic and bigenic mice developed intraneuronal accumulation of APP/Abeta, although only APP(E693Q) X PS1Delta9 bigenic mice developed amyloid plaques. The APP(E693Q) mice did not develop amyloid plaques at any age studied, up to 30 months. APP(E693Q) mice were tested for spatial learning and memory, and only 12-month-old APP(E693Q) mice with readily detectable oAbeta/ADDLs displayed a significant delay in acquisition of the MWM task when compared to nontransgenic littermates. These data suggest that cerebral oAbeta/ADDL assemblies generated in brain in situ from human APP transgenes may be associated with cognitive impairment. We propose that a DTC analysis may be a sensitive method for assessing the cognitive impact in mice of endogenously generated oligomeric human Abeta assemblies. ANN NEUROL 2010.",
"The amyloid-beta(1-42) (Abeta42) peptide rapidly aggregates to form oligomers, protofibils and fibrils en route to the deposition of amyloid plaques associated with Alzheimer's disease. We show that low-temperature and low-salt conditions can stabilize disc-shaped oligomers (pentamers) that are substantially more toxic to mouse cortical neurons than protofibrils and fibrils. We find that these neurotoxic oligomers do not have the beta-sheet structure characteristic of fibrils. Rather, the oligomers are composed of loosely aggregated strands whose C termini are protected from solvent exchange and which have a turn conformation, placing Phe19 in contact with Leu34. On the basis of NMR spectroscopy, we show that the structural conversion of Abeta42 oligomers to fibrils involves the association of these loosely aggregated strands into beta-sheets whose individual beta-strands polymerize in a parallel, in-register orientation and are staggered at an intermonomer contact between Gln15 and Gly37.",
"Alzheimer disease (AD) is a disease of lost memories. Mushroom postsynaptic spines play a key role in memory storage, and loss of mushroom spines has been proposed to be linked to memory loss in AD. Generation of amyloidogenic peptides and accumulation of amyloid plaques is one of the pathological hallmarks of AD. It is important to evaluate effects of amyloid on stability of mushroom spines. In this study we used in vitro and in vivo models of amyloid synaptotoxicity to investigate effects of amyloid peptides on hippocampal mushroom spines. We discovered that application of Aβ42 oligomers to hippocampal cultures or injection of Aβ42 oligomers directly into hippocampal region resulted in reduction of mushroom spines and activity of synaptic calcium-calmodulin-dependent kinase II (CaMKII). We further discovered that expression of STIM2 protein rescued CaMKII activity and protected mushroom spines from amyloid toxicity in vitro and in vivo. Obtained results suggest that downregulation of STIM2-dependent stability of mushroom spines and reduction in activity of synaptic CaMKII is a mechanism of hippocampal synaptic loss in AD model of amyloid synaptotoxicity and that modulators/activators of this pathway may have a potential therapeutic value for treatment of AD.",
"Since their first description by Ramon y Cajal at the end of the 19th century, dendritic spines have been proposed as important sites of neuronal contacts and it has been suggested that changes in the activity of neurons directly affect spine morphology. In fact, since then it has been shown that about 90% of excitatory synapses end on spines. Recent data indicate that spines are highly dynamic structures and that spine shape correlates with the strength of synaptic transmission. Furthermore, several mental disorders including Alzheimer's disease (AD) are associated with spine pathology suggesting that spine alterations play a central role in mental deficits. The aim of this review is to provide an overview about the current knowledge on spine morphology and function as well as about different experimental models to analyze spine changes and dynamics. The second part concentrates on disease-relevant factors that are associated with AD and which lead to spine alterations. In particular, data that provide evidence that Abeta oligomers or fibrillar Abeta deposits influence spine morphology and function will be presented and the contribution of tau pathology will be discussed. The review ends with the discussion of potential mechanisms how disease-relevant factors influence dendritic spines and whether and how spine changes could be therapeutically suppressed or reversed.",
"Neurons express multiple types of voltage-gated calcium (Ca2+) channels. Two subtypes of neuronal L-type Ca2+ channels are encoded by CaV1.2 and CaV1.3 pore-forming subunits. Both CaV1.2 and CaV1.3 subunits contain class I PDZ (postsynaptic density-95/Discs large/zona occludens-1) domain-binding consensus at their C termini. In yeast two-hybrid screen of rat brain cDNA library with the C-terminal bait of CaV1.3a (long C-terminal splice variant) L-type Ca2+ channel subunit, we isolated multiple clones of postsynaptic adaptor protein Shank. We demonstrated a specific association of CaV1.3a C termini, but not of CaV1.2 C termini, with Shank PDZ domain in vitro. We further demonstrated that the proline-rich region present in C termini of CaV1.3a subunit binds to Shank Src homology 3 domain. We established that CaV1.3a and Shank localized to postsynaptic locations in cultured rat hippocampal neurons. By expressing epitope-tagged recombinant CaV1.3 subunits in rat hippocampal neuronal cultures, we demonstrated that the presence of Shank-binding motifs in CaV1.3a sequence is both necessary and sufficient for synaptic clustering of CaV1.3 L-type Ca2+ channels. In experiments with dominant-negative peptides and dihydropyridine-resistant CaV1.3a mutants, we demonstrated an importance of Shank-binding motif in CaV1.3a sequence for phosphorylated cAMP response element-binding protein (pCREB) signaling in cultured hippocampal neurons. Our results directly link CaV1.3 neuronal L-type Ca2+ channels to macromolecular signaling complex formed by Shank and other modular adaptor proteins at postsynaptic density and provide novel information about the role played by CaV1.3 L-type Ca2+ channels in pCREB signaling.",
"Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder. Familial AD (FAD) mutations in presenilins have been linked to calcium (Ca(2+)) signaling abnormalities. To explain these results, we previously proposed that presenilins function as endoplasmic reticulum (ER) passive Ca(2+) leak channels. To directly investigate the role of presenilins in neuronal ER Ca(2+) homeostasis, we here performed a series of Ca(2+) imaging experiments with primary neuronal cultures from conditional presenilin double-knock-out mice (PS1(dTAG/dTAG), PS2(-/-)) and from triple-transgenic AD mice (KI-PS1(M146V), Thy1-APP(KM670/671NL), Thy1-tau(P301L)). Obtained results provided additional support to the hypothesis that presenilins function as ER Ca(2+) leak channels in neurons. Interestingly, we discovered that presenilins play a major role in ER Ca(2+) leak function in hippocampal but not in striatal neurons. We further discovered that, in hippocampal neurons, loss of presenilin-mediated ER Ca(2+) leak function was compensated by an increase in expression and function of ryanodine receptors (RyanRs). Long-term feeding of the RyanR inhibitor dantrolene to amyloid precursor protein-presenilin-1 mice (Thy1-APP(KM670/671NL), Thy1-PS1(L166P)) resulted in an increased amyloid load, loss of synaptic markers, and neuronal atrophy in hippocampal and cortical regions. These results indicate that disruption of ER Ca(2+) leak function of presenilins may play an important role in AD pathogenesis.",
"Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by insidious cognitive decline and memory dysfunction. Synapse loss is the best pathological correlate of cognitive decline in AD and mounting evidence suggests that AD is primarily a disease of synaptic dysfunction. Soluble oligomeric forms of amyloid beta (Aβ), the peptide that aggregates to form senile plaques in the brain of AD patients, have been shown to be toxic to neuronal synapses both in vitro and in vivo. Aβ oligomers inhibit long-term potentiation (LTP) and facilitate long-term depression (LTD), electrophysiological correlates of memory formation. Furthermore, oligomeric Aβ has also been shown to induce synapse loss and cognitive impairment in animals. The molecular underpinnings of these observations are now being elucidated, and may provide clear therapeutic targets for effectively treating the disease. Here, we review recent findings concerning AD pathogenesis with a particular focus on how Aβ impacts synapses.",
"A fundamental challenge in understanding how dendritic spine morphology controls learning and memory has been quantifying three-dimensional (3D) spine shapes with sufficient precision to distinguish morphologic types, and sufficient throughput for robust statistical analysis. The necessity to analyze large volumetric data sets accurately, efficiently, and in true 3D has been a major bottleneck in deriving reliable relationships between altered neuronal function and changes in spine morphology. We introduce a novel system for automated detection, shape analysis and classification of dendritic spines from laser scanning microscopy (LSM) images that directly addresses these limitations. The system is more accurate, and at least an order of magnitude faster, than existing technologies. By operating fully in 3D the algorithm resolves spines that are undetectable with standard two-dimensional (2D) tools. Adaptive local thresholding, voxel clustering and Rayburst Sampling generate a profile of diameter estimates used to classify spines into morphologic types, while minimizing optical smear and quantization artifacts. The technique opens new horizons on the objective evaluation of spine changes with synaptic plasticity, normal development and aging, and with neurodegenerative disorders that impair cognitive function.",
"A fundamental feature of Alzheimer disease (AD) is the accumulation of beta-amyloid (Abeta), a peptide generated from the amyloid precursor protein (APP). Emerging evidence suggests that soluble Abeta oligomers adversely affect synaptic function, which leads to cognitive failure associated with AD. The Abeta-induced synaptic dysfunction has been attributed to the synaptic removal of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors (AMPARs); however, it is unclear how Abeta induces the loss of AMPARs at the synapses. In this study we have examined the potential involvement of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), a signaling molecule critical for AMPAR trafficking and function. We found that the synaptic pool of CaMKII was significantly decreased in cortical neurons from APP transgenic mice, and the density of CaMKII clusters at synapses was significantly reduced by Abeta oligomer treatment. In parallel, the surface expression of GluR1 subunit as well as AMPAR-mediated synaptic response and ionic current was selectively decreased in APP transgenic mice and Abeta-treated cultures. Moreover, the reducing effect of Abeta on AMPAR current density was mimicked and occluded by knockdown of CaMKII and blocked by overexpression of CaMKII. These results suggest that the Abeta-induced change in CaMKII subcellular distribution may underlie the removal of AMPARs from synaptic membrane by Abeta."
] |
Contrast-induced nephropathy and the RenalGuard system
|
Contrast-induced nephropathy (CIN) is a well-recognized complication of coronary angiography that is associated with poor outcomes. Several small randomized controlled trials (RCTs) have recently shown that in patients with chronic kidney disease (CKD), furosemide-induced forced diuresis with matched hydration using the RenalGuard system can prevent its occurrence. However, individual studies have been underpowered and thus cannot show significant differences in major clinical endpoints.
|
[
"Flaws in the design, conduct, analysis, and reporting of randomised trials can cause the effect of an intervention to be underestimated or overestimated. The Cochrane Collaboration’s tool for assessing risk of bias aims to make the process clearer and more accurate",
"Guidelines are inconsistent in how they rate the quality of evidence and the strength of recommendations. This article explores the advantages of the GRADE system, which is increasingly being adopted by organisations worldwide",
"We sought to develop a simple risk score of contrast-induced nephropathy (CIN) after percutaneous coronary intervention (PCI). Although several risk factors for CIN have been identified, the cumulative risk rendered by their combination is unknown. A total of 8,357 patients were randomly assigned to a development and a validation dataset. The baseline clinical and procedural characteristics of the 5,571 patients in the development dataset were considered as candidate univariate predictors of CIN (increase >or=25% and/or >or=0.5 mg/dl in serum creatinine at 48 h after PCI vs. baseline). Multivariate logistic regression was then used to identify independent predictors of CIN with a p value <0.0001. Based on the odds ratio, eight identified variables (hypotension, intra-aortic balloon pump, congestive heart failure, chronic kidney disease, diabetes, age >75 years, anemia, and volume of contrast) were assigned a weighted integer; the sum of the integers was a total risk score for each patient. The overall occurrence of CIN in the development set was 13.1% (range 7.5% to 57.3% for a low [<or=5] and high [>or=16] risk score, respectively); the rate of CIN increased exponentially with increasing risk score (Cochran Armitage chi-square, p < 0.0001). In the 2,786 patients of the validation dataset, the model demonstrated good discriminative power (c statistic = 0.67); the increasing risk score was again strongly associated with CIN (range 8.4% to 55.9% for a low and high risk score, respectively). The risk of CIN after PCI can be simply assessed using readily available information. This risk score can be used for both clinical and investigational purposes.",
"Despite the efficacy of combination antiretroviral therapy (ART) and the improvement in prognosis of those living with HIV/AIDS, a large proportion of individuals on ART does not achieve or maintain adequate virological suppression. Several tools have been proposed to enhance ART outcomes, including therapeutic drug monitoring (TDM) of antiretrovirals (ARVs). The aim of ARV TDM is to identify elevated (potentially toxic) or low (potentially sub-therapeutic) ARV concentrations. ARV TDM may thus optimise efficacy and minimise toxicity of ART. To evaluate whether ARV TDM reduces mortality and morbidity of adult patients on ART. The primary outcome measures that have been assessed include death (all cause); occurrence of HIV-related events (death or AIDS-defining illness) and the proportion of patients achieving and maintaining an undetectable viral load, as defined by the authors. We conducted a comprehensive search including both published and unpublished studies in all languages in MEDLINE, EMBASE and The Cochrane Library, between January 1980 and January 2008. Databases listing conference abstracts and reference lists of articles were searched. Additional data were sought from relevant authors; however, no additional data were provided. Only randomized controlled trials conducted subsequent to the introduction of combination ART were included in this systematic review. Participants could be on either a protease inhibitor (PI)-based regimen or non-nucleoside reverse transcriptase (NNRTI)-based regimen and be either ARV-naive or -experienced. Two reviewers independently assessed and extracted data for analysis. Meta-analysis was conducted where appropriate. Where study outcomes could not be combined, a narrative review was performed. Outcome measures for dichotomous data were reported as a relative risk with 95% confidence intervals. Stratified analyses were conducted by ARV regimen and treatment groups. Heterogeneity between studies was anticipated; therefore, random effects models were chosen to generate pooled effects. Differences in the findings were assessed by the chi square test for heterogeneity (p <0.1) that was quantified by the Higgins I(2) statistic. Identified were 1408 records, and eight trials with a total of 1181 participants were included in the review. Trials were conducted in higher income earning countries between 2002 and 2007. Sample sizes ranged between 40 and 230. The methodological quality of the studies was judged to be generally good, although allocation concealment was reported in only three of the eight studies. A meta-analysis including three studies did not show any significant effect on virological suppression below 500 HIV-RNA copies/mL at one year (RR 1.28; [0.86, 1.92] chi(2) = 11.55 (P = 0.003), I(2) = 83%). Two trials including participants predominantly treated with unboosted PI-based regimens reported a 49% increased likelihood of achieving a HIV-RNA viral load below 500 copies/mL at 52 weeks (RR 1.49 [1.20, 1.83] chi(2) = 0.69 (P = 0.4), I(2) = 0%). Safety outcomes were reported in four studies and were similar between TDM and standard of care. Uptake of expert advice based on TDM results was good in two trials (>70%), but low (<35%) in the remaining three studies that reported uptake of the recommendations. Our review does not support routine use of ARV TDM in ARV-naive or -experienced patients on either boosted PI or NNRTI ART regimens. TDM in treatment-naive participants on a PI-based ART regimen, particularly if unboosted by ritonavir, may improve virological outcomes. Trials were underpowered with small sample sizes, short durations of follow-up and generally poor uptake of TDM recommendations. As these trials were conducted in higher income earning countries, results may not be generalisable to resource-limited countries where the burden of HIV is heaviest.",
"Results from better quality studies should in some sense be more valid or more accurate than results from other studies, and as a consequence should tend to be distributed differently from results of other studies. To date, however, quality scores have been poor predictors of study results. We discuss possible reasons and remedies for this problem. It appears that 'quality' (whatever leads to more valid results) is of fairly high dimension and possibly non-additive and nonlinear, and that quality dimensions are highly application-specific and hard to measure from published information. Unfortunately, quality scores are often used to contrast, model, or modify meta-analysis results without regard to the aforementioned problems, as when used to directly modify weights or contributions of individual studies in an ad hoc manner. Even if quality would be captured in one dimension, use of quality scores in summarization weights would produce biased estimates of effect. Only if this bias were more than offset by variance reduction would such use be justified. From this perspective, quality weighting should be evaluated against formal bias-variance trade-off methods such as hierarchical (random-coefficient) meta-regression. Because it is unlikely that a low-dimensional appraisal will ever be adequate (especially over different applications), we argue that response-surface estimation based on quality items is preferable to quality weighting. Quality scores may be useful in the second stage of a hierarchical response-surface model, but only if the scores are reconstructed to maximize their correlation with bias.",
"The purpose of this study was to investigate the effect of the RenalGuard System (PLC Medical Systems, Milford, Massachusetts) on prevention of acute kidney injury (AKI) in patients undergoing transcatheter aortic valve replacement (TAVR). TAVR is associated with varying degrees of post-procedural AKI. The RenalGuard System is a dedicated device designed for contrast-induced AKI prevention. Whether this device is also effective in patients with severe aortic stenosis undergoing TAVR is unexplored. The present is an investigator-driven, single-center, prospective, open-label, registry-based randomized study that used the TAVR institutional registry of the Ferrarotto Hospital in Catania, Italy, as the platform for randomization, data collection, and follow-up assessment. A total of 112 consecutive patients undergoing TAVR were randomly assigned to hydration with normal saline solution controlled by the RenalGuard system and furosemide (RenalGuard group) or normal saline solution (control group). The primary endpoint was the incidence of Valve Academic Research Consortium-defined AKI in the first 72 h after the procedure. The AKI rate was lower in the RenalGuard group than in the control group (n = 3 [5.4%] vs. n =14 [25.0%], respectively, p = 0.014). The majority of patients (5.4% vs. 23.2%) developed a mild AKI (stage 1); severe damage (stage 3) occurred only in 1 patient in the control group (0.0% vs. 1.8%). No case of in-hospital renal failure requiring dialysis was reported. No significant differences in terms of mortality, cerebrovascular events, bleeding, and hospitalization for heart failure were noted in both groups at 30 days. Furosemide-induced diuresis with matched isotonic intravenous hydration using the RenalGuard system is an effective therapeutic tool to reduce the occurrence of AKI in patients undergoing TAVR."
] |
Oral melatonin as an alternative sedation for MRI in preschool children with juvenile idiopathic arthritis.
|
Magnetic resonance imaging (MRI) in preschool children is often challenging due to excessive motion artifacts. Sedation or general anesthesia (GA) is commonly used to prevent children from moving in the MRI scanner, with increased risk for cardiopulmonary complications and requirement for skilled personnel. Herein, we investigated whether oral melatonin, a natural hormone implicated in circadian rhythm regulation, could be used as an alternative sedation method prior to the MRI in preschool children with musculoskeletal problems. Fifteen children with suspected juvenile idiopathic arthritis underwent a total of 16 MRI examinations following administration of 10 mg of oral melatonin; satisfactory images were obtained in all but one case, with no adverse events.
|
[
"Sedation can be used to relieve anxiety and manage behaviour in children, unfortunately it is difficult to determine from published research which agents, dosages and techniques are effective. To evaluate the relative efficacy of the various conscious sedation techniques and dosages for behaviour management in paediatric dentistry. DESIGN AND KEY METHODS: Relevant databases and reference lists from articles were searched up to December 2005. Studies were selected if they met the following criteria: randomised controlled trials of conscious sedation comparing two or more drugs/techniques/placebo undertaken by the dentist or one of the dental team in anxious children up to 16 years of age. Sixty-five studies were included with 3372 subjects in total. Overall quality of studies was found to be disappointing with poor reporting often the main problem. Authors were not able to reach any definitive conclusion on which was the most effective drug or method of sedation used for anxious children due to issues with the quality and validity of published studies to date.",
"To achieve diagnostic images during MRI examinations, small children need to lie still to avoid movement artefact. To reduce patient motion, obviate the need for voluntary immobilisation or breath-holding and therefore obtain high-quality images, MRI of infants is frequently carried out under sedation or general anaesthesia, but this is not without risk and expense. However, many other techniques are available for preparing children for MRI, which have not been fully evaluated. Here, we evaluate the advantages and disadvantage of sedation and anaesthesia for MRI. We then evaluate the alternatives, which include neonatal comforting techniques, sleep manipulation, and appropriate adaptation of the physical environment. We summarize the evidence for their use according to an established hierarchy. Lastly, we discuss several factors that will influence the choice of imaging preparation, including patient factors, imaging factors and service provision. The choice of approach to paediatric MRI is multi-factorial, with limited scientific evidence for many of the current approaches. These considerations may enable others to image children using MRI under different circumstances.",
"Complicated upper and lower endoscopic procedures of the gastrointestinal tract are performed in children for a variety of diagnostic and therapeutic reasons. Unlike adult patients, who receive conscious sedation, children usually require deep sedation (DS) or general anesthesia (GA). The aim of this study is to assess the safety parameters of complicated endoscopic procedures under DS or GA performed in children in the endoscopy suite rather than in the operating theatre. Between May 1997 and December 2002, 296 patients (mean age 4.5 years, range 3 weeks to 16 years), defined as ASA I-III, underwent either DS or GA for endoscopic foreign body extraction, endoscopic retrograde cholangiopancreatography (ERCP) or percutaneous endoscopic gastrostomy (PEG) insertion. ASA physical status I was found in 15%, II in 57% and III in 28%. The pathologies included neuromuscular diseases, genetic syndromes, nesidioblastosis, biliary atresia, hematologic, respiratory (cystic fibrosis) and cardiac disorders. Propofol was the drug of choice (63%) followed by a combination of propofol and midazolam (16%). Transient desaturation (O2 saturation <90%) was the only complication recorded in 21/296 (7.09%) patients. Only two patients with severe respiratory underlying disease were hospitalized for follow-up for a 24-h period. The use of DS and GA for complicated endoscopies in a moderately high-risk pediatric population was found to be safe. The very low complication rate found in this study suggests that complicated pediatric patients can be managed successfully outside the operating theatre, provided that all the safety criteria for ambulatory DS or anesthesia are present.",
"To determine the efficacy of 25% oral sucrose in the reduction of pain during a heel lance procedure in sick hospitalized infants. In a blinded randomized- controlled trial, hospitalized infants were given either 1 mL 25% sucrose or 1 mL water 2 min prior to a heel lance procedure. Pain assessment comprised a four-point facial expression score, incidence and duration of crying, heart rate and oxygen saturation changes. A total of 128 infants were included. Facial scores immediately upon heel lance, and at 1 and 2 min in the recovery period were reduced in the treatment (sucrose) group compared to the placebo (water) group (P < 0.05). At other observation points, the differences in facial scores between the two groups of infants did not reach statistical significance. Neither incidence nor duration of crying whilst the blood collection was in progress was significantly reduced by sucrose. In the 3-min recovery period following completion of the blood collection, incidence and duration of crying were significantly less in the treatment group of infants (P < 0.05). Physiological responses of heart rate and oxygen saturation were not attenuated by sucrose at any time point during or following the blood collection. Oral sucrose was effective in reducing behavioural responses to pain upon heel lance and in the period following completion of a heel lance procedure in this group of sick hospitalized infants. This simple strategy can be promoted in institutions caring for sick babies, as a method of reducing behavioural responses to procedural pain."
] |
-Synuclein Aggregation and Neurotoxicity in Parkinson's Disease
|
Genetics and neuropathology strongly link α-synuclein aggregation and neurotoxicity to the pathogenesis of Parkinson's disease and related α-synucleinopathies. Here we describe a new Drosophila model of α-synucleinopathy based on widespread expression of wild-type human α-synuclein, which shows robust neurodegeneration, early-onset locomotor deficits, and abundant α-synuclein aggregation. We use results of forward genetic screening and genetic analysis in our new model to demonstrate that α-synuclein expression promotes reorganization of the actin filament network and consequent mitochondrial dysfunction through altered Drp1 localization. Similar changes are present in a mouse α-synucleinopathy model and in postmortem brain tissue from patients with α-synucleinopathy. Importantly, we provide evidence that the interaction of α-synuclein with spectrin initiates pathological alteration of the actin cytoskeleton and downstream neurotoxicity. These findings suggest new therapeutic approaches for α-synuclein induced neurodegeneration.
|
[
"A comprehensive understanding of the brain requires the analysis of individual neurons. We used twin-spot mosaic analysis with repressible cell markers (twin-spot MARCM) to trace cell lineages at high resolution by independently labeling paired sister clones. We determined patterns of neurogenesis and the influences of lineage on neuron-type specification. Notably, neural progenitors were able to yield intermediate precursors that create one, two or more neurons. Furthermore, neurons acquired stereotyped projections according to their temporal position in various brain sublineages. Twin-spot MARCM also permitted birth dating of mutant clones, enabling us to detect a single temporal fate that required chinmo in a sublineage of six Drosophila central complex neurons. In sum, twin-spot MARCM can reveal the developmental origins of neurons and the mechanisms that underlie cell fate.",
"The Drosophila fruitless (fru) gene product Fru has been postulated to be a neural sex determination factor that directs development of the central nervous system (CNS), thereby producing male-typical courtship behaviour and inducing male-specific muscle. Male-specific Fru protein is expressed in small groups of neurons scattered throughout the CNS of male, but not female, Drosophila. Collectively, these observations suggest that Fru 'masculinizes' certain neurons, thereby establishing neural substrates for male-typical behaviour. However, specific differences between neurons resulting from the presence or absence of Fru are unknown. Previous studies have suggested that Fru might result in sexual differences in the CNS at the functional level, as no overt sexual dimorphism in CNS structure was discernible. Here we identify a subset of fru-expressing interneurons in the brain that show marked sexual dimorphism in their number and projection pattern. We also demonstrate that Fru supports the development of neurons with male-specific dendritic fields, which are programmed to die during female development as a result of the absence of Fru. Thus, Fru expression can produce a male-specific neural circuit, probably used during heterosexual courtship, by preventing cell death in identifiable neurons.",
"A transcription map of the qa gene cluster of Neurospora crassa has been constructed by using cloned DNA fragments as hybridization probes. The mRNAs encoded in the previously identified qa-2 (3-dehydroquinate hydro-lyase, EC 4.2.1.10), qa-4 (dehydroshikimate dehydratase), qa-3 (quinate:NAD+ 3-oxidoreductase, EC 1.1.1.24), and qa-1 (regulatory protein) genes have been characterized. In addition, mRNAs encoded in two new genes in this cluster (qa-x, qa-y) have been identified. Regulation of this system occurs at the level of transcription and is under the combined control of quinic acid and the qa-1 protein. The qa cluster represents a group of adjacent coding sequences which occupy approximately 18 kilobases in linkage group VII. The expression of the qa-1 gene appears to be constitutive but also autoregulated. mRNAs encoded in genes flanking the qa gene cluster have also been identified.",
"α-Synuclein is the major protein associated with Lewy body dementia, Parkinson's disease and multiple system atrophy. Since α-synuclein is present in the brain in physiological conditions as a presynaptic protein, it is crucial to characterize disease-associated modifications to develop an in vivo biomarker. With the aim to develop antibodies showing high specificity and sensitivity for disease-associated α-synuclein, synthetic peptides containing different amino acid sequences were used for immunization of mice. After generation of α-synuclein aggregates, ELISA and immunoblotting were used to test the specificity of antibodies. Tissue microarray sections originating from different human α-synucleinopathies were used to compare immunostaining with other, commercially available antibodies. Immunization of mice with the peptide TKEGVVHGVATVAE (amino acid 44-57 of α-synuclein) resulted in the generation of a monoclonal antibody (5G4), which was able to bind aggregated α-synuclein preparation in sandwich ELISA or coated on magnetic beads. 5G4 proved to be superior to other antibodies in comparative immunohistochemical studies by revealing more widespread and distinct α-synuclein pathology. Immunoblotting of human brain tissue revealed an additional band seen in dementia with Lewy bodies, whereas the band representing monomeric α-synuclein was very weak or lacking. In summary, the 5G4 antibody is most promising for re-evaluation of archival material and may offer new perspective for the development of in vivo diagnostic assays for detecting disease-associated α-synuclein in body fluids.",
"The structure and function of mitochondrial respiratory-chain enzyme proteins were studied postmortem in the substantia nigra of nine patients with Parkinson's disease and nine matched controls. Total protein and mitochondrial mass were similar in the two groups. NADH-ubiquinone reductase (Complex I) and NADH cytochrome c reductase activities were significantly reduced, whereas succinate cytochrome c reductase activity was normal. These results indicated a specific defect of Complex I activity in the substantia nigra of patients with Parkinson's disease. This biochemical defect is the same as that produced in animal models of parkinsonism by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and adds further support to the proposition that Parkinson's disease may be due to an environmental toxin with action(s) similar to those of MPTP.",
"Mitochondrial abnormalities have been documented in Alzheimer's disease and related neurodegenerative disorders, but the causal relationship between mitochondrial changes and neurodegeneration, and the specific mechanisms promoting mitochondrial dysfunction, are unclear. Here, we find that expression of human tau results in elongation of mitochondria in both Drosophila and mouse neurons. Elongation is accompanied by mitochondrial dysfunction and cell cycle-mediated cell death, which can be rescued in vivo by genetically restoring the proper balance of mitochondrial fission and fusion. We have previously demonstrated that stabilization of actin by tau is critical for neurotoxicity of the protein. Here, we demonstrate a conserved role for actin and myosin in regulating mitochondrial fission and show that excess actin stabilization inhibits association of the fission protein DRP1 with mitochondria, leading to mitochondrial elongation and subsequent neurotoxicity. Our results thus identify actin-mediated disruption of mitochondrial dynamics as a direct mechanism of tau toxicity in neurons in vivo.",
"We describe a new repressible binary expression system based on the regulatory genes from the Neurospora qa gene cluster. This \"Q system\" offers attractive features for transgene expression in Drosophila and mammalian cells: low basal expression in the absence of the transcriptional activator QF, high QF-induced expression, and QF repression by its repressor QS. Additionally, feeding flies quinic acid can relieve QS repression. The Q system offers many applications, including (1) intersectional \"logic gates\" with the GAL4 system for manipulating transgene expression patterns, (2) GAL4-independent MARCM analysis, and (3) coupled MARCM analysis to independently visualize and genetically manipulate siblings from any cell division. We demonstrate the utility of the Q system in determining cell division patterns of a neuronal lineage and gene function in cell growth and proliferation, and in dissecting neurons responsible for olfactory attraction. The Q system can be expanded to other uses in Drosophila and to any organism conducive to transgenesis."
] |
Efferocytosis of eryptotic erythrocytes contributes to recovery from intracerebral hemorrhage
|
Macrophages are a source of both proinflammatory and restorative functions in damaged tissue through complex dynamic phenotypic changes. Here, we sought to determine whether monocyte-derived macrophages (MDMs) contribute to recovery after acute sterile brain injury. By profiling the transcriptional dynamics of MDMs in the murine brain after experimental intracerebral hemorrhage (ICH), we found robust phenotypic changes in the infiltrating MDMs over time and demonstrated that MDMs are essential for optimal hematoma clearance and neurological recovery. Next, we identified the mechanism by which the engulfment of erythrocytes with exposed phosphatidylserine directly modulated the phenotype of both murine and human MDMs. In mice, loss of receptor tyrosine kinases AXL and MERTK reduced efferocytosis of eryptotic erythrocytes and hematoma clearance, worsened neurological recovery, exacerbated iron deposition, and decreased alternative activation of macrophages after ICH. Patients with higher circulating soluble AXL had poor 1-year outcomes after ICH onset, suggesting that therapeutically augmenting efferocytosis may improve functional outcomes by both reducing tissue injury and promoting the development of reparative macrophage responses. Thus, our results identify the efferocytosis of eryptotic erythrocytes through AXL/MERTK as a critical mechanism modulating macrophage phenotype and contributing to recovery from ICH.
|
[
"The liver has a remarkable capacity to regenerate after injury; yet, the role of macrophages (MF) in this process remains controversial mainly due to difficulties in distinguishing between different MF subsets. In this study, we used a murine model of acute liver injury induced by overdose of N-acetyl-p-aminophenol (APAP) and defined three distinct MF subsets that populate the liver following injury. Accordingly, resident Kupffer cells (KC) were significantly reduced upon APAP challenge and started recovering by self-renewal at resolution phase without contribution of circulating Ly6C(hi) monocytes. The latter were recruited in a CCR2- and M-CSF-mediated pathway at the necroinflammatory phase and differentiated into ephemeral Ly6C(lo) MF subset at resolution phase. Moreover, their inducible ablation resulted in impaired recovery. Microarray-based molecular profiling uncovered high similarity between steady-state KC and those recovered at the resolution phase. In contrast, KC and monocyte-derived MF displayed distinct prorestorative genetic signature at the resolution phase. Finally, we show that infiltrating monocytes acquire a prorestorative polarization manifested by unique expression of proangiogenesis mediators and genes involved with inhibition of neutrophil activity and recruitment and promotion of their clearance. Collectively, our results present a novel phenotypic, ontogenic, and molecular definition of liver-MF compartment following acute injury.",
"Healing after myocardial infarction involves the biphasic accumulation of inflammatory lymphocyte antigen 6C (Ly-6C)(high) and reparative Ly-6C(low) monocytes/macrophages (Mo/MΦ). According to 1 model, Mo/MΦ heterogeneity in the heart originates in the blood and involves the sequential recruitment of distinct monocyte subsets that differentiate to distinct macrophages. Alternatively, heterogeneity may arise in tissue from 1 circulating subset via local macrophage differentiation and polarization. The orphan nuclear hormone receptor, nuclear receptor subfamily 4, group a, member 1 (Nr4a1), is essential to Ly-6C(low) monocyte production but dispensable to Ly-6C(low) macrophage differentiation; dependence on Nr4a1 can thus discriminate between systemic and local origins of macrophage heterogeneity. This study tested the role of Nr4a1 in myocardial infarction in the context of the 2 Mo/MΦ accumulation scenarios. We show that Ly-6C(high) monocytes infiltrate the infarcted myocardium and, unlike Ly-6C(low) monocytes, differentiate to cardiac macrophages. In the early, inflammatory phase of acute myocardial ischemic injury, Ly-6C(high) monocytes accrue in response to a brief C-C chemokine ligand 2 burst. In the second, reparative phase, accumulated Ly-6C(high) monocytes give rise to reparative Ly-6C(low) F4/80(high) macrophages that proliferate locally. In the absence of Nr4a1, Ly-6C(high) monocytes express heightened levels of C-C chemokine receptor 2 on their surface, avidly infiltrate the myocardium, and differentiate to abnormally inflammatory macrophages, which results in defective healing and compromised heart function. Ly-6C(high) monocytes orchestrate both inflammatory and reparative phases during myocardial infarction and depend on Nr4a1 to limit their influx and inflammatory cytokine expression.",
"The mechanisms underlying neural injury in intracerebral hemorrhage (ICH) remain uncertain. The present two-part study investigated cell death in the region of ICH and its association with caspase-3 activation. ICH was produced in adult rats by injection of 100 microl of autologous blood or saline into the right basal ganglia. The animals' brains were removed at 6 hours or at 1, 3, 7, or 14 days after hemorrhage. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin in situ nick end-labeling (TUNEL) was used to detect deoxyribonucleic acid (DNA) fragmentation. TUNEL-positive cells were quantified. Caspase-3 activation was measured by Western blotting and immunohistochemistry. Double labeling was used to compare TUNEL with caspase-3 distribution and to identify the cell types affected. TUNEL-positive cells were also quantified at 6 hours, 1 day, and 3 days after injection of 5 U of thrombin into the right basal ganglion. At 6 hours, TUNEL-positive cells appeared in the ICH model (but not in the saline control brains) and were present for more than 2 weeks after ICH, peaking at 3 days. Western blot analysis revealed that the increase in immunoreactivity for the activated caspase-3 precedes that of DNA fragmentation, peaking at 1 day after ICH and declining thereafter. Immunohistochemistry analysis showed nucleus translocation of caspase-3 after ICH. Double-labeling studies demonstrated that both neurons and astrocytes surrounding the clot were TUNEL-positive. In addition, TUNEL and caspase-3 were colocalized in the same cells. Intracerebral thrombin injection elicited DNA fragmentation similar to that observed after the injection of blood. Double-strand breaks in genomic DNA and induction of caspase-3 were demonstrated adjacent to parenchymal hematoma in the animals' brains. These results provide evidence that cell loss after ICH is associated with activation of caspase-3.",
"Phagocytosis is necessary to eliminate the hematoma after intracerebral hemorrhage (ICH); however, release of proinflammatory mediators and free radicals during phagocyte activation is toxic to neighboring cells, leading to secondary brain injury. Promotion of phagocytosis in a timely and efficient manner may limit the toxic effects of persistent blood products on surrounding tissue and may be important for recovery after ICH. Intrastriatal blood injection in rodents and primary microglia in culture exposed to red blood cells were used to model ICH and to study mechanisms of hematoma resolution and phagocytosis regulation by peroxisome proliferator-activated receptor gamma (PPARgamma) in microglia/macrophages. Our study demonstrated that the PPARgamma agonist, rosiglitazone, promoted hematoma resolution, decreased neuronal damage, and improved functional recovery in a mouse ICH model. Microglia isolated from murine brains showed more efficient phagocytosis in response to PPARgamma activators. PPARgamma activators significantly increased PPARgamma-regulated gene (catalase and CD36) expression, whereas reducing proinflammatory gene (tumor necrosis factor-alpha, interleukin-1beta, matrix metalloproteinase-9, and inducible nitric oxide synthase) expression, extracellular H(2)O(2) level, and neuronal damage. Phagocytosis by microglia was significantly inhibited by PPARgamma gene knockdown or neutralizing anti-CD36 antibody, whereas it was enhanced by exogenous catalase. PPARgamma in macrophages acts as an important factor in promoting hematoma absorption and protecting other brain cells from ICH-induced damage.",
"Intracerebral hemorrhage (ICH) is a devastating type of stroke that lacks a specific treatment. An intense immune response develops after ICH, which contributes to neuronal injury, disability, and death. However, the specific mediators of inflammation-induced injury remain unclear. The objective of the present study was to determine whether blood-derived CCR2+ Ly6C(hi) inflammatory monocytes contribute to disability. ICH was induced in mice and the resulting inflammatory response was quantified using flow cytometry, confocal microscopy, and neurobehavioral testing. Importantly, blood-derived monocytes were distinguished from resident microglia by differential CD45 staining and by using bone marrow chimeras with fluorescent leukocytes. After ICH, blood-derived CCR2+ Ly6C(hi) inflammatory monocytes trafficked into the brain, outnumbered other leukocytes, and produced tumor necrosis factor. Ccr2(-/-) mice, which have few circulating inflammatory monocytes, exhibited better motor function following ICH than control mice. Chimeric mice with wild-type CNS cells and Ccr2(-/-) hematopoietic cells also exhibited early improvement in motor function, as did wild-type mice after inflammatory monocyte depletion. These findings suggest that blood-derived inflammatory monocytes contribute to acute neurological disability. To determine the translational relevance of our experimental findings, we examined CCL2, the principle ligand for the CCR2 receptor, in ICH patients. Serum samples from 85 patients were collected prospectively at two hospitals. In patients, higher CCL2 levels at 24 h were independently associated with poor functional outcome at day 7 after adjusting for potential confounding variables. Together, these findings suggest that inflammatory monocytes worsen early disability after murine ICH and may represent a therapeutic target for patients.",
"Accumulating evidence indicates that inflammatory responses cause secondary injury after intracerebral hemorrhage (ICH). We recently demonstrated the involvement of toll-like receptor 4 (TLR4) signaling in these processes. The purpose of the current study was to investigate the protective effect and mechanism of TAK-242 (Ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl) sulfamoyl] cyclohex-1-ene-1 -carboxylate, Takeda), a TLR4 antagonist, in an ICH mouse model. TAK-242 was intraperitoneally injected 6 hours after ICH once daily for 5 successive days. We assessed neurological deficit scores; changes in brain water content; and levels of inflammatory factors, DNA damage, and neuronal degeneration in perihematomal region 1, 3, and 5 days after ICH. Peripheral inflammatory cell infiltration was determined using flow cytometry; and the expression of TLR4 downstream signaling molecules was assessed by Western blot. TAK-242 significantly reduced brain water content, neurological deficit scores, and levels of inflammatory factors. The levels of DNA damage and neuronal degeneration were also significantly decreased, as was peripheral inflammatory cell infiltration. The expression of TLR4 downstream signaling molecules, including myeloid differentiation primary response gene 88, toll/IR-1(TIR)-domain-containing adaptor protein inducing interferon-beta IκBα, nuclear factor-κBp65, and phosphorylated nuclear factor-κBp65, was significantly downregulated. The results suggest that TLR4 antagonist reduced inflammatory injury and neurological deficits in a mouse model of ICH. The mechanism may involve decreased expression of signaling molecules downstream of TLR4.",
"Three members of the NR4A1/Nur77/ NGFIB orphan nuclear hormone receptor subfamily (NR4A1, NR4A2, and NR4A3) belong to the steroid nuclear hormone receptor superfamily. They share similar structural features and have no known natural ligand. They constitute immediate early genes that are induced by serum, growth factors and receptor engagement and are thus implicated in cell mitogenic responses. These nuclear receptors are transcription factors that exert their functions through activation and subsequent induction of the downstream pathways. They have been shown to play a role in complex pathways of cell survival and apoptosis. Although the expression of these genes have been shown to be pro-survival, it has also been reported that NR4A1 expression can cause apoptosis. These two opposite effects apparently result from distinct mechanisms: either transcriptional activation of genes responsible for cell survival or cell apoptosis, or translocation into the cytoplasm where they target the mitochondria and cause cell apoptosis via Bcl-2 binding. The latter mechanism constitutes a new paradigm of cellular apoptosis. In vitro functional studies using over-expression (gain of function) or gene inactivation (loss of function) type assays, combined with transgenic or knockout animal data in vivo, have revealed these effects and their physiological roles, including thymocyte development for NR4A1/3 and pro-survival in CNS for NR4A2. Recent studies have also suggested an important role of these receptors in cell transformation and tumorigenicity via both their anti-apoptotic and pro-apoptotic functions. In particular, the recent identification of a functional ligand for NR4A1 suggests that these members could potentially serve as drug targets for disease indications such as cancer. While many aspects of these receptors have been previously reviewed, this article focuses on new experimentation and discovery of their apoptotic and carcinogenic roles, and discusses their potential roles as therapeutic targets."
] |
Gamma oscillations in the beta band represent quantitative information about somatosensory features during stimulus retention
|
Ample evidence suggests that oscillations in the beta band represent quantitative information about somatosensory features during stimulus retention. Visual and auditory working memory (WM) research, on the other hand, has indicated a predominant role of gamma oscillations for active WM processing. Here we reconciled these findings by recording whole-head magnetoencephalography during a vibrotactile frequency comparison task. A Braille stimulator presented healthy subjects with a vibration to the left fingertip that was retained in WM for comparison with a second stimulus presented after a short delay. During this retention interval spectral power in the beta band from the right intraparietal sulcus and inferior frontal gyrus (IFG) monotonically increased with the to-be-remembered vibrotactile frequency. In contrast, induced gamma power showed the inverse of this pattern and decreased with higher stimulus frequency in the right IFG. Together, these results expand the previously established role of beta oscillations for somatosensory WM to the gamma band and give further evidence that quantitative information may be processed in a fronto-parietal network.
|
[
"The anterior part of the parietal association area in the cerebral cortex of primates has been implicated in the integration of somatosensory signals, which generate neural images of body parts and apposed objects and provide signals for sensorial guidance of movements. Here we show that this area is active in primates performing numerically based behavioural tasks. We required monkeys to select and perform movement A five times, switch to movement B for five repetitions, and return to movement A, in a cyclical fashion. Cellular activity in the superior parietal lobule reflected the number of self-movement executions. For the most part, the number-selective activity was also specific for the type of movement. This type of numerical representation of self-action was seen less often in the inferior parietal lobule, and rarely in the primary somatosensory cortex. Such activity in the superior parietal lobule is useful for processing numerical information, which is necessary to provide a foundation for the forthcoming motor selection.",
"How single neurons represent information about the magnitude of a stimulus remains controversial. Neurons encoding purely sensory magnitude typically show monotonic response functions (\"summation coding\"), and summation units are usually implemented in models of numerosity representation. In contrast, cells representing numerical quantity exhibit nonmonotonic tuning functions that peak at their preferred numerosity (\"labeled-line code\"), but the restricted range of tested quantities in these studies did not permit a definite answer. Here, we analyzed both behavioral and neuronal representations of a broad range of numerosities from 1 to 30 in the prefrontal cortex of monkeys. Numerosity-selective neurons showed a clear and behaviorally relevant labeled-line code for all numerosities. Moreover, both the behavioral and neuronal tuning functions obeyed the Weber-Fechner Law and were best represented on a nonlinearly compressed scale. Our single-cell study is in good agreement with functional imaging data reporting peaked tuning functions in humans, demonstrating neuronal precursors for human number competence in a nonhuman primate. Our findings also emphasize that the manner in which neurons encode and maintain magnitude information may depend on the precise task at hand as well as the type of magnitude to represent and memorize.",
"We consider the potential role of oscillations in the prefrontal cortex (PFC) in mediating attention, working memory and memory consolidation. Activity in the theta, beta, and gamma bands is related to communication between PFC and different brain areas. While gamma/beta oscillations mediate bottom-up and top-down interactions between PFC and visual cortices, related to attention, theta rhythms are engaged by hippocampal/PFC interplay. These interactions are dynamic, depending on the nature and relevance of the information currently being processed. The profound modifications of the PFC neuronal network associated with changes in oscillatory coherence are controlled by neuromodulators such as dopamine, which thereby allow or prevent the formation of cell assemblies for information encoding and storage.",
"Successful working memory (WM) requires the engagement of relevant brain areas but possibly also the disengagement of irrelevant areas. We used magnetoencephalography (MEG) to elucidate the temporal dynamics of areas involved in a somatosensory WM task. We found an increase in gamma band activity in the primary and secondary somatosensory areas during encoding and retention, respectively. This was accompanied by an increase of alpha band activity over task-irrelevant regions including posterior and ipsilateral somatosensory cortex. Importantly, the alpha band increase was strongest during successful WM performance. Furthermore, we found frontal gamma band activity that correlated both with behavioral performance and the alpha band increase. We suggest that somatosensory gamma band activity reflects maintenance and attention-related components of WM operations, whereas alpha band activity reflects frontally controlled disengagement of task-irrelevant regions. Our results demonstrate that resource allocation involving the engagement of task-relevant and disengagement of task-irrelevant regions is needed for optimal task execution.",
"Neuroimaging studies in the last 20 years have tried to unravel the neural correlates of number processing across formats in humans and non-human primates. Results point to the intraparietal sulcus as the core area for an abstract representation of numerical quantity. On the other hand, there exist a variety of behavioral and neuroimaging data that are difficult to reconcile with the existence of such an abstract representation. In this study, we addressed this issue by applying multi-voxel pattern analysis (MVPA) to functional Magnetic Resonance Imaging (fMRI) data to unravel the neural representations of symbolic (digits) and non-symbolic (dots) numbers and their possible overlap on three different spatial scales (entire lobules, smaller regions of interest and a searchlight analysis with 2-voxel radius). Results showed that numbers in both formats are decodable in occipital, frontal, temporal and parietal regions. However, there were no overlapping representations between dots and digits on any of the spatial scales. These data suggest that the human brain does not contain an abstract representation of numerical magnitude.",
"Although humans are the only species to possess language-driven abstract mathematical capacities, we share with many other animals a nonverbal capacity for estimating quantities or numerosity. For some time, researchers have clearly differentiated between small numbers of items--less than about four--referred to as the subitizing range, and larger numbers, where counting or estimation is required. In this review, we examine more recent evidence suggesting a further division, between sets of items greater than the subitizing range, but sparse enough to be individuated as single items; and densely packed stimuli, where they crowd each other into what is better considered as a texture. These two different regimes are psychophysically discriminable in that they follow distinct psychophysical laws and show different dependencies on eccentricity and on luminance levels. But provided the elements are not too crowded (less than about two items per square degree in central vision, less in the periphery), there is little evidence that estimation of numerosity depends on mechanisms responsive to texture. The distinction is important, as the ability to discriminate numerosity, but not texture, correlates with formal maths skills.",
"Neural oscillations at different frequencies have recently been related to a wide range of basic and higher cognitive processes. One possible role of oscillatory activity is to assure the maintenance of information in working memory (WM). Here we review the possibility that rhythmic activity at theta, alpha, and gamma frequencies serve distinct functional roles during WM maintenance. Specifically, we propose that gamma-band oscillations are generically involved in the maintenance of WM information. By contrast, alpha-band activity reflects the active inhibition of task-irrelevant information, whereas theta-band oscillations underlie the organization of sequentially ordered WM items. Finally, we address the role of cross-frequency coupling (CFC) in enabling alpha-gamma and theta-gamma codes for distinct WM information.",
"Neuropsychologists commonly use the Wisconsin Card Sorting Test as a test of the integrity of frontal lobe functions. However, an account of its range of validity and of the neuronal mechanisms involved is lacking. We analyze the test at 3 different levels. First, the different versions of the test are described, and the results obtained with normal subjects and brain-lesioned patients are reviewed. Second, a computational analysis is used to reveal what algorithms may pass the test, and to predict their respective performances. At this stage, 3 cognitive components are isolated that may critically contribute to performance: the ability to change the current rule when negative reward occurs, the capacity to memorize previously tested rules in order to avoid testing them twice, and the possibility of rejecting some rules a priori by reasoning. Third, a model neuronal network embodying these 3 components is described. The coding units are clusters of neurons organized in layers, or assemblies. A sensorimotor loop enables the network to sort the input cards according to several criteria (color, form, etc.). A higher-level assembly of rule-coding clusters codes for the currently tested rule, which shifts when negative reward is received. Internal testing of the possible rules, analogous to a reasoning process, also occurs, by means of an endogenous auto-evaluation loop. When lesioned, the model reproduces the behavior of frontal lobe patients. Plausible biological or molecular implementations are presented for several of its components.",
"Building on evidence for working memory (WM) coding of vibrotactile frequency information in monkey prefrontal cortex, recent electroencephalography studies found frequency processing in human WM to be reflected by quantitative modulations of prefrontal upper beta activity (20-30 Hz) as a function of the to-be-maintained stimulus attribute. This kind of stimulus-dependent activity has been observed across different sensory modalities, suggesting a generalized role of prefrontal beta during abstract WM processing of quantitative magnitude information. However, until now the available empirical evidence for such quantitative WM representation remains critically limited to the retention of periodic stimulus frequencies. In the present experiment, we used retrospective cueing to examine the quantitative WM processing of stationary (intensity) and temporal (duration) attributes of a previously presented tactile stimulus. We found parametric modulations of prefrontal beta activity during cued WM processing of each type of quantitative information, in a very similar manner as had before been observed only for periodic frequency information. In particular, delayed prefrontal beta modulations systematically reflected the magnitude of the retrospectively selected stimulus attribute and were functionally linked to successful behavioral task performance. Together, these findings converge on a generalized role of stimulus-dependent prefrontal beta-band oscillations during abstract scaling of analog quantity information in human WM.",
"Switching flexibly between behavioral goals is a hallmark of executive control and requires integration of external and internal information. We recorded single-neuron correlates of different numerical representations (sensory-, working memory-, and rule-related activity) in the dorsal premotor area (PMd), the cingulate motor areas (CMA), and the ventral intraparietal sulcus (VIP) and compared them to previous recordings in the lateral prefrontal cortex (PFC). Two monkeys were trained to encode and memorize numerosities and flexibly switch between two abstract quantitative rules based on rule cues. Almost 20% of randomly selected PFC and PMd neurons significantly represented the numerical rule in a behaviorally relevant manner, approximately twice as many as in the CMA and VIP. Rule selectivity was significantly better for PMd neurons than for PFC cells. Seemingly at the expense of rule selectivity, however, sensory- and memory-related numerosity activity was greatly diminished compared with previous delayed match-to-numerosity studies. These findings suggest the involvement of the frontal premotor areas in strategic planning such as rule following. Moreover, the results emphasize that the coding capacities of neurons in association cortical areas are far more dynamic depending on task demands than previously thought."
] |
Cortico-ponto-cerebellar tract injury following mild traumatic brain injury
|
We report on a patient with injury of the cortico-ponto-cerebellar tract (CPCT) following mild traumatic brain injury (TBI), diagnosed by diffusion tensor tractography (DTT).
|
[
"Clinical diagnosis of olivopontocerebellar atrophy (OPCA) must be confirmed by radiologic demonstration of atrophy in an appropriate distribution. OPCA may be associated with degeneration of other systems in multisystem atrophy (MSA). The authors report 23 cases of OPCA, eight of which were associated with MSA. Atrophy involved the cerebellum, pons, and middle cerebellar peduncles in all cases. On intermediate and T2-weighted magnetic resonance (MR) images, abnormal signal intensity was always observed in the transverse pontine fibers, middle cerebellar peduncles, and cerebellum, structures known from pathologic study to degenerate in OPCA. Pyramidal tracts and superior cerebellar peduncles stood out because of their normal signal intensity. Of the eight patients with MSA, four also had variable abnormal signal intensities in the putamen. The authors believe that the combination of atrophy and abnormal signal intensity in the appropriate distribution strongly supports the diagnosis of OPCA. In some cases, MR imaging may demonstrate involvement of different systems, thus confirming the diagnosis of MSA.",
"Concussion is the most common form of traumatic brain injury (TBI), but diagnosis remains controversial because the brain appears quite normal in conventional computed tomography and magnetic resonance imaging (MRI). These conventional tools are not sensitive enough to detect diffuse traumatic axonal injury, and cannot depict aberrations in mild TBIs. Advanced MRI modalities including diffusion tensor imaging (DTI), and magnetic resonance spectroscopy (MRS), make it possible to detect brain injuries in TBI. The purpose of this review is to provide the latest information regarding the visualization and quantification of important abnormalities in TBI and new insights into their clinical significance. Advanced imaging modalities allow the discovery of biomarkers of injury and the detection of changes in brain injury over time. Such tools will likely be used to evaluate treatment efficacy in research. Combining multiple imaging modalities would not only provide greater insight into the underlying physiological changes in TBI, but also improve diagnostic accuracy in predicting outcomes. In this review we present evidence of brain abnormalities in TBI based on investigations using MRI, including DTI and MRS. Our review provides a summary of some of the important studies published from 2002 to 2012 on the topic of MRI findings in head trauma. With the growing realization that even mild head injury can lead to neurocognitive deficits, medical imaging has assumed preeminence for detecting abnormalities associated with TBI. Advanced MRI modalities such as DTI and MRS have an important role in the diagnosis of lesions for TBI patients.",
"We sought to determine whether diffusion-tensor imaging (DTI) can detect in vivo axonal damage in the corticopontocerebellar pathway of patients with adult-onset ataxic neurodegenerative disease. Conventional MRI and DTI were performed on 18 patients with adult-onset ataxic neurodegenerative disease and 28 age-matched control subjects. Fractional anisotropy (FA) and the mean diffusivity (MD) were measured in the ventral, central, and dorsal pons, middle cerebellar peduncle (MCP) and internal capsule to evaluate corticopontocerebellar projection. Changes in FA and MD values were compared between patients and controls. Clinical disability was assessed according to the International Cooperative Ataxia Rating Scale (ICARS). The relationship between DTI measurements and ICARS was studied. Follow-up MRI was performed in five patients approximately 1 year later. FA values were significantly lower in the ventral and central portions of the pons, MCP, and internal capsules than in these areas in control subjects (P < 0.05) with the lower FA values correlating with poorer ICARS (r > -0.57, P < 0.05). MD values were elevated in these areas, but the differences were smaller than for the FA values. No relationship was observed between the MD and ICARS. In the five patients who underwent the follow-up study, there were significant decreases between the initial study and the follow-up DTI study for FA in the MCP and internal capsule (P < 0.05). DTI can demonstrate a degenerated corticopontocerebellar pathway in patients, and FA values can be correlated with ataxia severity. DTI may be a clinically useful tool as a quantitative surrogate marker for monitoring disease progression.",
"Magnetic resonance tractography based on diffusion-tensor imaging was first introduced to the medical imaging community a decade ago. It has been successfully applied to a number of neurological conditions and most commonly used for preoperative planning for brain tumors and vascular malformations. Areas of active research include stroke, and dementia, where it provides valuable information not available through other imaging techniques. This technique was first introduced using the deterministic streamline algorithm and has evolved to use more sophisticated probabilistic approaches. We will review the past, present, and future of tractography, focusing primarily on its clinical applications.",
"MRI of the brain was performed in 53 patients with a variety of degenerative ataxias and related disorders and 96 control subjects. Atrophy of intracranial structures was not seen in patients with the pure type of hereditary spastic paraplegia, or in early cases of Friedreich's ataxia. In advanced Friedreich's ataxia there was atrophy of the vermis and medulla. The MRI features of early onset cerebellar ataxia with retained reflexes were variable, and suggest heterogeneity. In autosomal dominant cerebellar ataxias, most patients had cerebellar and brainstem atrophy, probably reflecting the pathological process of olivopontocerebellar atrophy; there was no clearly defined group with both clinical and imaging features of isolated cerebellar involvement. The MRI abnormalities in idiopathic late onset cerebellar ataxia were predominantly those of cerebellar and brainstem atrophy or pure cerebellar atrophy. The clinical and imaging features of brainstem abnormalities were discordant in several patients. Pure cerebellar atrophy was associated with slower progression of disability. Cerebral atrophy was common in the late onset ataxias. Cerebral white matter lesions, although usually few in number, were observed in significantly more patients than controls, particularly those aged over 50 years.",
"Despite the involvement of cerebellar ataxia in a large variety of conditions and its frequent association with other neurological symptoms, the quantification of the specific core of the cerebellar syndrome is possible and useful in Neurology. Recent studies have shown that cerebellar ataxia might be sensitive to various types of pharmacological agents, but the scales used for assessment were all different. With the long-term goal of double-blind controlled trials-multicentric and international-an ad hoc Committee of the World Federation of Neurology has worked to propose a one-hundred-point semi-quantitative International Cooperative Ataxia Rating Scale (ICARS). The scale proposed involves a compartimentalized quantification of postural and stance disorders, limb ataxia, dysarthria and oculomotor disorders, in order that a subscore concerning these symptoms may be separately studied. The weight of each symptomatologic compartment has been carefully designed. The members of the Committee agreed upon precise definitions of the tests, to minimize interobserver variations. The validation of this scale is in progress.",
"Using proton magnetic resonance spectroscopic imaging, we studied the cerebellum of 9 patients with cerebellar degeneration and of 9 age-matched normal control subjects. This technique permits the simultaneous measurement of N-acetylaspartate, choline-containing compounds, creatine/phosphocreatine, and lactate signal intensities from four 15-mm slices divided into 0.84-ml single-volume elements. Because patients with cerebellar degeneration often show substantial atrophy on magnetic resonance imaging (MRI), we specifically chose to analyze the spectroscopic signals only from tissue that did not have an atrophic appearance on the MRI. The spectroscopic findings showed a significant reduction of N-acetylaspartate in all parts of the cerebellum, a significant correlation with MRI scores of cerebellar atrophy, and a significant correlation with clinical rating scores of cerebellar disturbance. Our method of analysis suggests the presence of a neurodegenerative process in cerebellar areas that do not appear to be atrophic on the MRI. Some limitations of proton magnetic resonance spectroscopic imaging in the present study were related to the partial field inhomogeneity characteristics of the posterior fossa, the anatomical location of the cerebellum, and the particularly severe cerebellar atrophy in some of the patients."
] |
A heuristic for improving traffic congestion modeling
|
Traffic congestion is an important problem faced by Intelligent Transportation Systems (ITS), requiring models that allow predicting the impact of different solutions on urban traffic flow. Such an approach typically requires the use of simulations, which should be as realistic as possible. However, achieving high degrees of realism can be complex when the actual traffic patterns, defined through an Origin/Destination (O-D) matrix for the vehicles in a city, remain unknown. Thus, the main contribution of this paper is a heuristic for improving traffic congestion modeling. In particular, we propose a procedure that, starting from real induction loop measurements made available by traffic authorities, iteratively refines the output of DFROUTER, which is a module provided by the SUMO (Simulation of Urban MObility) tool. This way, it is able to generate an O-D matrix for traffic that resembles the real traffic distribution and that can be directly imported by SUMO. We apply our technique to the city of Valencia, and we then compare the obtained results against other existing traffic mobility data for the cities of Cologne (Germany) and Bologna (Italy), thereby validating our approach. We also use our technique to determine what degree of congestion is expectable if certain conditions cause additional traffic to circulate in the city, adopting both a uniform pattern and a hotspot-based pattern for traffic injection to demonstrate how to regulate the overall number of vehicles in the city. This study allows evaluating the impact of vehicle flow changes on the overall traffic congestion levels.
|
[
"The emergence of large, fine-grained mobility datasets offers significant opportunities for the development and application of new methodologies for transportation analysis. In this paper, the link between routing behaviour and traffic patterns in urban areas is examined, introducing a method to derive estimates of traffic patterns from a large collection of fine-grained routing data. Using this dataset, the interconnectivity between road network junctions is extracted in the form of a Markov chain. This representation encodes the probability of the successive usage of adjacent road junctions, encoding routes as flows between decision points rather than flows along road segments. This network of functional interactions is then integrated within a modified Markov chain Monte Carlo (MCMC) framework, adapted for the estimation of urban traffic patterns. As part of this approach, the data-derived links between major junctions influence the movement of directed random walks executed across the network to model origin-destination journeys. The simulation process yields estimates of traffic distribution across the road network. The paper presents an implementation of the modified MCMC approach for London, United Kingdom, building an MCMC model based on a dataset of nearly 700000 minicab routes. Validation of the approach clarifies how each element of the MCMC framework contributes to junction prediction performance, and finds promising results in relation to the estimation of junction choice and minicab traffic distribution. The paper concludes by summarising the potential for the development and extension of this approach to the wider urban modelling domain.",
"This paper proposes a two-stage algorithm to simultaneously estimate origin-destination (OD) matrix, link choice proportion, and dispersion parameter using partial traffic counts in a congested network. A non-linear optimization model is developed which incorporates a dynamic dispersion parameter, followed by a two-stage algorithm in which Generalized Least Squares (GLS) estimation and a Stochastic User Equilibrium (SUE) assignment model are iteratively applied until the convergence is reached. To evaluate the performance of the algorithm, the proposed approach is implemented in a hypothetical network using input data with high error, and tested under a range of variation coefficients. The root mean squared error (RMSE) of the estimated OD demand and link flows are used to evaluate the model estimation results. The results indicate that the estimated dispersion parameter theta is insensitive to the choice of variation coefficients. The proposed approach is shown to outperform two established OD estimation methods and produce parameter estimates that are close to the ground truth. In addition, the proposed approach is applied to an empirical network in Seattle, WA to validate the robustness and practicality of this methodology. In summary, this study proposes and evaluates an innovative computational approach to accurately estimate OD matrices using link-level traffic flow data, and provides useful insight for optimal parameter selection in modeling travelers' route choice behavior.",
"Most existing network sensor location problem (NSLP) models are designed to identify the number of sensors with fixed costs and installation locations, and sensors are assumed to be installed permanently. However, sometimes sensors are carried by individuals to collect traffic data measurements manually at fixed locations. Hence, their duration of operation for which traffic data measurements are collected is limited, and their costs are not fixed as they are correlated with the duration of operation. This paper proposes a NSLP model that integrates optimal heterogeneous sensor deployment and operation strategies for the dynamic O-D demand estimates under budget constraints. The deployment strategy consists of the numbers of link and node sensors and their installation locations. The operation strategy includes sensors' start time and duration of operation, which has not been addressed in previous studies. An algorithm is developed to solve the proposed model. Numerical experiments performed on a network from a part of Chennai, India show that the proposed model can identify the optimal heterogeneous sensor deployment and operation strategies with the maximum dynamic O-D demand estimation accuracy."
] |
Impact of HIV-1 envelope glycoprotein conformation on antibody-dependent cellular cytotoxicity responses
|
Recent analysis of HIV-1 envelope glycoproteins (Env) dynamics showed that the unliganded Env trimer can potentially sample three conformations: a metastable "closed" conformation (State 1), an "open" CD4-bound conformation (State 3), and an intermediate "partially open" conformation (State 2). HIV-1 evolved several mechanisms to avoid "opening" its Env in order to evade immune responses such as antibody-dependent cellular cytotoxicity (ADCC), which preferentially targets Envs in the CD4-bound conformation on the surface of infected cells. Here we took advantage of a well-characterized single-residue change in the gp120 trimer association domain to modify Env conformation and evaluate its impact on ADCC responses. We found that cells infected with viruses expressing Env stabilized in States 2/3 become highly susceptible to ADCC responses by sera from HIV-1-infected individuals. Our results indicate that the conformations spontaneously sampled by the Env trimer at the surface of infected cells has a significant impact on ADCC responses.
|
[
"The cytoplasmic tails of human and simian immunodeficiency virus (HIV and SIV, respectively) envelope glycoproteins contain a highly conserved, membrane-proximal endocytosis motif that prevents the accumulation of Env on the surface of infected cells prior to virus assembly. Using an assay designed to measure the killing of virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC), we show that substitutions in this motif increase the susceptibility of HIV-1- and SIV-infected cells to ADCC in a manner that directly correlates with elevated Env levels on the surface of virus-infected cells. In the case of HIV-1, this effect is additive with a deletion in vpu recently shown to enhance the susceptibility of HIV-1-infected cells to ADCC as a result of tetherin-mediated retention of budding virions on the cell surface. These results reveal a previously unappreciated role for the membrane-proximal endocytosis motif of gp41 in protecting HIV-1- and SIV-infected cells from antibody responses by regulating the amount of Env present on the cell surface. This study reveals an unappreciated role for the membrane-proximal endocytosis motif of gp41 in protecting HIV-1- and SIV-infected cells from elimination by Env-specific antibodies. Thus, strategies designed to interfere with this mechanism of Env internalization may improve the efficacy of antibody-based vaccines and antiretroviral therapies designed to enhance the immunological control of HIV-1 replication in chronically infected individuals.",
"Antibodies can have an impact on HIV-1 infection in multiple ways, including antibody-dependent cellular cytotoxicity (ADCC), a correlate of protection observed in the RV144 vaccine trial. One of the most potent ADCC-inducing epitopes on HIV-1 Env is recognized by the C11 antibody. Here, we present the crystal structure, at 2.9 Å resolution, of the C11-like antibody N12-i3, in a quaternary complex with the HIV-1 gp120, a CD4-mimicking peptide M48U1, and an A32-like antibody, N5-i5. Antibody N12-i3 recognizes an epitope centered on the N-terminal \"eighth strand\" of a critical β sandwich, which our analysis indicates to be emblematic of a late-entry state, after the gp120 detachment. In prior entry states, this sandwich comprises only seven strands, with the eighth strand instead pairing with a portion of the gp120 C terminus. The conformational gymnastics of HIV-1 gp120 thus includes altered β-strand pairing, possibly to reduce immunogenicity, although nevertheless still recognized by the human immune system.",
"Antibody-dependent cellular cytotoxicity (ADCC) is a potentially effective adaptive immune response to human immunodeficiency virus (HIV) infection. The study of ADCC responses has been hampered by the lack of simple methods to quantify these responses and map effective epitopes. We serendipitously observed that standard intracellular cytokine assays on fresh whole blood from a cohort of 26 HIV-infected subjects identified non-T lymphocytes expressing gamma interferon (IFN-gamma) in response to overlapping linear peptides spanning HIV-1 proteins. The effector cells were CD3(-) CD4(-) CD8(-) CD14(-) CD2(+) CD56(+/-) NK lymphocytes and degranulated granzyme B and perforin in response to antigen stimulation. Serum transfer assays demonstrated that the specific response was mediated by immunoglobulin G. Fresh blood samples from half of the HIV-infected cohort demonstrated robust HIV peptide-specific IFN-gamma expression by NK cells, predominately to Env, Pol, and Vpu HIV-1 proteins. Responses were readily mapped to define minimal epitopes utilizing this assay. Antibody-dependent, HIV-specific NK cell recognition, involving components of both innate and adaptive immune systems, represents a potentially effective immune response to induce by vaccination.",
"Tetherin (BST2/CD317) has emerged as a key host cell defense molecule, inhibiting the release and spread of diverse enveloped virions from infected cells. In this chapter, I review the molecular and cellular basis for tetherin's antiviral activities and the function of virally encoded countermeasures that disrupt its function. I further describe recent advances in our understanding of tetherin's associated role in viral pattern recognition and the evidence for its role in limiting viral pathogenesis in vivo.",
"Anti-HIV-1 envelope glycoprotein (Env) antibodies without broadly neutralizing activity correlated with protection in the RV144 clinical trial, stimulating interest in other protective mechanisms involving antibodies, such as antibody-dependent cell-mediated cytotoxicity (ADCC). Env epitopes targeted by many antibodies effective at mediating ADCC are poorly exposed on the unliganded Env trimer. Here we investigated the mechanism of exposure of ADCC epitopes on Env and showed that binding of Env and CD4 within the same HIV-1-infected cell effectively exposes these epitopes. Env capacity to transit to the CD4-bound conformation is required for ADCC epitope exposure. Importantly, cell surface CD4 downregulation by Nef and Vpu accessory proteins and Vpu-mediated BST-2 antagonism modulate exposure of ADCC-mediating epitopes and reduce the susceptibility of infected cells to this effector function in vitro. Significantly, Env conformational changes induced by cell surface CD4 are conserved among Env from HIV-1 and HIV-2/SIVmac lineages. Altogether, our observations describe a highly conserved mechanism required to expose ADCC epitopes that might help explain the evolutionary advantage of downregulation of cell surface CD4 by the HIV-1 Vpu and Nef proteins. HIV-1 envelope epitopes targeted by many antibodies effective at mediating antibody-dependent cell-mediated cytotoxicity (ADCC) are poorly exposed on the unliganded envelope trimer. Here we investigated the mechanism of exposure of these epitopes and found that envelope interaction with the HIV-1 CD4 receptor is required to expose some of these epitopes. Moreover, our results suggest that HIV-1 CD4 downregulation might help avoid the killing of HIV-1-infected cells by this immune mechanism.",
"The type I interferon-inducible factor tetherin retains virus particles on the surfaces of cells infected with vpu-deficient human immunodeficiency virus type 1 (HIV-1). While this mechanism inhibits cell-free viral spread, the immunological implications of tethered virus have not been investigated. We found that surface tetherin expression increased the antibody opsonization of vpu-deficient HIV-infected cells. The absence of Vpu also stimulated NK cell-activating FcγRIIIa signaling and enhanced NK cell degranulation and NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). The deletion of vpu in HIV-1-infected primary CD4(+) T cells enhanced the levels of antibody binding and Fc receptor signaling mediated by HIV-positive-patient-derived antibodies. The magnitudes of antibody binding and Fc signaling were both highly correlated to the levels of tetherin on the surfaces of infected primary CD4 T cells. The affinity of antibody binding to FcγRIIIa was also found to be critical in mediating efficient Fc activation. These studies implicate Vpu antagonism of tetherin as an ADCC evasion mechanism that prevents antibody-mediated clearance of virally infected cells. The ability of the HIV-1 accessory factor to antagonize tetherin has been considered to primarily function by limiting the spread of virus by preventing the release of cell-free virus. This study supports the hypothesis that a major function of Vpu is to decrease the recognition of infected cells by anti-HIV antibodies at the cell surface, thereby reducing recognition by antibody-dependent clearance by natural killer cells.",
"Carriage of the natural killer (NK) receptor genotype KIR3DL1*h/*y with its HLA-B*57 ligand (*h/*y+B*57) is associated with slow time to AIDS and low viral load (VL). To provide a functional basis for these epidemiological observations, we assessed whether HIV-1-infected slow progressors (SP) carrying the *h/*y+B*57 compound genotype would have increased NK cell polyfunctional potential in comparison to SP with other killer immunoglobulin-like receptor (KIR)/HLA compound genotypes and whether this enhanced polyfunctionality was dependent upon the coexpression of both KIR3DL1*h/*y and HLA-B*57. The functional potential of NK cells was investigated by stimulating peripheral blood mononuclear cells with HLA-devoid targets or single HLA transfectants. Multiparametric flow cytometry was used to detect NK cells with seven functional profiles representing all permutations of CD107a expression and gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) secretion. NK cells from individuals carrying KIR3DL1 receptor-HLA-Bw4 ligand pairs had greater trifunctional responses than those from KIR3DL1 homozygotes (hmz), who were Bw6 homozygotes. NK cells from subjects carrying the *h/*y+B*57 genotypes exhibited the highest trifunctional potential, and this was dependent on cocarriage of the NK receptor and its ligand. Trifunctional cells secreted more of each function tested on a per-cell basis than each corresponding monofunctional NK subset. Although VL influenced NK functionality, individuals with defined KIR/HLA genotypes exhibited differences in NK cell polyfunctionality that could not be accounted for by VL alone. The protective effect of HLA-B*57 on slow progression to AIDS and low VL may be mediated through its interaction with KIR3DL1 alleles to educate NK cells for potent activity upon stimulation."
] |
Laplacian Regularized Sparse Subspace Learning for MiRNA-Disease Association Prediction
|
Predicting novel microRNA (miRNA)-disease associations is clinically significant due to miRNAs' potential roles of diagnostic biomarkers and therapeutic targets for various human diseases. Previous studies have demonstrated the viability of utilizing different types of biological data to computationally infer new disease-related miRNAs. Yet researchers face the challenge of how to effectively integrate diverse datasets and make reliable predictions. In this study, we presented a computational model named Laplacian Regularized Sparse Subspace Learning for MiRNA-Disease Association prediction (LRSSLMDA), which projected miRNAs/diseases' statistical feature profile and graph theoretical feature profile to a common subspace. It used Laplacian regularization to preserve the local structures of the training data and a L1-norm constraint to select important miRNA/disease features for prediction. The strength of dimensionality reduction enabled the model to be easily extended to much higher dimensional datasets than those exploited in this study. Experimental results showed that LRSSLMDA outperformed ten previous models: the AUC of 0.9178 in global leave-one-out cross validation (LOOCV) and the AUC of 0.8418 in local LOOCV indicated the model's superior prediction accuracy; and the average AUC of 0.9181+/-0.0004 in 5-fold cross validation justified its accuracy and stability. In addition, three types of case studies further demonstrated its predictive power. Potential miRNAs related to Colon Neoplasms, Lymphoma, Kidney Neoplasms, Esophageal Neoplasms and Breast Neoplasms were predicted by LRSSLMDA. Respectively, 98%, 88%, 96%, 98% and 98% out of the top 50 predictions were validated by experimental evidences. Therefore, we conclude that LRSSLMDA would be a valuable computational tool for miRNA-disease association prediction.
|
[
"It has been reported that increasingly microRNAs are associated with diseases. However, the patterns among the microRNA-disease associations remain largely unclear. In this study, in order to dissect the patterns of microRNA-disease associations, we performed a comprehensive analysis to the human microRNA-disease association data, which is manually collected from publications. We built a human microRNA associated disease network. Interestingly, microRNAs tend to show similar or different dysfunctional evidences for the similar or different disease clusters, respectively. A negative correlation between the tissue-specificity of a microRNA and the number of diseases it associated was uncovered. Furthermore, we observed an association between microRNA conservation and disease. Finally, we uncovered that microRNAs associated with the same disease tend to emerge as predefined microRNA groups. These findings can not only provide help in understanding the associations between microRNAs and human diseases but also suggest a new way to identify novel disease-associated microRNAs.",
"Recent work has revealed the existence of a class of small non-coding RNA species, known as microRNAs (miRNAs), which have critical functions across various biological processes. Here we use a new, bead-based flow cytometric miRNA expression profiling method to present a systematic expression analysis of 217 mammalian miRNAs from 334 samples, including multiple human cancers. The miRNA profiles are surprisingly informative, reflecting the developmental lineage and differentiation state of the tumours. We observe a general downregulation of miRNAs in tumours compared with normal tissues. Furthermore, we were able to successfully classify poorly differentiated tumours using miRNA expression profiles, whereas messenger RNA profiles were highly inaccurate when applied to the same samples. These findings highlight the potential of miRNA profiling in cancer diagnosis.",
"Recently, microRNAs (miRNA), small noncoding RNAs, have taken center stage in the field of human molecular oncology. However, their roles in tumor biology remain largely unknown. According to the assumption that miRNAs implicated in a specific tumor phenotype will show aberrant regulation of their target genes, we introduce an approach based on the miRNA target-dysregulated network (MTDN) to prioritize novel disease miRNAs. Target genes have predicted binding sites for any miRNA. The MTDN is constructed by combining computational target prediction with miRNA and mRNA expression profiles in tumor and nontumor tissues. Application of the proposed method to prostate cancer reveals that known prostate cancer miRNAs are characterized by a greater number of dysregulations and coregulators and the tendency to coregulate with each other and that they share a higher proportion of targets with other prostate cancer miRNAs. Support vector machine classifier, based on these features and changes in miRNA expression, is constructed and gives an average overall prediction accuracy of 0.8872 in cross-validation tests. The classifier is then applied to miRNAs in the MTDN. Functions enriched by dysregulated targets of novel predicted miRNAs are closely associated with oncogenesis. In addition, predicted cancer miRNAs within families or from different families show combinatorial dysregulation of target genes, as revealed by analysis of the MTDN modular organization. Finally, 3 miRNA target regulations are verified to hold in prostate cancer cells by transfection assays. These results show that the network-centric method could prioritize novel disease miRNAs and model how oncogenic lesions are mediated by miRNAs, providing important insights into tumorigenesis.",
"The aim of this study was to find a novel molecular network involved in renal cell carcinoma (RCC) development through investigating the functions of miR-1 and miR-133a and their target genes. We checked the expression levels of miR-1 and miR-133a in RCC cell lines and specimens (N=40) using real time RT-PCR. MiR-1 and miR-133a transfectants were subjected to a gain-of-function study to identify the functions of the miRNAs. To find the target genes of the miRNAs, we analysed the gene expression profile of their transfectants and performed a luciferase reporter assay. mRNA expression levels of the candidate target gene in the clinical specimens were examined, and loss-of-function studies were performed. The expression levels of miR-1 and miR-133a were significantly suppressed in RCC cell lines and specimens. Ectopic restoration of miR-1 and miR-133a showed significant inhibition of cell proliferation and invasion, and moreover, revealed induction of apoptosis and cell cycle arrest. The luciferase assay revealed transgelin-2 (TAGLN2), selected as a target gene for miR-1 and miR-133a on the basis of the gene expression profile, to be directly regulated by both miR-1 and miR-133a. The loss-of-function studies showed significant inhibitions of cell proliferation and invasion in the si-TAGLN2 transfectant. The expression level of TAGLN2 mRNA was significantly up-regulated in the RCC specimens; in addition, there was a statistically significant inverse correlation between TAGLN2 and miR-1 and miR-133a expression. Our data indicate that up-regulation of the oncogenic TAGLN2 was due to down-regulation of tumour-suppressive miR-1 and miR-133a in human RCC.",
"MicroRNAs (miRNAs) are endogenous noncoding RNAs, about 22 nucleotides in length, that mediate post-transcriptional gene silencing by annealing to inexactly complementary sequences in the 3'-untranslated regions of target mRNAs. Our current understanding of the functions of miRNAs relies mainly on their tissue-specific or developmental stage-dependent expression and their evolutionary conservation, and therefore is primarily limited to their involvement in developmental regulation and oncogenesis. Of more than 300 miRNAs that have been identified, miR-1 and miR-133 are considered to be muscle specific. Here we show that miR-1 is overexpressed in individuals with coronary artery disease, and that when overexpressed in normal or infarcted rat hearts, it exacerbates arrhythmogenesis. Elimination of miR-1 by an antisense inhibitor in infarcted rat hearts relieved arrhythmogenesis. miR-1 overexpression slowed conduction and depolarized the cytoplasmic membrane by post-transcriptionally repressing KCNJ2 (which encodes the K(+) channel subunit Kir2.1) and GJA1 (which encodes connexin 43), and this likely accounts at least in part for its arrhythmogenic potential. Thus, miR-1 may have important pathophysiological functions in the heart, and is a potential antiarrhythmic target.",
"Exosomal microRNAs (miRNAs) have been attracting major interest as potential diagnostic biomarkers of cancer. The aim of this study was to characterize the miRNA profiles of serum exosomes and to identify those that are altered in colorectal cancer (CRC). To evaluate their use as diagnostic biomarkers, the relationship between specific exosomal miRNA levels and pathological changes of patients, including disease stage and tumor resection, was examined. Microarray analyses of miRNAs in exosome-enriched fractions of serum samples from 88 primary CRC patients and 11 healthy controls were performed. The expression levels of miRNAs in the culture medium of five colon cancer cell lines were also compared with those in the culture medium of a normal colon-derived cell line. The expression profiles of miRNAs that were differentially expressed between CRC and control sample sets were verified using 29 paired samples from post-tumor resection patients. The sensitivities of selected miRNAs as biomarkers of CRC were evaluated and compared with those of known tumor markers (CA19-9 and CEA) using a receiver operating characteristic analysis. The expression levels of selected miRNAs were also validated by quantitative real-time RT-PCR analyses of an independent set of 13 CRC patients. The serum exosomal levels of seven miRNAs (let-7a, miR-1229, miR-1246, miR-150, miR-21, miR-223, and miR-23a) were significantly higher in primary CRC patients, even those with early stage disease, than in healthy controls, and were significantly down-regulated after surgical resection of tumors. These miRNAs were also secreted at significantly higher levels by colon cancer cell lines than by a normal colon-derived cell line. The high sensitivities of the seven selected exosomal miRNAs were confirmed by a receiver operating characteristic analysis. Exosomal miRNA signatures appear to mirror pathological changes of CRC patients and several miRNAs are promising biomarkers for non-invasive diagnosis of the disease.",
"MicroRNAs circulating in the blood, stabilized by complexation with proteins and/or additionally by encapsulation in lipid vesicles, are currently being evaluated as biomarkers. The consequences of their differential association with lipids/vesicles for their stability and use as biomarkers are largely unexplored and are subject of the present study. The levels of a set of selected microRNAs were determined by quantitative reverse-transcription PCR after extraction from sera or vesicle- and non-vesicle fractions prepared from sera. The stability of these microRNAs after incubation with RNase A or RNase inhibitor, an inhibitor of RNase A family enzymes was studied. The levels of microRNA-1 and microRNA-122, but not those of microRNA-16, microRNA-21 and microRNA-142-3p, declined significantly during a 5-h incubation of the sera. RNase inhibitor prevented the loss of microRNAs in serum as well as the degradation of microRNA-122, a microRNA not expressed in blood cells, in whole blood. Stabilization of microRNA-122 was also achieved by hemolysis. Prolonged incubation of the sera led to enrichment of vesicle-associated relative to non-vesicle-associated microRNAs. Vesicle-associated microRNAs were more resistant to RNase A treatment than the respective microRNAs not associated with vesicles. Serum microRNAs showed differential stability upon prolonged incubation. RNase inhibitor might be useful to robustly preserve the pattern of cell-free circulating microRNAs. In the case of microRNAs not expressed in blood cells this can also be achieved by hemolysis. Vesicle-associated microRNAs appeared to be more stable than those not associated with vesicles, which might be useful to disclose additional biomarker properties of miRNAs."
] |
Fluorescence-RBT: Simultaneous Measurements of Molecular Dynamics
|
Single-molecule methods provide direct measurements of macromolecular dynamics, but are limited by the number of degrees of freedom that can be followed at one time. High-resolution rotor bead tracking (RBT) measures DNA torque, twist, and extension, and can be used to characterize the structural dynamics of DNA and diverse nucleoprotein complexes. Here, we extend RBT to enable simultaneous monitoring of additional degrees of freedom. Fluorescence-RBT (FluoRBT) combines magnetic tweezers, infrared evanescent scattering, and single-molecule FRET imaging, providing real-time multiparameter measurements of complex molecular processes. We demonstrate the capabilities of FluoRBT by conducting simultaneous measurements of extension and FRET during opening and closing of a DNA hairpin under tension, and by observing simultaneous changes in FRET and torque during a transition between right-handed B-form and left-handed Z-form DNA under controlled supercoiling. We discover unanticipated continuous changes in FRET with applied torque, and also show how FluoRBT can facilitate high-resolution FRET measurements of molecular states, by using a mechanical signal as an independent temporal reference for aligning and averaging noisy fluorescence data. By combining mechanical measurements of global DNA deformations with FRET measurements of local conformational changes, FluoRBT will enable multidimensional investigations of systems ranging from DNA structures to large macromolecular machines.
|
[
"We study the effect of dye-dye interactions in labeled double-stranded DNA molecules on the Förster resonance energy transfer (FRET) efficiency at the single-molecule level. An extensive analysis of internally labeled double-stranded DNA molecules in bulk and at the single-molecule level reveals that donor-acceptor absolute distances can be reliably extracted down to approximately 3-nm separation, provided that dye-dye quenching is accounted for. At these short separations, we find significant long-lived fluorescence fluctuations among discrete levels originating from the simultaneous and synchronous quenching of both dyes. By comparing four different donor-acceptor dye pairs (TMR-ATTO647N, Cy3-ATTO647N, TMR-Cy5, and Cy3-Cy5), we find that this phenomenon depends on the nature of the dye pair used, with the cyanine pair Cy3-Cy5 showing the least amount of fluctuations. The significance of these results is twofold: First, they illustrate that when dye-dye quenching is accounted for, single-molecule FRET can be used to accurately measure inter-dye distances, even at short separations. Second, these results are useful when deciding which dye pairs to use for nucleic acids analyses using FRET.",
"The technically challenging field of single-molecule biophysics has established itself in the last decade by granting access to detailed information about the fate of individual biomolecules, unattainable in traditional biochemical assays. The appeal of single-molecule methods lies in the directness of the information obtained from individual biomolecules. Technological improvements in single-molecule methods have made it possible to combine optical tweezers, fluorescence microscopy, and microfluidic flow systems. Such a combination of techniques has opened new possibilities to study complex biochemical reactions on the single-molecule level. In this chapter, we provide general considerations for the development of a combined optical trapping, fluorescence microscopy, and microfluidics instrument, along with methods to solve technical issues that are critical for designing successful experiments. Finally, we present several experiments to illustrate the power of this combination of techniques.",
"Changes in global DNA linking number can be accommodated by localized changes in helical structure. We have used single-molecule torque measurements to investigate sequence-specific strand separation and Z-DNA formation. By controlling the boundary conditions at the edges of sequences of interest, we have confirmed theoretical predictions of distinctive boundary-dependent backbending patterns in torque-twist relationships. Abrupt torque jumps are associated with the formation and collapse of DNA bubbles, permitting direct observations of DNA breathing dynamics.",
"While the statistical mechanical description of DNA has a long tradition, renewed interest in DNA melting from a physics perspective is nourished by measurements of the fluctuation dynamics of local denaturation bubbles by single molecule spectroscopy. The dynamical opening of DNA bubbles (DNA breathing) is supposedly crucial for biological functioning during, for instance, transcription initiation and DNA's interaction with selectively single-stranded DNA binding proteins. Motivated by this, we consider the bubble breathing dynamics in a heteropolymer DNA based on a (2+1)-variable master equation and complementary stochastic Gillespie simulations, providing the bubble size and the position of the bubble along the sequence as a function of time. We utilize new experimental data that independently obtain stacking and hydrogen bonding contributions to DNA stability. We calculate the spectrum of relaxation times and the experimentally measurable autocorrelation function of a fluorophore-quencher tagged basepair, and demonstrate good agreement with fluorescence correlation experiments. A significant dependence of opening probability and waiting time between bubble events on the local DNA sequence is revealed and quantified for a promoter sequence of the T7 phage. The strong dependence on sequence, temperature and salt concentration for the breathing dynamics of DNA found here points at a good potential for nanosensing applications by utilizing short fluorophore-quencher dressed DNA constructs.",
"Despite its fundamental importance in cellular processes and abundant use in biotechnology, we lack a detailed understanding of the kinetics of nucleic acid hybridization. In particular, the identity of the transition state, which determines the kinetics of the two-state reaction, remains poorly characterized. Here, we used optical tweezers with single-molecule fluorescence to observe directly the binding and unbinding of short oligonucleotides (7-12 nt) to a complementary strand held under constant force. Binding and unbinding rate constants measured across a wide range of forces (1.5-20 pN) deviate from the exponential force dependence expected from Bell's equation. Using a generalized force dependence model, we determined the elastic behavior of the transition state, which we find to be similar to that of the pure single-stranded state. Our results indicate that the transition state for hybridization is visited before the strands form any significant amount of native base pairs. Such a transition state supports a model in which the rate-limiting step of the hybridization reaction is the alignment of the two strands prior to base pairing.",
"Riboswitches regulate genes through structural changes in ligand-binding RNA aptamers. With the use of an optical-trapping assay based on in situ transcription by a molecule of RNA polymerase, single nascent RNAs containing pbuE adenine riboswitch aptamers were unfolded and refolded. Multiple folding states were characterized by means of both force-extension curves and folding trajectories under constant force by measuring the molecular contour length, kinetics, and energetics with and without adenine. Distinct folding steps correlated with the formation of key secondary or tertiary structures and with ligand binding. Adenine-induced stabilization of the weakest helix in the aptamer, the mechanical switch underlying regulatory action, was observed directly. These results provide an integrated view of hierarchical folding in an aptamer, demonstrating how complex folding can be resolved into constituent parts, and supply further insights into tertiary structure formation.",
"Preservation of genetic information in DNA relies on shielding the nucleobases from damage within the double helix. Thermal fluctuations lead to infrequent events of the Watson-Crick basepair opening, or DNA \"breathing\", thus making normally buried groups available for modification and interaction with proteins. Fluctuational basepair opening implies the disruption of hydrogen bonds between the complementary bases and flipping of the base out of the helical stack. Prediction of sequence-dependent basepair opening probabilities in DNA is based on separation of the two major contributions to the stability of the double helix: lateral pairing between the complementary bases and stacking of the pairs along the helical axis. The partition function calculates the basepair opening probability at every position based on the loss of two stacking interactions and one base-pairing. Our model also includes a term accounting for the unfavorable positioning of the exposed base, which proceeds through a formation of a highly constrained small loop, or a ring. Quantitatively, the ring factor is found as an adjustable parameter from the comparison of the theoretical basepair opening probabilities and the experimental data on short DNA duplexes measured by NMR spectroscopy. We find that these thermodynamic parameters suggest nonobvious sequence dependent basepair opening probabilities.",
"We have developed a technique that allows mechanical and ligand-binding events in a single myosin molecule to be monitored simultaneously. We describe how steps in the ATPase reaction are temporally related to mechanical events at the single molecule level. The results show that the force generation does not always coincide with the release of bound nucleotide, presumably ADP. Instead the myosin head produces force several hundreds of milliseconds after bound nucleotide is released. This finding does not support the widely accepted view that force generation is directly coupled to the release of bound ligands. It suggests that myosin has a hysteresis or memory state, which stores chemical energy from ATP hydrolysis.",
"Bacteria make extensive use of riboswitches to sense metabolites and control gene expression, and typically do so by modulating premature transcription termination or translation initiation. The most widespread riboswitch class known in bacteria responds to the coenzyme thiamine pyrophosphate (TPP), which is a derivative of vitamin B1. Representatives of this class have also been identified in fungi and plants, where they are predicted to control messenger RNA splicing or processing. We examined three TPP riboswitches in the filamentous fungus Neurospora crassa, and found that one activates and two repress gene expression by controlling mRNA splicing. A detailed mechanism involving riboswitch-mediated base-pairing changes and alternative splicing control was elucidated for precursor NMT1 mRNAs, which code for a protein involved in TPP metabolism. These results demonstrate that eukaryotic cells employ metabolite-binding RNAs to regulate RNA splicing events that are important for the control of key biochemical processes.",
"The thiamine pyrophosphate (TPP) riboswitch is a cis-regulatory element in mRNA that modifies gene expression in response to TPP concentration. Its specificity is dependent upon conformational changes that take place within its aptamer domain. Here, the role of tertiary interactions in ligand binding was studied at the single-molecule level by combined force spectroscopy and Förster resonance energy transfer (smFRET), using an optical trap equipped for simultaneous smFRET. The 'Force-FRET' approach directly probes secondary and tertiary structural changes during folding, including events associated with binding. Concurrent transitions observed in smFRET signals and RNA extension revealed differences in helix-arm orientation between two previously-identified ligand-binding states that had been undetectable by spectroscopy alone. Our results show that the weaker binding state is able to bind to TPP, but is unable to form a tertiary docking interaction that completes the binding process. Long-range tertiary interactions stabilize global riboswitch structure and confer increased ligand specificity."
] |
Human Charcot-Marie-Tooth disease type 1A fibroblasts exhibit a phenotype similar to Schwann cells from neuropathic mice
|
A common form of hereditary autosomal dominant demyelinating neuropathy known as Charcot-Marie-Tooth disease type 1A (CMT1A) is linked with duplication of the peripheral myelin protein 22 (PMP22) gene. Although studies from animal models have led to better understanding of the pathobiology of these neuropathies, there continues to be a gap in the translation of findings from rodents to humans. Because PMP22 was originally identified in fibroblasts as growth arrest specific gene 3 (gas3) and is expressed broadly in the body, it was tested whether skin cells from neuropathic patients would display the cellular pathology observed in Schwann cells from rodent models. Dermal fibroblasts from two CMT1A pedigrees with confirmed PMP22 gene duplication were studied. Samples from age-matched non-neuropathic individuals were used as controls. CMT1A patient-derived cultures contain approximately 1.5-fold elevated levels of PMP22 mRNA, exhibit reduced mitotic potential, and display intracellular protein aggregates as compared to cells from unaffected individuals. The presence of cytosolic PMP22 coincides with a decrease in proteasome activity and an increase in autophagy-lysosomal proteins, including LC3-II and LAMP1. These results indicate that the abnormalities in the subcellular processing of excess PMP22 elicit a detectable response in human CMT1A fibroblasts, a phenotype that resembles Schwann cells from neuropathic mice. These findings support the use of human CMT1A fibroblasts as a platform for therapy testing.
|
[
"Charcot-Marie-Tooth disease type 1A (CMT1A) is caused by a 1.4 Mb duplication on chromosome 17p11.2, which contains the peripheral myelin protein-22 (PMP22) gene. Increased levels of PMP22 in compact myelin of peripheral nerves have been demonstrated and presumed to cause the phenotype of CMT1A. The objective of the present study was to determine whether an extra copy of the PMP22 gene in CMT1A disrupts the normally coordinated expression of PMP22 protein in peripheral nerve myelin and to evaluate PMP22 over-expression in patients with CMT1A and determine whether levels of PMP22 are molecular markers of disease severity. PMP22 expression was measured by taking skin biopsies from patients with CMT1A (n = 20) and both healthy controls (n = 7) and patients with Hereditary Neuropathy with liability to Pressure Palsies (HNPP) (n = 6), in which patients have only a single copy of PMP22. Immunological electron microscopy was performed on the skin biopsies to quantify PMP22 expression in compact myelin. Similar biopsies were analysed by real time PCR to measure PMP22 mRNA levels. Results were also correlated with impairment in CMT1A, as measured by the validated CMT Neuropathy Score. Most, but not all patients with CMT1A, had elevated PMP22 levels in myelin compared with the controls. The levels of PMP22 in CMT1A were highly variable, but not in HNPP or the controls. However, there was no correlation between neurological disabilities and the level of over-expression of PMP22 protein or mRNA in patients with CMT1A. The extra copy of PMP22 in CMT1A results in disruption of the tightly regulated expression of PMP22. Thus, variability of PMP22 levels, rather than absolute level of PMP22, may play an important role in the pathogenesis of CMT1A.",
"Peripheral myelin protein 22 (PMP22) is an integral membrane protein that is essential for the normal formation and maintenance of peripheral myelin. Duplications, deletions, or mutations in the PMP22 gene account for a set of dominantly inherited peripheral neuropathies. The heterozygous Trembler-J (TrJ) genotype in mice is similar genetically to a Charcot-Marie-Tooth disease type 1A pedigree in humans, whereas the homozygous TrJ condition leads to the most severe form of PMP22-associated neuropathies. To characterize the consequences of the TrJ mutation, we labeled wild-type (wt-) and TrJ-PMP22 in the third loop of the protein with different epitope tags and expressed them separately or together in COS7 cells and primary Schwann cells. Here we show that the transport of the mutant TrJ-PMP22 is interrupted in the intermediate compartment, preventing its insertion into the plasma membrane and affecting the morphology of the endoplasmic reticulum. In addition, TrJ-PMP22 forms a heterodimer with the wt-PMP22. This interaction causes a fraction of the wt-PMP22 to be retained with TrJ-PMP22 in the intermediate compartment of COS7 and Schwann cells. The relative stability of a wt-mutant PMP22 heterodimer as compared with the wt-wt PMP22 homodimer may determine whether a particular mutation is semidominant or dominant. The neuropathy itself appears to result both from decreased trafficking of wt-PMP22 to the plasma membrane and from a toxic gain of function via the accumulation of wt- and TrJ-PMP22 in the intermediate compartment.",
"Although the Gas3/PMP22 protein is expressed at highest levels in differentiated Schwann cells, its presence, albeit at lower levels, in non-neuronal tissues and in NIH-3T3 growth-arrested fibroblasts argues for a more general function of this protein that is uncoupled to myelin structure. We show that gas3/PMP22 overexpression in NIH-3T3 growing cells leads to an apoptotic-like phenotype, which is suppressed by antioxidants and characterized by typical membrane blebbing, rounding up, and chromatin condensation, but with no evidence of DNA fragmentation. REF-52 fibroblasts seem to be completely refractive to gas3/PMP22 overexpression. Recently, several point mutations of the human gas3/PMP22 gene have been associated with Charcot-Marie-Tooth type 1A (CMT1A), a common hereditary demyelinating neuropathy. When gas3/PMP22 point mutations (L16P, S79C, T118M, and G150D) are similarly overexpressed in NIH-3T3 cells, the induced apoptotic-like phenotype as compared to the wild-type is significantly reduced. Both of the dominant mutations (L16P, S79C) for CMT1A behave as dominant negatives with respect to the wild type, whereas T118M, the only recessive mutant described, behaves as recessive under the same coexpression experiments. These data suggest a role for altered Schwann cell apoptosis in the pathogenesis of CMT1A.",
"Charcot-Marie-Tooth disease type 1A (CMT1A) is a hereditary demyelinating neuropathy linked with duplication of the peripheral myelin protein 22 (PMP22) gene. Transgenic C22 mice, a model of CMT1A, display many features of the human disease, including slowed nerve conduction velocity and demyelination of peripheral nerves. How overproduction of PMP22 leads to compromised myelin and axonal pathology is not fully understood, but likely involves subcellular alterations in protein homoeostatic mechanisms within affected Schwann cells. The subcellular response to abnormally localized PMP22 includes the recruitment of the ubiquitin-proteasome system (UPS), autophagosomes and heat-shock proteins (HSPs). Here we assessed biochemical markers of these protein homoeostatic pathways in nerves from PMP22-overexpressing neuropathic mice between the ages of 2 and 12 months to ascertain their potential contribution to disease progression. In nerves of 3-week-old mice, using endoglycosidases and Western blotting, we found altered processing of the exogenous human PMP22, an abnormality that becomes more prevalent with age. Along with the ongoing accrual of misfolded PMP22, the activity of the proteasome becomes compromised and proteins required for autophagy induction and lysosome biogenesis are up-regulated. Moreover, cytosolic chaperones are consistently elevated in nerves from neuropathic mice, with the most prominent change in HSP70. The gradual alterations in protein homoeostatic response are accompanied by Schwann cell de-differentiation and macrophage infiltration. Together, these results show that while subcellular protein quality control mechanisms respond appropriately to the presence of the overproduced PMP22, with aging they are unable to prevent the accrual of misfolded proteins.",
"A set of growth arrest-specific (gas) genes whose expression is negatively regulated by serum has recently been identified. We report on the detailed analysis of one of these genes (gas3). The kinetics of regulation by the presence and absence of serum were investigated, and it was found that this gene is regulated at the post-transcriptional level. The encoded protein deduced from the nucleotide sequence showed some similarity to a mitochondrial oxyreductase, and in vitro translation established that the protein product is a transmembrane glycoprotein.",
"Charcot-Marie-Tooth disease type 1A (CMT1A) is associated with increased gene dosage for PMP22. Therapeutic approaches are currently aiming at correcting PMP22 over-expression. It is unknown whether PMP22 can be used as a biological marker of disease progression and therapy efficacy. We performed quantitative real-time polymerase chain reaction on skin biopsies of 45 patients with CMT1A, obtained at study entry and after 24-months of treatment either with ascorbic acid or placebo. Data of a subgroup of patients were also compared with matched healthy subjects. Finally, we analysed PMP22 messenger RNA levels in sural nerve biopsies. We did not find significant differences in the levels of any known PMP22 transcripts in treated or untreated patients with CMT1A, thus confirming that ascorbic acid does not impact on the molecular features of CMT1A. Most importantly, we did not observe any correlation between PMP22 messenger RNA levels and the different clinical and electrophysiological outcome measures, underscoring the weakness of PMP22 to mirror the phenotypic variability of patients with CMT1A. We did not find increased PMP22 messenger RNA levels in skin and sural nerve biopsies of patients with CMT1A compared with relative controls. In conclusion, this study shows that ascorbic acid does not impact on PMP22 transcriptional regulation and PMP22 is not a suitable biomarker for CMT1A.",
"Peripheral myelin protein 22 (PMP22) is a tetraspan membrane glycoprotein, the misexpression of which is associated with hereditary demyelinating neuropathies. Myelinating Schwann cells (SCs) produce the highest levels of PMP22, yet the function of the protein in peripheral nerve biology is unresolved. To investigate the potential roles of PMP22, we engineered a novel knock-out (-/-) mouse line by replacing the first two coding exons of pmp22 with the lacZ reporter. PMP22-deficient mice show strong beta-galactosidase reactivity in peripheral nerves, cartilage, intestines, and lungs, whereas phenotypically they display the characteristics of tomaculous neuropathy. In the absence of PMP22, myelination of peripheral nerves is delayed, and numerous axon-SC profiles show loose basal lamina, suggesting altered interactions of the glial cells with the extracellular matrix. The levels of beta4 integrin, a molecule involved in the linkage between SCs and the basal lamina, are severely reduced in nerves of PMP22-deficient mice. During early stages of myelination, PMP22 and beta4 integrin are coexpressed at the cell surface and can be coimmunoprecipitated together with laminin and alpha6 integrin. In agreement, in clone A colonic carcinoma cells, epitope-tagged PMP22 forms a complex with beta4 integrin. Together, these data indicate that PMP22 is a binding partner in the integrin/laminin complex and is involved in mediating the interaction of SCs with the extracellular environment."
] |
NAMPT is a novel therapeutic target in Glioma progression and relapse
|
Gliomas are the most prevalent primary malignant brain tumors associated with poor prognosis. NAMPT, a rate-limiting enzyme that boosts the nicotinamide adenine dinucleotide (NAD) regeneration in the salvage pathway, is commonly expressed in these tumors. NAD metabolism is required to maintain tissue homeostasis. To maintain metabolism, cancer cells require a stable NAD regeneration circuit. However, high levels of NAD confer resistance to therapy to these tumors, usually treated with Temozolomide (TMZ). We report that NAMPT overexpression in glioma cell lines increases tumorigenic properties controlling stem cell pathways and enriching the cancer-initiating cell (CIC) population. Furthermore, NAMPT expression correlated with high levels of Nanog, CD133 and CIC-like cells in glioblastoma directly extracted from patients. Meta-analysis reveals that NAMPT is also a key factor inducing cancer stem pathways in glioma cells. Furthermore, we report a novel NAMPT-driven signature which stratify prognosis within tumor staging. NAMPT signature also correlates directly with EGFR positive and IDH negative tumors. Finally, NAMPT inhibition increases sensitivity to apoptosis in both NAMPT-expressing cells and tumorspheres. Therefore, NAMPT represents a novel therapeutic target in Glioma progression and relapse.
|
[
"Colony-forming human epidermal cells are heterogeneous in their capacity for sustained growth. Once a clone has been derived from a single cell, its growth potential can be estimated from the colony types resulting from a single plating, and the clone can be assigned to one of three classes. The holoclone has the greatest reproductive capacity: under standard conditions, fewer than 5% of the colonies formed by the cells of a holoclone abort and terminally differentiate. The paraclone contains exclusively cells with a short replicative lifespan (not more than 15 cell generations), after which they uniformly abort and terminally differentiate. The third type of clone, the meroclone, contains a mixture of cells of different growth potential and is a transitional stage between the holoclone and the paraclone. The incidence of the different clonal types is affected by aging, since cells originating from the epidermis of older donors give rise to a lower proportion of holoclones and a higher proportion of paraclones.",
"Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step in NAD synthesis and is up-regulated in several human malignancies, including breast, colon, prostate, thyroid, gastric, and several hematopoietic malignancies. In some malignancies, such as gastric, thyroid, and prostate carcinomas, higher NAMPT expression correlates with deeper tumor invasion, increased metastatic potential and chemotherapy resistance. We employed tissue microarray immunohistochemistry to examine NAMPT expression in benign skeletal and smooth muscle, leiomyomas, leiomyosarcomas (graded low-, intermediate-, and high-grade), and spindle, embryonal, pleomorphic, and alveolar rhabdomyosarcomas. We found low to intermediate NAMPT expression in benign tissue, leiomyomas, leiomyosarcomas (low- and intermediate-grades), and spindle cell rhabdomyosarcomas. In contrast, high-grade leiomyosarcomas and embryonal, alveolar, and pleomorphic rhabdomyosarcomas showed high NAMPT expression. Herein we show for the first time that NAMPT is overexpressed in certain sarcoma types and the level of NAMPT expression correlates with tumor behavior.",
"IDH1-mutant gliomas are dependent upon the canonical coenzyme NAD+ for survival. It is known that PARP activation consumes NAD+ during base excision repair (BER) of chemotherapy-induced DNA damage. We therefore hypothesized that a strategy combining NAD+ biosynthesis inhibitors with the alkylating chemotherapeutic agent temozolomide could potentiate NAD+ depletion-mediated cytotoxicity in mutant IDH1 cancer cells. To investigate the impact of temozolomide on NAD+ metabolism, patient-derived xenografts and engineered mutant IDH1-expressing cell lines were exposed to temozolomide, in vitro and in vivo, both alone and in combination with nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, which block NAD+ biosynthesis. The acute time period (<3 hours) after temozolomide treatment displayed a burst of NAD+ consumption driven by PARP activation. In IDH1-mutant-expressing cells, this consumption reduced further the abnormally lowered basal steady-state levels of NAD+, introducing a window of hypervulnerability to NAD+ biosynthesis inhibitors. This effect was selective for IDH1-mutant cells and independent of methylguanine methyltransferase or mismatch repair status, which are known rate-limiting mediators of adjuvant temozolomide genotoxic sensitivity. Combined temozolomide and NAMPT inhibition in an in vivo IDH1-mutant cancer model exhibited enhanced efficacy compared with each agent alone. Thus, we find IDH1-mutant cancers have distinct metabolic stress responses to chemotherapy-induced DNA damage and that combination regimens targeting nonredundant NAD+ pathways yield potent anticancer efficacy in vivo Such targeting of convergent metabolic pathways in genetically selected cancers could minimize treatment toxicity and improve durability of response to therapy. Cancer Res; 77(15); 4102-15. ©2017 AACR.",
"Sclerosing and spindle cell rhabdomyosarcoma (RMS) are rare types of RMS recently reclassified as a stand-alone pathologic entity, separate from embryonal RMS (ERMS). Although sclerosing and spindle cell RMS share clinical and morphologic features, a pathogenetic link based on shared molecular alterations has not been established. Spindle cell RMS in children have been associated with a less aggressive clinical course compared to adults. Recently, recurrent MYOD1 mutations were described in 44% of adult spindle cell RMS, but no pediatric tumors or sclerosing RMS were studied for comparison. Thus, we investigated 16 RMS (5 sclerosing and 11 spindle cell) in children and adults for the presence of MYOD1 mutations by targeted Polymerase Chain Reaction (PCR). Remarkably, all 5 sclerosing RMS and 4 of 11 spindle cell RMS showed the MYOD1 p.L122R hot-spot mutation. Of the five pediatric tumors, 2/2 sclerosing RMS and 2/3 spindle cell RMS showed MYOD1 mutations. Three of nine MYOD1-mutant RMS showed coexistent PIK3CA mutations, while no MDM2 amplifications were identified. All four pediatric MYOD1-mutated RMS patients died of the disease at 12-35 months following diagnosis. In conclusion, spindle cell and sclerosing RMS show recurrent MYOD1 mutations, in keeping with a single pathologic entity, regardless of age at presentation. This group however, is distinct from the infantile RMS associated with NCOA2 fusions. Although our study suggests that pediatric MYOD1-mutant RMS follow an aggressive behavior with high mortality, further studies are required to confirm this finding.",
"Nicotinamide phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme in regenerating nicotinamide adenine dinucleotide (NAD(+)) from nicotinamide in mammals. NAMPT has crucial roles for many cellular functions by regulating NAD(+)-dependent SIRT1 deacetylase. However, roles of NAMPT in cancer are poorly defined. In this study, we show that NAMPT is prominently overexpressed in human prostate cancer cells along with SIRT1. Elevation of NAMPT expression occurs early for the prostate neoplasia. Inhibition of NAMPT significantly suppresses cell growth in culture, soft agar colony formation, cell invasion and growth of xenografted prostate cancer cells in mice. NAMPT knockdown sensitizes prostate cancer cells to oxidative stress caused by H(2)O(2) or chemotherapeutic treatment. Overexpression of NAMPT increases prostate cancer cell resistance to oxidative stress, which is partially blocked by SIRT1 knockdown. We demonstrate that in addition to modulating SIRT1 functions, the NAMPT inhibition reduces forkhead box, class 'O' (FOXO)3a protein expression and its downstream anti-oxidant genes catalase and manganese superoxide dismutase. Our results suggest important roles of concomitant upregulation of NAMPT and SIRT1 along with increased FOXO3a protein level for prostate carcinogenesis and their contribution to oxidative stress resistance of prostate cancer cells. These findings may have implications for exploring the NAMPT pathway for prostate cancer prevention and treatment.",
"Nicotinamide phosphoribosyltransferase (NAMPT) is a promising anticancer target. Using high throughput screening system targeting NAMPT, we obtained a potent NAMPT inhibitor MS0 (China Patent ZL201110447488.9) with excellent in vitro activity (IC50 = 9.87 ± 1.15 nM) and anti-proliferative activity against multiple human cancer cell lines including stem-like cancer cells. Structure-activity relationship studies yielded several highly effective analogues. These inhibitors specifically bound NAMPT, rather than downstream NMNAT. We provided the first chemical case using cellular thermal shift assay to explain the difference between in vitro and cellular activity; MS7 showed best in vitro activity (IC50 = 0.93 ± 0.29 nM) but worst cellular activity due to poor target engagement in living cells. Site-directed mutagenesis studies identified important residues for NAMPT catalytic activity and inhibitor binding. The present findings contribute to deep understanding the action mode of NAMPT inhibitors and future development of NAMPT inhibitors as anticancer agents.",
"Stat3 has been classified as a proto-oncogene and constitutive Stat3 signaling appears to be involved in oncogenesis of human cancers. However, whether constitutive Stat3 signaling plays a role in the survival and growth of osteosarcomas, rhabdomyosarcomas, and soft-tissue sarcomas is still unclear. To examine whether Stat3 is activated in osteosarcomas, rhabdomyosarcomas and other soft-tissue sarcomas we analyzed sarcoma tissue microarray slides and sarcoma cell lines using immunohistochemistry and Western blot analysis, respectively, with a phospho-specific Stat3 antibody. To examine whether the activated Stat3 pathway is important for sarcoma cell growth and survival, adenovirus-mediated expression of a dominant-negative Stat3 (Y705F) and a small molecule inhibitor (termed STA-21) were used to inhibit constitutive Stat3 signaling in human sarcoma cell lines expressing elevated levels of Stat3 phosphorylation. Cell viability was determined by MTT assays and induction of apoptosis was analyzed by western blotting using antibodies that specifically recognize cleaved caspases-3, 8, and 9. Stat3 phosphorylation is elevated in 19% (21/113) of osteosarcoma, 27% (17/64) of rhabdomyosarcoma, and 15% (22/151) of other soft-tissue sarcoma tissues as well as in sarcoma cell lines. Expression of the dominant-negative Stat3 and treatment of STA-21 inhibited cell viability and growth and induced apoptosis through caspases 3, 8 and 9 pathways in human sarcoma cell lines expressing elevated levels of phosphorylated Stat3. This study demonstrates that Stat3 phosphorylation is elevated in human rhabdomyosarcoma, osteosarcomas and soft-tissue sarcomas. Furthermore, the activated Stat3 pathway is important for cell growth and survival of human sarcoma cells."
] |
Gender inequitability in health systems
|
This editorial discusses a collection of papers examining gender across a range of health policy and systems contexts, from access to services, governance, health financing, and human resources for health. The papers interrogate differing health issues and core health systems functions using a gender lens. Together they produce new knowledge on the multiple impacts of gender on health experiences and demonstrate the importance of gender analyses and gender sensitive interventions for promoting well-being and health systems strengthening. The findings from these papers collectively show how gender intersects with other axes of inequity within specific contexts to shape experiences of health and health seeking within households, communities and health systems; illustrate how gender power relations affect access to important resources; and demonstrate that gender norms, poverty and patriarchy interplay to limit women's choices and chances both within household interactions and within the health sector. Health systems researchers have a responsibility to promote the incorporation of gender analyses into their studies in order to inform more strategic, effective and equitable health systems interventions, programmes, and policies. Responding to gender inequitable systems, institutions, and services in this sector requires an 'all hands-on deck' approach. We cannot claim to take a 'people-centred approach' to health systems if the status quo continues.
|
[
"To examine associations between war experiences, mental health, and gender in a sample of male and female Sierra Leonean former child soldiers. A total of 273 former child soldiers (29% females) were assessed for depression and anxiety by using the Hopkins Symptoms Checklist, and for hostility, confidence, and prosocial attitudes by using an instrument developed for use with Sierra Leonean child soldiers. The former child soldiers had witnessed and perpetrated violence at largely comparable rates, although females experienced higher rates of rape (p < .0001). More females scored within clinical ranges for depression (p = .008) and anxiety (p < .0001). In multiple regression analyses, female gender was a significant predictor of lower levels of confidence but not of mental health problems. Children who perpetrated injury or killing reported greater levels of depression (p < .0001), anxiety (p < .0001), and hostility (p < .0001). Surviving rape was associated with increased anxiety (p < .05) and hostility (p < .05), in males. Surviving rape was also related to higher confidence levels (p < .05) and prosocial attitudes (p < .05). Male former child soldiers who lost caregivers were also more vulnerable to depression (p < .05) and anxiety (p < .05), strong and significant effects noted among male child soldiers. In our sample, female and male child soldiers experienced comparable levels of most war exposures. Female soldiers reported higher rates of rape and lower levels of adaptive outcomes. Toxic forms of violence (killing or injuring; rape) were associated with particularly poor outcomes. Although all boys and girls who experience rape and loss of caregivers are generally at risk for mental health problems, boys in our sample demonstrated increased vulnerability; these findings indicate a need for more inclusive mental health services.",
"Adaptive and adequately resourced health systems are necessary to achieve good health outcomes in post-conflict settings, however domains beyond the health system are also critical to ensure broader wellbeing. This paper focuses on the importance of psychosocial support services for adolescent girls in fragile contexts. Its starting point is that adolescence is a pivotal time in the life course but given the physical, cognitive and emotional changes triggered by the onset of puberty, it can also be a period of heightened sensitivity and vulnerability to trauma, social isolation, bullying by peers, a lack of supportive adults and gender-based and sexual violence. Our findings highlight why humanitarian and biomedical approaches in their current form are inadequate to address these complexities. Drawing on qualitative fieldwork (consisting of in-depth and key informant interviews as well as group discussions in Gaza, Liberia and Sri Lanka involving a total of 386 respondents across the three countries), we argue that going beyond biomedical approaches and considering the social determinants of health, including approaches to tackle discriminatory gendered norms and barriers to service access, are critical for achieving broader health and wellbeing. While all three case study countries are classified as post-conflict, the political economy dynamics vary with associated implications for experiences of psychosocial vulnerabilities and the service environment. The study concludes by reflecting on actions to address psychosocial vulnerabilities facing adolescent girls. These include: tailoring services to ensure gender and age-sensitivity; investing in capacity building of service providers to promote service uptake; and enhancing strategies to regulate and coordinate actors providing mental health and psychosocial support services."
] |
Classification of cancer, miRNA, and mRNA expression profiles using multiobjective optimization and stack-based ensemble
|
MicroRNA (miRNA) plays vital roles in biological processes like RNA splicing and regulation of gene expression. Studies have revealed that there might be possible links between oncogenesis and expression profiles of some miRNAs, due to their differential expression between normal and tumor tissues. However, the automatic classification of miRNAs into different categories by considering the similarity of their expression values has rarely been addressed. This article proposes a solution framework for solving some real-life classification problems related to cancer, miRNA, and mRNA expression datasets. In the first stage, a multiobjective optimization based framework, non-dominated sorting genetic algorithm II, is proposed to automatically determine the appropriate classifier type, along with its suitable parameter and feature combinations, pertinent for classifying a given dataset. In the second page, a stack-based ensemble technique is employed to get a single combinatorial solution from the set of solutions obtained in the first stage. The performance of the proposed two-stage approach is evaluated on several cancer and RNA expression profile datasets. Compared to several state-of-the-art approaches for classifying different datasets, our method shows supremacy in the accuracy of classification.
|
[
"Over the last few years, microRNAs (miRNA)-controlled cancer stem cells have drawn enormous attention. Cancer stem cells are a small population of tumor cells that possess the stem cell property of self-renewal. Recent data shows that miRNA regulates this small population of stem cells. In the present review, we explained different characteristics of cancer stem cells as well as miRNA regulation of self-renewal and differentiation in cancer stem cells. We also described the migration and tumor formation. Finally, we described the different miRNAs that regulate various types of cancer stem cells, such as prostate cancer stem cells, head and neck cancer stem cells, breast cancer stem cells, colorectal cancer stem cells, lung cancer stem cells, gastric cancer stem cells, pancreatic cancer stem cells, etc. Extensive research is needed in order to employ miRNA-based therapeutics to control cancer stem cell population in various cancers in the future.",
"There has been an increased focus on the region adjacent to the lateral ventricles (LV) as a potential source of malignant tumors and/or more aggressive disease. We set out to determine if glioblastoma multiforme (GBM) bordering the LV was associated with decreased survival as compared to non-LV GBM. We reviewed the clinical records of 69 consecutive patients undergoing craniotomy for GBM at a single academic institution. Twenty-six patients were identified with contrast-enhancing lesions (CEL) bordering the LV (LV CEL). These 26 patients were matched with 26 patients with CEL not bordering the LV (non-LV CEL). These cohorts were matched for factors consistently shown to be associated with survival, which were age, tumor size, Karnofsky performance score, extent of resection, Gliadel implantation, and Temodar chemotherapy. Overall survival was compared between the cohorts via Log-rank analysis. Despite similarities in pre-operative clinical status, tumor size, peri-operative outcome, and treatment regimens, the median survival for patients with LV CEL was significantly decreased as compared to patients with non-LV CEL (8 months vs. 11 months), P = 0.02. Additionally, survival analysis in patients stratified by primary and secondary resection also demonstrated a strong trend towards decreased survival after resection of LV CEL. After primary and secondary resection, patients with LV CEL versus non-LV CEL had a median survival of 11 months vs. 14 months (P = 0.10) and 7 months vs. 10 months (P = 0.11), respectively. While the causal factors underlying this observation are not provided with this observational study, GBM bordering the LV may carry a prognostic significance.",
"MicroRNAs (miRNAs), have been shown to play important roles in gene regulation and various biological processes. Recent studies have revealed that abnormal expression of some specific miRNAs often results in the development of cancer. Microarray datasets containing the expression profiles of several miRNAs are being used for identification of miRNAs which are differentially expressed in normal and malignant tissue samples. In this article, a multiobjective feature selection approach is proposed for this purpose. The proposed method uses Genetic Algorithm for multiobjective optimization and support vector machine (SVM) classifier as a wrapper for evaluating the chromosomes that encode feature subsets. The performance has been demonstrated on real-life miRNA datasets for and the identified miRNA markers are reported. Moreover biological significance tests have been carried out for the obtained markers.",
"Cancers may arise from rare self-renewing tumor-initiating cells (T-IC). However, how T-IC self renewal, multipotent differentiation, and tumorigenicity are maintained remains obscure. Because miRNAs can regulate cell-fate decisions, we compared miRNA expression in self-renewing and differentiated cells from breast cancer lines and in breast T-IC (BT-IC) and non-BT-IC from 1 degrees breast cancers. let-7 miRNAs were markedly reduced in BT-IC and increased with differentiation. Infecting BT-IC with let-7-lentivirus reduced proliferation, mammosphere formation, and the proportion of undifferentiated cells in vitro and tumor formation and metastasis in NOD/SCID mice, while antagonizing let-7 by antisense oligonucleotides enhanced in vitro self renewal of non-T-IC. Increased let-7 paralleled reduced H-RAS and HMGA2, known let-7 targets. Silencing H-RAS in a BT-IC-enriched cell line reduced self renewal but had no effect on differentiation, while silencing HMGA2 enhanced differentiation but did not affect self renewal. Therefore let-7 regulates multiple BT-IC stem cell-like properties by silencing more than one target.",
"Acute myeloid leukaemia (AML) is thought to be maintained by a small population of leukemic progenitor cells. To define the phenotype of such cells with long-term proliferative capacity in vitro and in vivo, we have used the production of leukemic clonogenic cells (CFU) after 2 to 8 weeks in suspension culture as a measure of these cells in vitro and compared their phenotype with that of cells capable of engrafting nonobese diabetic severe combined immune deficient (NOD/SCID) mice. Leukemic blast peripheral blood cells were evaluated for expression of CD34 and Thy-1 (CD90) antigens. The majority of AML blast cells at diagnosis lacked expression of Thy-1. Most primary CFU-blast and the CFU detected at up to 8 weeks from suspension cultures were CD34+/Thy-1-. AML cells that were capable of engrafting NOD/SCID mice were also found to have the CD34+/Thy-1- phenotype. However, significant engraftment was achieved using both CD34+/Thy-1- and CD34- subfractions from one AML M5 patient. These results suggest that while heterogeneity exists between individual patients, the leukemic progenitor cells that are capable of maintaining the disease in vitro and in vivo differ from normal hematopoietic progenitor cells in their lack of expression of Thy-1.",
"Dicer is the enzyme that cleaves double-stranded RNA (dsRNA) into 21-25-nt-long species responsible for sequence-specific RNA-induced gene silencing at the transcriptional, post-transcriptional, or translational level. We disrupted the dicer-1 (dcr-1) gene in mouse embryonic stem (ES) cells by conditional gene targeting and generated Dicer-null ES cells. These cells were viable, despite being completely defective in RNA interference (RNAi) and the generation of microRNAs (miRNAs). However, the mutant ES cells displayed severe defects in differentiation both in vitro and in vivo. Epigenetic silencing of centromeric repeat sequences and the expression of homologous small dsRNAs were markedly reduced. Re-expression of Dicer in the knockout cells rescued these phenotypes. Our data suggest that Dicer participates in multiple, fundamental biological processes in a mammalian organism, ranging from stem cell differentiation to the maintenance of centromeric heterochromatin structure and centromeric silencing.",
"Recent evidence supports the hypothesis that cancer stem cells are responsible for tumour initiation and formation. Using flow cytometry, we isolated a population of CD44+CD24(-) prostate cells that display stem cell characteristics as well as gene expression patterns that predict overall survival in prostate cancer patients. CD44+CD24(-) cells form colonies in soft agar and form tumours in NOD/SCID mice when as few as 100 cells are injected. Furthermore, CD44+CD24(-) cells express genes known to be important in stem cell maintenance, such as BMI-1 and Oct-3/4. Moreover, we can maintain CD44+CD24(-) prostate stem-like cells as nonadherent spheres in serum-replacement media without substantially shifting gene expression. Addition of serum results in adherence to plastic and shifts gene expression patterns to resemble the differentiated parental cells. Thus, we propose that CD44+CD24(-) prostate cells are stem-like cells responsible for tumour initiation and we provide a genomic definition of these cells and the differentiated cells they give rise to. Furthermore, gene expression patterns of CD44+CD24(-) cells have a genomic signature that is predictive of poor patient prognosis. Therefore, CD44+CD24(-) LNCaP prostate cells offer an attractive model system to both explore the biology important to the maintenance and differentiation of prostate cancer stem cells as well as to develop the therapeutics, as the gene expression pattern in these cells is consistent with poor survival in prostate cancer patients."
] |
Exploring integron gene cassettes from polluted Indian river sediments
|
Over the past 75 years, human pathogens have acquired antibiotic resistance genes (ARGs), often from environmental bacteria. Integrons play a major role in the acquisition of antibiotic resistance genes. We therefore hypothesized that focused exploration of integron gene cassettes from microbial communities could be an efficient way to find novel mobile resistance genes. DNA from polluted Indian river sediments were amplified using three sets of primers targeting class 1 integrons and sequenced by long- and short-read technologies to maintain both accuracy and context.
|
[
"Gene cassettes are small mobile elements, consisting of little more than a single gene and recombination site, which are captured by larger elements called integrons. Several cassettes may be inserted into the same integron forming a tandem array. The discovery of integrons in the chromosome of many species has led to the identification of thousands of gene cassettes, mostly of unknown function, while integrons associated with transposons and plasmids carry mainly antibiotic resistance genes and constitute an important means of spreading resistance. An updated compilation of gene cassettes found in sequences of such 'mobile resistance integrons' in GenBank was facilitated by a specially developed automated annotation system. At least 130 different (<98% identical) cassettes that carry known or predicted antibiotic resistance genes were identified, along with many cassettes of unknown function. We list exemplar GenBank accession numbers for each and address some nomenclature issues. Various modifications to cassettes, some of which may be useful in tracking cassette epidemiology, are also described. Despite potential biases in the GenBank dataset, preliminary analysis of cassette distribution suggests interesting differences between cassettes and may provide useful information to direct more systematic studies.",
"The integron/gene cassette systems identified in bacteria comprise a class of genetic elements that allow adaptation by acquisition of gene cassettes. Integron gene cassettes have been shown to facilitate the spread of drug resistance in human pathogens but their role outside a clinical setting has not been explored extensively. We sequenced 2145 integron gene cassettes from four marine sediment samples taken from the vicinity of Halifax Nova Scotia, Canada, increasing the number of gene cassettes obtained from environmental microbial communities by 10-fold. Sequence analyses reveals that the majority of these cassettes encode novel proteins and that this study is consistent with previous claims of high cassette diversity as we estimate a Chao1 diversity index of approximately 3000 cassettes from these samples. The functional distribution of environmental cassettes recovered in this study, when compared with that of cassettes from the only other source with significant sampling (Vibrio genomes) suggests that alternate selection regimes might be acting on these two gene pools. The majority of cassettes recovered in this study encode novel, unknown proteins. In instances where we obtained multiple alleles of a novel protein we demonstrate that non-synonymous versus synonymous substitution rates ratios suggest relaxed selection. Cassette-encoded proteins with known homologues represent a variety of functions and prevalent among these are isochorismatases; proteins involved in iron scavenging. Phylogenetic analysis of these isochorismatases as well as of cassette-encoded acetyltransferases reveals a patchy distribution, suggesting multiple sources for the origin of these cassettes. Finally, the two most environmentally similar sample sites considered in this study display the greatest overlap of cassette types, consistent with the hypothesis that cassette genes encode adaptive proteins.",
"Acinetobacter baumannii is a species of nonfermentative gram-negative bacteria commonly found in water and soil. This organism was susceptible to most antibiotics in the 1970s. It has now become a major cause of hospital-acquired infections worldwide due to its remarkable propensity to rapidly acquire resistance determinants to a wide range of antibacterial agents. Here we use a comparative genomic approach to identify the complete repertoire of resistance genes exhibited by the multidrug-resistant A. baumannii strain AYE, which is epidemic in France, as well as to investigate the mechanisms of their acquisition by comparison with the fully susceptible A. baumannii strain SDF, which is associated with human body lice. The assembly of the whole shotgun genome sequences of the strains AYE and SDF gave an estimated size of 3.9 and 3.2 Mb, respectively. A. baumannii strain AYE exhibits an 86-kb genomic region termed a resistance island--the largest identified to date--in which 45 resistance genes are clustered. At the homologous location, the SDF strain exhibits a 20 kb-genomic island flanked by transposases but devoid of resistance markers. Such a switching genomic structure might be a hotspot that could explain the rapid acquisition of resistance markers under antimicrobial pressure. Sequence similarity and phylogenetic analyses confirm that most of the resistance genes found in the A. baumannii strain AYE have been recently acquired from bacteria of the genera Pseudomonas, Salmonella, or Escherichia. This study also resulted in the discovery of 19 new putative resistance genes. Whole-genome sequencing appears to be a fast and efficient approach to the exhaustive identification of resistance genes in epidemic infectious agents of clinical significance.",
"Metagenomic studies have shown that antibiotic resistance genes are ubiquitous in the environment, which has led to the suggestion that there is a high risk that these genes will spread to bacteria that cause human infections. If this is true, estimating the real risk of dissemination of resistance genes from environmental reservoirs to human pathogens is therefore very difficult. In this Opinion article, we analyse the current definitions of antibiotic resistance and antibiotic resistance genes, and we describe the bottlenecks that affect the transfer of antibiotic resistance genes to human pathogens. We propose rules for estimating the risks associated with genes that are present in environmental resistomes by evaluating the likelihood of their introduction into human pathogens, and the consequences of such events for the treatment of infections.",
"The quality of automated gene prediction in microbial organisms has improved steadily over the past decade, but there is still room for improvement. Increasing the number of correct identifications, both of genes and of the translation initiation sites for each gene, and reducing the overall number of false positives, are all desirable goals. With our years of experience in manually curating genomes for the Joint Genome Institute, we developed a new gene prediction algorithm called Prodigal (PROkaryotic DYnamic programming Gene-finding ALgorithm). With Prodigal, we focused specifically on the three goals of improved gene structure prediction, improved translation initiation site recognition, and reduced false positives. We compared the results of Prodigal to existing gene-finding methods to demonstrate that it met each of these objectives. We built a fast, lightweight, open source gene prediction program called Prodigal http://compbio.ornl.gov/prodigal/. Prodigal achieved good results compared to existing methods, and we believe it will be a valuable asset to automated microbial annotation pipelines.",
"Antibiotic resistance genes (ARGs) are emerging contaminants posing a potential worldwide human health risk. Intensive animal husbandry is believed to be a major contributor to the increased environmental burden of ARGs. Despite the volume of antibiotics used in China, little information is available regarding the corresponding ARGs associated with animal farms. We assessed type and concentrations of ARGs at three stages of manure processing to land disposal at three large-scale (10,000 animals per year) commercial swine farms in China. In-feed or therapeutic antibiotics used on these farms include all major classes of antibiotics except vancomycins. High-capacity quantitative PCR arrays detected 149 unique resistance genes among all of the farm samples, the top 63 ARGs being enriched 192-fold (median) up to 28,000-fold (maximum) compared with their respective antibiotic-free manure or soil controls. Antibiotics and heavy metals used as feed supplements were elevated in the manures, suggesting the potential for coselection of resistance traits. The potential for horizontal transfer of ARGs because of transposon-specific ARGs is implicated by the enrichment of transposases--the top six alleles being enriched 189-fold (median) up to 90,000-fold in manure--as well as the high correlation (r(2) = 0.96) between ARG and transposase abundance. In addition, abundance of ARGs correlated directly with antibiotic and metal concentrations, indicating their importance in selection of resistance genes. Diverse, abundant, and potentially mobile ARGs in farm samples suggest that unmonitored use of antibiotics and metals is causing the emergence and release of ARGs to the environment.",
"Veterinary antibiotics can enter the environment due to the common practice of land application of manure from treated animals. The environmental fate of tetracyclines in swine manure after composting and field application remains largely unknown. This study analyzed the concentrations of tetracyclines in manure, manure-based compost and compost amended soil in selected swine farms from Beijing, Jiaxing and Putian, China to determine the dilution effects of antibiotics when released into the soil environment. The results demonstrate that residues of antibiotics were detected in all samples and chlortetracycline as well as its degradation products should be regarded critically concerning their potential ecotoxicity. Application of manure-based compost to soil could reduce the possible risk posed by antibiotic contamination, but the trigger value of 100 microg/kg was still exceeded in soil samples (776.1 microg/kg dw) from Putian City after application of compost. Field studies such as the present one can help to improve the routine administration of antibiotic-containing composted manure."
] |
Methamphetamine and modafinil elicit different cognitive outcomes in the novel object recognition test
|
Methamphetamine (METH) and modafinil are psychostimulants with different long-term cognitive profiles: METH is addictive and leads to cognitive decline, whereas modafinil has little abuse liability and is a cognitive enhancer. Increasing evidence implicates epigenetic mechanisms of gene regulation behind the lasting changes that drugs of abuse and other psychotropic compounds induce in the brain, like the control of gene expression by histones 3 and 4 tails acetylation (H3ac and H4ac) and DNA cytosine methylation (5-mC). Mice were treated with a seven-day repeated METH, modafinil or vehicle protocol and evaluated in the novel object recognition (NOR) test or sacrificed 4days after last injection for molecular assays. We evaluated total H3ac, H4ac and 5-mC levels in the medial prefrontal cortex (mPFC), H3ac and H4ac promotor enrichment (ChIP) and mRNA expression (RT-PCR) of neurotransmitter systems involved in arousal, wakefulness and cognitive control, like dopaminergic (Drd1 and Drd2), α-adrenergic (Adra1a and Adra1b), orexinergic (Hcrtr1 and Hcrtr2), histaminergic (Hrh1 and Hrh3) and glutamatergic (AMPA Gria1 and NMDA Grin1) receptors. Repeated METH and modafinil treatment elicited different cognitive outcomes in the NOR test, where modafinil-treated mice performed as controls and METH-treated mice showed impaired recognition memory. METH-treated mice also showed i) decreased levels of total H3ac and H4ac, and increased levels of 5-mC, ii) decreased H3ac enrichment at promoters of Drd2, Hcrtr1/2, Hrh1 and Grin1, and increased H4ac enrichment at Drd1, Hrh1 and Grin1, iii) increased mRNA of Drd1a, Grin1 and Gria1. Modafinil-treated mice shared none of these effects and showed increased H3ac enrichment and mRNA expression at Adra1b. Modafinil and METH showed similar effects linked to decreased H3ac in Hrh3, increased H4ac in Hcrtr1, and decreased mRNA expression of Hcrtr2. The specific METH-induced epigenetic and transcriptional changes described here may be related to the long-term cognitive decline effects of the drug and its detrimental effects on mPFC function. The lack of similar epigenetic effects of chronic modafinil administration supports this notion.
|
[
"Chronic use of methamphetamine (METH) leads to long-lasting cognitive dysfunction in humans and in animal models. Modafinil is a wake-promoting compound approved for the treatment of sleeping disorders. It is also prescribed off label to treat METH dependence. In the present study, we investigated whether modafinil could improve cognitive deficits induced by sub-chronic METH treatment in mice by measuring visual retention in a Novel Object Recognition (NOR) task. After sub-chronic METH treatment (1 mg/kg, once a day for 7 days), mice performed the NOR task, which consisted of habituation to the object recognition arena (5 min a day, 3 consecutive days), training session (2 equal objects, 10 min, day 4), and a retention session (1 novel object, 5 min, day 5). One hour before the training session, mice were given a single dose of modafinil (30 or 90 mg/kg). METH-treated mice showed impairments in visual memory retention, evidenced by equal preference of familiar and novel objects during the retention session. The lower dose of modafinil (30 mg/kg) had no effect on visual retention scores in METH-treated mice, while the higher dose (90 mg/kg) rescued visual memory retention to control values. We also measured extracellular signal-regulated kinase (ERK) phosphorylation in medial prefrontal cortex (mPFC), hippocampus, and nucleus accumbens (NAc) of METH- and vehicle-treated mice that received modafinil 1 h before exposure to novel objects in the training session, compared to mice placed in the arena without objects. Elevated ERK phosphorylation was found in the mPFC of vehicle-treated mice, but not in METH-treated mice, exposed to objects. The lower dose of modafinil had no effect on ERK phosphorylation in METH-treated mice, while 90 mg/kg modafinil treatment restored the ERK phosphorylation induced by novelty in METH-treated mice to values comparable to controls. We found neither a novelty nor treatment effect on ERK phosphorylation in hippocampus or NAc of vehicle- and METH-treated mice receiving acute 90 mg/kg modafinil treatment. Our results showed a palliative role of modafinil against METH-induced visual cognitive impairments, possibly by normalizing ERK signaling pathways in mPFC. Modafinil may be a valuable pharmacological tool for the treatment of cognitive deficits observed in human METH abusers as well as in other neuropsychiatric conditions. This article is part of the Special Issue entitled 'CNS Stimulants'.",
"Considerable evidence indicates that amphetamine derivatives can deplete brain monoaminergic neurotransmitters. However, the behavioral and cognitive consequences of neurochemical depletions induced by amphetamines are not well established. In this study, mice were exposed to dosing regimens of 3,4-methylenedioxymethamphetamine (MDMA), methamphetamine (METH), or parachloroamphetamine (PCA) known to deplete the monoamine neurotransmitters dopamine and serotonin, and the effects of these dosing regimens on learning and memory were assessed. In the same animals, we determined deficits in learning and memory via passive avoidance (PA) behavior and changes in tissue content of monoamine neurotransmitters and their primary metabolites in the striatum, frontal cortex, cingulate, hippocampus, and amygdala via ex vivo high-pressure liquid chromatography. Exposure to METH and PCA impaired PA performance and resulted in significant depletions of dopamine, serotonin, and their metabolites in several brain regions. Multiple linear regression analysis revealed that the tissue concentration of dopamine in the anterior striatum was the strongest predictor of PA performance, with an additional significant contribution by the tissue concentration of the serotonin metabolite 5-hydroxyindoleacetic acid in the cingulate. In contrast to the effects of METH and PCA, exposure to MDMA did not deplete anterior striatal dopamine levels or cingulate levels of 5-hydroxyindoleacetic acid, and it did not impair PA performance. These studies demonstrate that certain amphetamines impair PA performance in mice and that these impairments may be attributable to specific neurochemical depletions.",
"A growing body of evidence indicates that protracted use of methamphetamine (mAMPH) causes long-term impairments in cognitive function in humans. Aside from the widely reported problems with attention, mAMPH users exhibit learning and memory deficits, particularly on tasks requiring response control. Although binge mAMPH administration to animals results in cognitive deficits, few studies have attempted to test behavioral flexibility in animals after mAMPH exposure. The aim of this study was to evaluate whether mAMPH would produce impairments in two tasks assessing flexible responding in rats: a touchscreen-based discrimination-reversal learning task and an attentional set shift task (ASST) based on a hallmark test of executive function in humans, the Wisconsin Card Sort. We treated male Long-Evans rats with a regimen of four injections of 2 mg/kg mAMPH (or vehicle) within a single day, a dosing regimen shown earlier to produce object recognition impairments. We then tested them on (1) reversal learning after pretreatment discrimination learning or (2) the ASST. Early reversal learning accuracy was impaired in mAMPH-treated rats. MAMPH pretreatment also selectively impaired reversal performance during ASST testing, leaving set-shifting performance intact. Postmortem analysis of [(125)I]RTI-55 binding revealed small (10-20%) but significant reductions in striatal dopamine transporters produced by this mAMPH regimen. Together, these results lend new information to the growing field documenting impaired cognition after mAMPH exposure, and constitute a rat model of the widely reported decision-making deficits resulting from mAMPH abuse seen in humans.",
"Hypothalamic hypocretin (orexin) peptides mediate arousal, attention, and reward processing. Fibers containing orexins project to brain structures that govern motivated behavior, including the ventral tegmental area (VTA). A number of psychiatric conditions, including attention deficit hyperactivity disorder (ADHD) and substance use disorders, are characterized by deficits in impulse control, however the relationship between orexin and impulsive behavior is incompletely characterized. The effects of systemic or centrally administered orexin receptor (OXR) antagonists on measures of impulsive-like behavior in rats were evaluated using the five-choice serial reaction time task (5-CSRTT) and delay discounting procedures. These paradigms were also used to test the capacity of OXR antagonists to attenuate acute cocaine-evoked impulsivity. Finally, immunohistochemistry and calcium imaging were used to assess potential cellular mechanisms by which OXR blockade may influence motor impulsivity. Suvorexant, a dual (OX1/2R) orexin receptor antagonist, reduced cocaine-evoked premature responses in 5-CSRTT when administered systemically or directly into VTA. Neither suvorexant nor OX1R- or OX2R-selective compounds (SB334867 or TCS-OX2-29, respectively) altered delay discounting. Finally, suvorexant did not alter Fos-immunoreactivity within tyrosine hydroxylase-immunolabeled neurons of VTA, but did attenuate cocaine- and orexin-induced increases in calcium transient amplitude within neurons of VTA. Results from the present studies suggest potential therapeutic utility of OXR antagonists in reducing psychostimulant-induced motor impulsivity. These findings also support the view that orexin transmission is closely involved in executive function in normal and pathological conditions."
] |
Telomeric chromatin dynamics during cell division and early development in mammals
|
Telomeres are specialized structures that evolved to protect the end of linear chromosomes from the action of the cell DNA damage machinery. They are composed of tandem arrays of repeated DNA sequences with a specific heterochromatic organization. The length of telomeric repeats is dynamically regulated and can be affected by changes in the telomere chromatin structure. When telomeres are not properly controlled, the resulting chromosomal alterations can induce genomic instability and ultimately the development of human diseases, such as cancer. Therefore, proper establishment, regulation, and maintenance of the telomere chromatin structure are required for cell homeostasis. Here, we review the current knowledge on telomeric chromatin dynamics during cell division and early development in mammals, and how its proper regulation safeguards genome stability.
|
[
"A fraction of cancer cells maintain telomeres through the telomerase-independent, 'Alternative Lengthening of Telomeres' (ALT) pathway. ALT relies on homologous recombination (HR) between telomeric sequences; yet, what makes ALT telomeres recombinogenic remains unclear. Here we show that the RNA endonuclease RNaseH1 regulates the levels of RNA-DNA hybrids between telomeric DNA and the long noncoding RNA TERRA, and is a key mediator of telomere maintenance in ALT cells. RNaseH1 associated to telomeres specifically in ALT cells and its depletion led to telomeric hybrid accumulation, exposure of single-stranded telomeric DNA, activation of replication protein A at telomeres and abrupt telomere excision. Conversely, overexpression of RNaseH1 weakened the recombinogenic nature of ALT telomeres and led to telomere shortening. Altering cellular RNaseH1 levels did not perturb telomere homoeostasis in telomerase-positive cells. RNaseH1 maintains regulated levels of telomeric RNA-DNA hybrids at ALT telomeres to trigger HR without compromising telomere integrity too severely.",
"Chromatin assembly factor-1 (CAF-1), a complex consisting of p150, p60, and p48 subunits, is highly conserved from yeast to humans and facilitates nucleosome assembly of newly replicated DNA in vitro. To investigate roles of CAF-1 in vertebrates, we generated two conditional DT40 mutants, respectively, devoid of CAF-1p150 and p60. Depletion of each of these CAF-1 subunits led to delayed S-phase progression concomitant with slow DNA synthesis, followed by accumulation in late S/G2 phase and aberrant mitosis associated with extra centrosomes, and then the final consequence was cell death. We demonstrated that CAF-1 is necessary for rapid nucleosome formation during DNA replication in vivo as well as in vitro. Loss of CAF-1 was not associated with the apparent induction of phosphorylations of S-checkpoint kinases Chk1 and Chk2. To elucidate the precise role of domain(s) in CAF-1p150, functional dissection analyses including rescue assays were preformed. Results showed that the binding abilities of CAF-1p150 with CAF-1p60 and DNA polymerase sliding clamp proliferating cell nuclear antigen (PCNA) but not with heterochromatin protein HP1-gamma are required for cell viability. These observations highlighted the essential role of CAF-1-dependent nucleosome assembly in DNA replication and cell proliferation through its interaction with PCNA.",
"We have investigated the role of the enzyme telomerase in highly proliferative organs in successive generations of mice lacking telomerase RNA. Late-generation animals exhibited defective spermatogenesis, with increased programmed cell death (apoptosis) and decreased proliferation in the testis. The proliferative capacity of haematopoietic cells in the bone marrow and spleen was also compromised. These progressively adverse effects coincided with substantial erosion of telomeres (the termini of eukaryotic chromosomes) and fusion and loss of chromosomes. These findings indicate an essential role for telomerase, and hence telomeres, in the maintenance of genomic integrity and in the long-term viability of high-renewal organ systems.",
"A striking proportion of human cleavage-stage embryos exhibit chromosome instability (CIN). Notably, until now, no experimental model has been described to determine the origin and mechanisms of complex chromosomal rearrangements. Here, we examined mouse embryos deficient for the chromatin remodeling protein ATRX to determine the cellular mechanisms activated in response to CIN. We demonstrate that ATRX is required for silencing of major satellite transcripts in the maternal genome, where it confers epigenetic asymmetry to pericentric heterochromatin during the transition to the first mitosis. This stage is also characterized by a striking kinetochore size asymmetry established by differences in CENP-C protein between the parental genomes. Loss of ATRX results in increased centromeric mitotic recombination, a high frequency of sister chromatid exchanges and double strand DNA breaks, indicating the formation of mitotic recombination break points. ATRX-deficient embryos exhibit a twofold increase in transcripts for aurora kinase B, the centromeric cohesin ESCO2, DNMT1, the ubiquitin-ligase (DZIP3) and the histone methyl transferase (EHMT1). Thus, loss of ATRX activates a pathway that integrates epigenetic modifications and DNA repair in response to chromosome breaks. These results reveal the cellular response of the cleavage-stage embryo to CIN and uncover a mechanism by which centromeric fission induces the formation of large-scale chromosomal rearrangements. Our results have important implications to determine the epigenetic origins of CIN that lead to congenital birth defects and early pregnancy loss, as well as the mechanisms involved in the oocyte to embryo transition.",
"How a more plastic chromatin state is maintained and reversed during development is unknown. Heterochromatin-mediated silencing of repetitive elements occurs in differentiated cells. Here, we used repetitive elements, including retrotransposons, as model loci to address how and when heterochromatin forms during development. RNA sequencing throughout early mouse embryogenesis revealed that repetitive-element expression is dynamic and stage specific, with most repetitive elements becoming repressed before implantation. We show that LINE-1 and IAP retrotransposons become reactivated from both parental genomes after fertilization. Chromatin immunoprecipitation for H3K4me3 and H3K9me3 in 2- and 8-cell embryos indicates that their developmental silencing follows loss of activating marks rather than acquisition of conventional heterochromatic marks. Furthermore, short LINE-1 RNAs regulate LINE-1 transcription in vivo. Our data indicate that reprogramming after mammalian fertilization comprises a robust transcriptional activation of retrotransposons and that repetitive elements are initially regulated through RNA.",
"For a long time, it has been assumed that the only role of sperm at fertilization is to introduce the male genome into the egg. Recently, ideas have emerged that the epigenetic state of the sperm nucleus could influence transcription in the embryo. However, conflicting reports have challenged the existence of epigenetic marks on sperm genes, and there are no functional tests supporting the role of sperm epigenetic marking on embryonic gene expression. Here, we show that sperm is epigenetically programmed to regulate embryonic gene expression. By comparing the development of sperm- and spermatid-derived frog embryos, we show that the programming of sperm for successful development relates to its ability to regulate transcription of a set of developmentally important genes. During spermatid maturation into sperm, these genes lose H3K4me2/3 and retain H3K27me3 marks. Experimental removal of these epigenetic marks at fertilization de-regulates gene expression in the resulting embryos in a paternal chromatin-dependent manner. This demonstrates that epigenetic instructions delivered by the sperm at fertilization are required for correct regulation of gene expression in the future embryos. The epigenetic mechanisms of developmental programming revealed here are likely to relate to the mechanisms involved in transgenerational transmission of acquired traits. Understanding how parental experience can influence development of the progeny has broad potential for improving human health.",
"Circumvention of the telomere length-dependent mechanisms that control the upper boundaries of cellular proliferation is necessary for the unlimited growth of cancer. Most cancer cells achieve cellular immortality by up-regulating the expression of telomerase to extend and maintain their telomere length. However, a small but significant number of cancers do so via the exchange of telomeric DNA between chromosomes in a pathway termed alternative lengthening of telomeres, or ALT. Although it remains to be clarified why a cell chooses the ALT pathway and how ALT is initiated, recently identified mutations in factors that shape the chromatin and epigenetic landscape of ALT telomeres are shedding light on these mechanisms. In this review, we examine these recent findings and integrate them into the current models of the ALT mechanism.",
"Mammalian oocytes can reprogram somatic cells into a totipotent state enabling animal cloning through somatic cell nuclear transfer (SCNT). However, the majority of SCNT embryos fail to develop to term due to undefined reprogramming defects. Here, we identify histone H3 lysine 9 trimethylation (H3K9me3) of donor cell genome as a major barrier for efficient reprogramming by SCNT. Comparative transcriptome analysis identified reprogramming resistant regions (RRRs) that are expressed normally at 2-cell mouse embryos generated by in vitro fertilization (IVF) but not SCNT. RRRs are enriched for H3K9me3 in donor somatic cells and its removal by ectopically expressed H3K9me3 demethylase Kdm4d not only reactivates the majority of RRRs, but also greatly improves SCNT efficiency. Furthermore, use of donor somatic nuclei depleted of H3K9 methyltransferases markedly improves SCNT efficiency. Our study thus identifies H3K9me3 as a critical epigenetic barrier in SCNT-mediated reprogramming and provides a promising approach for improving mammalian cloning efficiency.",
"How the chromatin regulatory landscape in the inner cell mass cells is established from differentially packaged sperm and egg genomes during preimplantation development is unknown. Here, we develop a low-input DNase I sequencing (liDNase-seq) method that allows us to generate maps of DNase I-hypersensitive site (DHS) of mouse preimplantation embryos from 1-cell to morula stage. The DHS landscape is progressively established with a drastic increase at the 8-cell stage. Paternal chromatin accessibility is quickly reprogrammed after fertilization to the level similar to maternal chromatin, while imprinted genes exhibit allelic accessibility bias. We demonstrate that transcription factor Nfya contributes to zygotic genome activation and DHS formation at the 2-cell stage and that Oct4 contributes to the DHSs gained at the 8-cell stage. Our study reveals the dynamic chromatin regulatory landscape during early development and identifies key transcription factors important for DHS establishment in mammalian embryos.",
"To assess telomerase as a cancer therapeutic target and determine adaptive mechanisms to telomerase inhibition, we modeled telomerase reactivation and subsequent extinction in T cell lymphomas arising in Atm(-/-) mice engineered with an inducible telomerase reverse transcriptase allele. Telomerase reactivation in the setting of telomere dysfunction enabled full malignant progression with alleviation of telomere dysfunction-induced checkpoints. These cancers possessed copy number alterations targeting key loci in human T cell lymphomagenesis. Upon telomerase extinction, tumor growth eventually slowed with reinstatement of telomere dysfunction-induced checkpoints, yet growth subsequently resumed as tumors acquired alternative lengthening of telomeres (ALT) and aberrant transcriptional networks centering on mitochondrial biology and oxidative defense. ALT+ tumors acquired amplification/overexpression of PGC-1β, a master regulator of mitochondrial biogenesis and function, and they showed marked sensitivity to PGC-1β or SOD2 knockdown. Genetic modeling of telomerase extinction reveals vulnerabilities that motivate coincidental inhibition of mitochondrial maintenance and oxidative defense mechanisms to enhance antitelomerase cancer therapy."
] |
ParB protein forms DNA bridging interactions
|
The ParB protein forms DNA bridging interactions around
|
[
"In bacteria, mitotic stability of plasmids and many chromosomes depends on replicon-specific systems, which comprise a centromere, a centromere-binding protein and an ATPase. Dynamic self-assembly of the ATPase appears to enable active partition of replicon copies into cell-halves, but for Walker-box partition ATPases the molecular mechanism is unknown. ATPase activity appears to be essential for this process. DNA and centromere-binding proteins are known to stimulate the ATPase activity but molecular details of the stimulation mechanism have not been reported. We have investigated the interactions which stimulate ATP hydrolysis by the SopA partition ATPase of plasmid F. By using SopA and SopB proteins deficient in DNA binding, we have found that the intrinsic ability of SopA to hydrolyze ATP requires direct DNA binding by SopA but not by SopB. Our results show that two independent interactions of SopA act in synergy to stimulate its ATPase. SopA must interact with (i) DNA, through its ATP-dependent nonspecific DNA binding domain and (ii) SopB, which we show here to provide an arginine-finger motif. In addition, the latter interaction stimulates ATPase maximally when SopB is part of the partition complex. Hence, our data demonstrate that DNA acts on SopA in two ways, directly as nonspecific DNA and through SopB as centromeric DNA, to fully activate SopA ATP hydrolysis.",
"The development of genetic competence in the Gram-positive eubacterium Bacillus subtilis is a complex postexponential process. Here we describe a new bicistronic operon, bdbDC, required for competence development, which was identified by the B. subtilis Systematic Gene Function Analysis program. Inactivation of either the bdbC or bdbD genes of this operon results in the loss of transformability without affecting recombination or the synthesis of ComK, the competence transcription factor. BdbC and BdbD are orthologs of enzymes known to be involved in extracytoplasmic disulfide bond formation. Consistent with this, BdbC and BdbD are needed for the secretion of the Escherichia coli disulfide bond-containing alkaline phosphatase, PhoA, by B. subtilis. Similarly, the amount of the disulfide bond-containing competence protein ComGC is severely reduced in bdbC or bdbD mutants. In contrast, the amounts of the competence proteins ComGA and ComEA remain unaffected by bdbDC mutations. Taken together, these observations imply that in the absence of either BdbC or BdbD, ComGC is unstable and that BdbC and BdbD catalyze the formation of disulfide bonds that are essential for the DNA binding and uptake machinery.",
"Indirect magnetization transfer increases the observed nuclear Overhauser enhancement (NOE) between two protons in many cases, leading to an underestimation of target distances. Wider distance bounds are necessary to account for this error. However, this leads to a loss of information and may reduce the quality of the structures generated from the inter-proton distances. Although several methods for spin diffusion correction have been published, they are often not employed to derive distance restraints. This prompted us to write a user-friendly and CPU-efficient method to correct for spin diffusion that is fully integrated in our program ambiguous restraints for iterative assignment (ARIA). ARIA thus allows automated iterative NOE assignment and structure calculation with spin diffusion corrected distances. The method relies on numerical integration of the coupled differential equations which govern relaxation by matrix squaring and sparse matrix techniques. We derive a correction factor for the distance restraints from calculated NOE volumes and inter-proton distances. To evaluate the impact of our spin diffusion correction, we tested the new calibration process extensively with data from the Pleckstrin homology (PH) domain of Mus musculus beta-spectrin. By comparing structures refined with and without spin diffusion correction, we show that spin diffusion corrected distance restraints give rise to structures of higher quality (notably fewer NOE violations and a more regular Ramachandran map). Furthermore, spin diffusion correction permits the use of tighter error bounds which improves the distinction between signal and noise in an automated NOE assignment scheme.",
"comK is a positive autoregulatory gene occupying a central position in the competence-signal-transduction network. All regulatory routes identified in this network converge at the level of comK expression. The ComK protein is required for the transcriptional induction of comK and the late competence genes, which specify morphogenetic and structural proteins necessary for construction of the DNA-binding and uptake apparatus. In this report we demonstrate that ComK specifically binds to DNA fragments containing promoter and upstream sequences of the genes it affects (comC, comE, comF, comG and comK). Using portions of the region upstream of comC we show that the ComK-binding sequences are essential for the expression of competence. Moreover, we demonstrate that the presence of ComK stimulates the expression of comF-lacZ and comG-lacZ translational fusions in vivo in Escherichia coli. These results indicate that the gene product of comK is identical to the previously inferred competence transcription factor (CTF).",
"The use of generous distance bounds has been the hallmark of NMR structure determination. However, bounds necessitate the estimation of data quality before the calculation, reduce the information content, introduce human bias, and allow for major errors in the structures. Here, we propose a new rapid structure calculation scheme based on Bayesian analysis. The minimization of an extended energy function, including a new type of distance restraint and a term depending on the data quality, results in an estimation of the data quality in addition to coordinates. This allows for the determination of the optimal weight on the experimental information. The resulting structures are of better quality and closer to the X-ray crystal structure of the same molecule. With the new calculation approach, the analysis of discrepancies from the target distances becomes meaningful. The strategy may be useful in other applications-for example, in homology modeling.",
"Generally, the presence of a consensus sequence in the promoter of a gene is taken as indication for regulation by the transcription factor that binds to this sequence. In light of the recent developments in genome research, we were interested to what extent this supposition is valid. We examined the relationship between the presence of a binding site for ComK, the competence transcription factor of Bacillus subtilis, and actual transcriptional activation by ComK. Bacillus subtilis contains 1062 putative ComK-binding sites (K-boxes) in its genome. We employed DNA macroarrays to identify ComK-activated genes, and found that the presence of a K-box is an unreliable predictor for regulation. Only approximately 8% of the genes containing a K-box in the putative promoter region are regulated by ComK. The predictive value of a K-box could be improved by taking into consideration the degree of deviation from the K-box consensus sequence, the presence of extra ComK-binding motifs and the positions of RNA polymerase-binding sites. Finally, many of the ComK-activated genes show no apparent function related to the competence process. Based on our findings, we propose that the ComK-dependent activation of several genes might serve no biological purpose and can be considered 'evolutionary noise'.",
"Sedimentation velocity analytical ultracentrifugation is an important tool in the characterization of macromolecules and nanoparticles in solution. The sedimentation coefficient distribution c(s) of Lamm equation solutions is based on the approximation of a single, weight-average frictional coefficient of all particles, determined from the experimental data, which scales the diffusion coefficient to the sedimentation coefficient consistent with the traditional s approximately M(2/3) power law. It provides a high hydrodynamic resolution, where diffusional broadening of the sedimentation boundaries is deconvoluted from the sedimentation coefficient distribution. The approximation of a single weight-average frictional ratio is favored by several experimental factors, and usually gives good results for chemically not too dissimilar macromolecules, such as mixtures of folded proteins. In this communication, we examine an extension to a two-dimensional distribution of sedimentation coefficient and frictional ratio, c(s,f(r)), which is representative of a more general set of size-and-shape distributions, including mass-Stokes radius distributions, c(M,R(S)), and sedimentation coefficient-molar mass distributions c(s,M). We show that this can be used to determine average molar masses of macromolecules and characterize macromolecular distributions, without the approximation of any scaling relationship between hydrodynamic and thermodynamic parameters."
] |
Effects of n-3 PUFA on CD4+ T cell plasma membrane organisation, mitochondrial bioenergetics and lymphoproliferation
|
Cell membrane fatty acids influence fundamental properties of the plasma membrane, including membrane fluidity, protein functionality, and lipid raft signalling. Evidence suggests that dietary n-3 PUFA may target the plasma membrane of immune cells by altering plasma membrane lipid dynamics, thereby regulating the attenuation of immune cell activation and suppression of inflammation. As lipid-based immunotherapy might be a promising new clinical strategy for the treatment of inflammatory disorders, we conducted in vitro and in vivo experiments to examine the effects of n-3 PUFA on CD4+ T cell membrane order, mitochondrial bioenergetics and lymphoproliferation. n-3 PUFA were incorporated into human primary CD4+ T cells phospholipids in vitro in a dose-dependent manner, resulting in a reduction in whole cell membrane order, oxidative phosphorylation and proliferation. At higher doses, n-3 PUFA induced unique phase separation in T cell-derived giant plasma membrane vesicles. Similarly, in a short-term human pilot study, supplementation of fish oil (4 g n-3 PUFA/d) for 6 weeks in healthy subjects significantly elevated EPA (20 : 5n-3) levels in CD4+ T cell membrane phospholipids, and reduced membrane lipid order. These results demonstrate that the dynamic reshaping of human CD4+ T cell plasma membrane organisation by n-3 PUFA may modulate down-stream clonal expansion.
|
[
"Naive T cells undergo metabolic reprogramming to support the increased energetic and biosynthetic demands of effector T cell function. However, how nutrient availability influences T cell metabolism and function remains poorly understood. Here we report plasticity in effector T cell metabolism in response to changing nutrient availability. Activated T cells were found to possess a glucose-sensitive metabolic checkpoint controlled by the energy sensor AMP-activated protein kinase (AMPK) that regulated mRNA translation and glutamine-dependent mitochondrial metabolism to maintain T cell bioenergetics and viability. T cells lacking AMPKα1 displayed reduced mitochondrial bioenergetics and cellular ATP in response to glucose limitation in vitro or pathogenic challenge in vivo. Finally, we demonstrated that AMPKα1 is essential for T helper 1 (Th1) and Th17 cell development and primary T cell responses to viral and bacterial infections in vivo. Our data highlight AMPK-dependent regulation of metabolic homeostasis as a key regulator of T cell-mediated adaptive immunity.",
"Membrane subdomains have been implicated in T cell signaling, although their properties and mechanisms of formation remain controversial. Here, we have used single-molecule and scanning confocal imaging to characterize the behavior of GFP-tagged signaling proteins in Jurkat T cells. We show that the coreceptor CD2, the adaptor protein LAT, and tyrosine kinase Lck cocluster in discrete microdomains in the plasma membrane of signaling T cells. These microdomains require protein-protein interactions mediated through phosphorylation of LAT and are not maintained by interactions with actin or lipid rafts. Using a two color imaging approach that allows tracking of single molecules relative to the CD2/LAT/Lck clusters, we demonstrate that these microdomains exclude and limit the free diffusion of molecules in the membrane but also can trap and immobilize specific proteins. Our data suggest that diffusional trapping through protein-protein interactions creates microdomains that concentrate or exclude cell surface proteins to facilitate T cell signaling.",
"Lateral heterogeneities in the classical fluid-mosaic model of cell membranes are now envisaged as domains or 'rafts' that are enriched in (glyco)sphingolipids, cholesterol, specific membrane proteins and glycosylphosphatidylinositol (GPI)-anchored proteins. These rafts dictate the sorting of associated proteins and/or provide sites for assembling cytoplasmic signalling molecules. However, there is no direct evidence that rafts exist in living cells. We have now measured the extent of energy transfer between isoforms of the folate receptor bound to a fluorescent analogue of folic acid, in terms of the dependence of fluorescence polarization on fluorophore densities in membranes. We find that the extent of energy transfer for the GPI-anchored folate-receptor isoform is density-independent, which is characteristic of organization in sub-pixel-sized domains at the surface of living cells; however, the extent of energy transfer for the transmembrane-anchored folate-receptor isoform was density-dependent, which is consistent with a random distribution. These domains are likely to be less than 70 nm in diameter and are disrupted by removal of cellular cholesterol. These results indicate that lipid-linked proteins are organized in cholesterol-dependent submicron-sized domains. Our methodology offers a new way of monitoring nanometre-scale association between molecules in living cells.",
"Lipid rafts are cholesterol and saturated lipid-rich, nanometer sized membrane domains that are hypothesized to play an important role in compartmentalization and spatiotemporal regulation of cellular signaling. Lipid rafts contribute to the plasma membrane order and to its spatial asymmetry, as well. The raft nanodomains on the surface of CD4(+) T lymphocytes coalesce during their interaction with antigen presenting cells (APCs). Sensing of foreign antigen by the antigen receptor on CD4(+) T cells occurs during these cell-cell interactions. In response to foreign antigen the CD4(+) T cells proliferate, allowing the expansion of few antigen-specific primary CD4(+) T cell clones. Proliferating CD4(+) T cells specialize in their function by undergoing differentiation into appropriate effectors tailored to mount an effective adaptive immune response against the invading pathogen. To investigate the role of lipid raft-based membrane order in the clonal expansion phase of primary CD4(+) T cells, we have disrupted membrane order by incorporating an oxysterol, 7-ketocholesterol (7-KC), into the plasma membrane of primary CD4(+) T cells expressing a T cell receptor specific to chicken ovalbumin323-339 peptide sequence and tested their antigen-specific response. We report that 7-KC, at concentrations that disrupt lipid rafts, significantly diminish the c-Ovalbumin323-339 peptide-specific clonal expansion of primary CD4(+) T cells. Our findings suggest that lipid raft-based membrane order is important for clonal expansion of CD4(+) T cells in response to a model peptide.",
"Powerful growth-inhibitory action has been shown for n-3 polyunsaturated fatty acids against colon cancer cells. We have previously described their ability to inhibit proliferation of colon epithelial cells in patients at high risk of colon cancer. In the work reported here we investigated the ability of docosahexaenoic acid (DHA) to potentiate the antineoplastic activity of 5-fluorouracil (5-FU) in p53-wildtype (LS-174 and Colo 320) and p53-mutant (HT-29 and Colo 205) human colon cancer cells. When in combination with DHA, 5-FU was used at concentrations ranging from 0.1 to 1.0 microM, much lower than those currently found in plasma patients after infusion of this drug. Similarly, the DHA concentrations (< or =10 microM) used in combination with 5-FU were lower than those widely used in vitro and known to cause peroxidative effects in vivo. Whereas the cells showed different sensitivity to the growth-inhibitory action of 5-FU, DHA reduced cell growth independently of p53 cellular status. DHA synergized with 5-FU in reducing colon cancer cell growth. The potentiating effect of DHA was attributable to the enhancement of the proapoptotic effect of 5-FU. DHA markedly increased the inhibitory effect of 5-FU on the expression of the antiapoptotic proteins BCL-2 and BCL-XL, and induced overexpression of c-MYC which has recently been shown to drive apoptosis and, when overexpressed, to sensitize cancer cells to the action of proapoptotic agents, including 5-FU. Our results indicate that DHA strongly increases the antineoplastic effects of low concentrations of 5-FU. Overall, the results suggest that combinations of low doses of the two compounds could represent a chemotherapeutic approach with low toxicity.",
"After activation, T lymphocytes restructure their cell surface to form membrane domains at T cell receptor (TCR)-signaling foci and immunological synapses (ISs). To address whether these rearrangements involve alteration in the structure of the plasma membrane bilayer, we used the fluorescent probe Laurdan to visualize its lipid order. We observed a condensation of the plasma membrane at TCR activation sites. The formation of ordered domains depends on the presence of the transmembrane protein linker for the activation of T cells and Src kinase activity. Moreover, these ordered domains are stabilized by the actin cytoskeleton. Membrane condensation occurs upon TCR stimulation alone but is prolonged by CD28 costimulation with TCR. In ISs, which are formed by conjugates of TCR transgenic T lymphocytes and cognate antigen-presenting cells, similar condensed membrane phases form first in central regions and later at the periphery of synapses. The formation of condensed membrane domains at T cell activation sites biophysically reflects membrane raft accumulation, which has potential implications for signaling at ISs.",
"Glycerol-3-phosphate acyltransferase-1 is the first rate limiting step in de novo glycerophospholipid synthesis. We have previously demonstrated that GPAT-1 deletion can significantly alter T cell function resulting in a T cell phenotype similar to that seen in aging. Recent studies have suggested that changes in the metabolic profile of T cells are responsible for defining specific effector functions and T cell subsets. Therefore, we determined whether T cell dysfunction in GPAT-1 (-/-) CD4(+) T cells could be explained by changes in cellular metabolism. We show here for the first time that GPAT-1 (-/-) CD4(+) T cells exhibit several key metabolic defects. Striking decreases in both the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) were observed in GPAT-1 (-/-) CD4(+) T cells following CD3/CD28 stimulation indicating an inherent cellular defect in energy production. In addition, the spare respiratory capacity (SRC) of GPAT-1 (-/-) CD4+ T cells, a key indicator of their ability to cope with mitochondrial stress was significantly decreased. We also observed a significant reduction in mitochondrial membrane potential in GPAT-1 (-/-) CD4(+) T cells compared to their WT counterparts, indicating that GPAT-1 deficiency results in altered or dysfunctional mitochondria. These data demonstrate that deletion of GPAT-1 can dramatically alter total cellular metabolism under conditions of increased energy demand. Furthermore, altered metabolic response following stimulation may be the defining mechanism underlying T cell dysfunction in GPAT-1 (-/-) CD4(+) T cells. Taken together, these results indicate that GPAT-1 is essential for the response to the increased metabolic demands associated with T cell activation.",
"CD2 mediates T cell adhesion via its ectodomain and signal transduction utilizing its 117-amino acid cytoplasmic tail. Here we show that a significant fraction of human CD2 molecules is inducibly recruited into lipid rafts upon CD2 cross-linking by a specific pair of mitogenic anti-CD2 monoclonal antibodies (anti-T11(2) + anti-T11(3)) or during cellular conjugate formation by CD58, the physiologic ligand expressed on antigen-presenting cells. Translocation to lipid microdomains is independent of the T cell receptor (TCR) and, unlike inducible TCR-raft association, requires no tyrosine phosphorylation. Structural integrity of rafts is necessary for CD2-stimulated elevation of intracellular free calcium and tyrosine phosphorylation of cellular substrates. Whereas murine CD2 contains two membrane-proximal intracellular cysteines, partitioning CD2 into cholesterol-rich lipid rafts constitutively, human CD2 has no cytoplasmic cysteines. Mapping studies using CD2 point mutation, deletion, and chimeric molecules suggest that conformational change in the CD2 ectodomain participates in inducible raft association and excludes the membrane-proximal N-linked glycans, the transmembrane segment, and the CD2 cytoplasmic region (residues 8-117) as necessary for translocation. Translocation of CD2 into lipid rafts may reorganize the membrane into an activation-ready state prior to TCR engagement by a peptide associated with a major histocompatibility complex molecule, accounting for synergistic T cell stimulation by CD2 and the TCR.",
"Ras proteins occupy dynamic plasma membrane nanodomains called nanoclusters. The significance of this spatial organization is unknown. Here we show, using in silico and in vivo analyses of mitogen-activated protein (MAP) kinase signalling, that Ras nanoclusters operate as sensitive switches, converting graded ligand inputs into fixed outputs of activated extracellular signal-regulated kinase (ERK). By generating Ras nanoclusters in direct proportion to ligand input, cells build an analogue-digital-analogue circuit relay that transmits a signal across the plasma membrane with high fidelity. Signal transmission is completely dependent on Ras spatial organization and fails if nanoclustering is abrogated. A requirement for high-fidelity signalling may explain the non-random distribution of other plasma membrane signalling complexes."
] |
Metabolic heterogeneity in human tumors
|
Glucose is a key metabolite used by cancer cells to generate ATP, maintain redox state and create biomass. Glucose can be catabolized to lactate in the cytoplasm, which is termed glycolysis, or alternatively can be catabolized to carbon dioxide and water in the mitochondria via oxidative phosphorylation. Metabolic heterogeneity exists in a subset of human tumors, with some cells maintaining a glycolytic phenotype while others predominantly utilize oxidative phosphorylation. Cells within tumors interact metabolically with transfer of catabolites from supporting stromal cells to adjacent cancer cells. The Reverse Warburg Effect describes when glycolysis in the cancer-associated stroma metabolically supports adjacent cancer cells. This catabolite transfer, which induces stromal-cancer metabolic coupling, allows cancer cells to generate ATP, increase proliferation, and reduce cell death. Catabolites implicated in metabolic coupling include the monocarboxylates lactate, pyruvate, and ketone bodies. Monocarboxylate transporters (MCT) are critically necessary for release and uptake of these catabolites. MCT4 is involved in the release of monocarboxylates from cells, is regulated by catabolic transcription factors such as hypoxia inducible factor 1 alpha (HIF1A) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and is highly expressed in cancer-associated fibroblasts. Conversely, MCT1 is predominantly involved in the uptake of these catabolites and is highly expressed in a subgroup of cancer cells. MYC and TIGAR, which are genes involved in cellular proliferation and anabolism, are inducers of MCT1. Profiling human tumors on the basis of an altered redox balance and intra-tumoral metabolic interactions may have important biomarker and therapeutic implications. Alterations in the redox state and mitochondrial function of cells can induce metabolic coupling. Hence, there is interest in redox and metabolic modulators as anticancer agents. Also, markers of metabolic coupling have been associated with poor outcomes in numerous human malignancies and may be useful prognostic and predictive biomarkers.
|
[
"The tumor microenvironment frequently displays abnormal cellular metabolism, which contributes to aggressive behavior. Metformin inhibits mitochondrial oxidative phosphorylation, altering metabolism. Though the mechanism is unclear, epidemiologic studies show an association between metformin use and improved outcomes in head and neck squamous cell carcinoma (HNSCC). We sought to determine if metformin alters metabolism and apoptosis in HNSCC tumors. Window of opportunity trial of metformin between diagnostic biopsy and resection. Participants were patients with newly diagnosed HNSCC. Fifty patients were enrolled, and 39 completed a full-treatment course. Metformin was titrated to standard diabetic dose (2,000 mg/day) for a course of 9 or more days prior to surgery. Immunohistochemistry (IHC) for the metabolic markers caveolin-1 (CAV1), B-galactosidase (GALB), and monocarboxylate transporter 4 (MCT4), as well as the Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis assay and Ki-67 IHC, were performed in pre- and postmetformin specimens. Exploratory mass spectroscopy imaging (MSI) to assess lactate levels also was performed in three subjects. Metformin was well tolerated. The average treatment course was 13.6 days. Posttreatment specimens showed a significant increase in stromal CAV1 (P < 0.001) and GALB (P < 0.005), as well as tumor cell apoptosis by TUNEL assay (P < 0.001). There was no significant change in stromal MCT4 expression or proliferation measured by Ki67. Lactate levels in carcinoma cells were increased 2.4-fold postmetformin (P < 0.05), as measured by MSI. Metformin increases markers of reduced catabolism and increases senescence in stromal cells as well as carcinoma cell apoptosis. This study demonstrates that metformin modulates metabolism in the HNSCC microenvironment. 4. Laryngoscope, 127:1808-1815, 2017.",
"Dysregulated metabolism is a hallmark of cancer cell lines, but little is known about the fate of glucose and other nutrients in tumors growing in their native microenvironment. To study tumor metabolism in vivo, we used an orthotopic mouse model of primary human glioblastoma (GBM). We infused (13)C-labeled nutrients into mice bearing three independent GBM lines, each with a distinct set of mutations. All three lines displayed glycolysis, as expected for aggressive tumors. They also displayed unexpected metabolic complexity, oxidizing glucose via pyruvate dehydrogenase and the citric acid cycle, and using glucose to supply anaplerosis and other biosynthetic activities. Comparing the tumors to surrounding brain revealed obvious metabolic differences, notably the accumulation of a large glutamine pool within the tumors. Many of these same activities were conserved in cells cultured ex vivo from the tumors. Thus GBM cells utilize mitochondrial glucose oxidation during aggressive tumor growth in vivo.",
"Otto Warburg pioneered quantitative investigations of cancer cell metabolism, as well as photosynthesis and respiration. Warburg and co-workers showed in the 1920s that, under aerobic conditions, tumour tissues metabolize approximately tenfold more glucose to lactate in a given time than normal tissues, a phenomenon known as the Warburg effect. However, this increase in aerobic glycolysis in cancer cells is often erroneously thought to occur instead of mitochondrial respiration and has been misinterpreted as evidence for damage to respiration instead of damage to the regulation of glycolysis. In fact, many cancers exhibit the Warburg effect while retaining mitochondrial respiration. We re-examine Warburg's observations in relation to the current concepts of cancer metabolism as being intimately linked to alterations of mitochondrial DNA, oncogenes and tumour suppressors, and thus readily exploitable for cancer therapy.",
"To investigate the clinicopathological significance and prognostic value of caveolin-1 (CAV-1) in both tumor and stromal cells in colorectal cancer (CRC). A total of 178 patients with CRC were included in this study. The correlation between CAV-1 expression and clinicopathologic features and survival was studied. CAV-1 expression was detected in tumor and stromal cells. The expression of stromal CAV-1 was closely associated with histological type (P=0.022), pathologic tumor-node-metastasis stage (P=0.047), pathologic N stage (P=0.035) and recurrence (P=0.000). However, tumor cell CAV-1 did not show any correlation with clinical parameters. Additionally, the loss of stromal CAV-1 expression was associated with shorter disease-free survival (P=0.000) and overall survival (P=0.000). Multivariate analysis revealed that the loss of stromal CAV-1 expression was an independent prognostic factor for both overall survival (P=0.014) and disease-free survival (P=0.006). The loss of stromal CAV-1 expression in CRC was associated with poor prognosis and could be a prognostic factor for CRC patients.",
"Mammalian cells fuel their growth and proliferation through the catabolism of two main substrates: glucose and glutamine. Most of the remaining metabolites taken up by proliferating cells are not catabolized, but instead are used as building blocks during anabolic macromolecular synthesis. Investigations of phosphoinositol 3-kinase (PI3K) and its downstream effector AKT have confirmed that these oncogenes play a direct role in stimulating glucose uptake and metabolism, rendering the transformed cell addicted to glucose for the maintenance of survival. In contrast, less is known about the regulation of glutamine uptake and metabolism. Here, we report that the transcriptional regulatory properties of the oncogene Myc coordinate the expression of genes necessary for cells to engage in glutamine catabolism that exceeds the cellular requirement for protein and nucleotide biosynthesis. A consequence of this Myc-dependent glutaminolysis is the reprogramming of mitochondrial metabolism to depend on glutamine catabolism to sustain cellular viability and TCA cycle anapleurosis. The ability of Myc-expressing cells to engage in glutaminolysis does not depend on concomitant activation of PI3K or AKT. The stimulation of mitochondrial glutamine metabolism resulted in reduced glucose carbon entering the TCA cycle and a decreased contribution of glucose to the mitochondrial-dependent synthesis of phospholipids. These data suggest that oncogenic levels of Myc induce a transcriptional program that promotes glutaminolysis and triggers cellular addiction to glutamine as a bioenergetic substrate.",
"The cancer stem cell (CSC) hypothesis suggests that neoplastic clones are maintained exclusively by a rare fraction of cells with stem cell properties. Although the existence of CSCs in human leukaemia is established, little evidence exists for CSCs in solid tumours, except for breast cancer. Recently, we prospectively isolated a CD133+ cell subpopulation from human brain tumours that exhibited stem cell properties in vitro. However, the true measures of CSCs are their capacity for self renewal and exact recapitulation of the original tumour. Here we report the development of a xenograft assay that identified human brain tumour initiating cells that initiate tumours in vivo. Only the CD133+ brain tumour fraction contains cells that are capable of tumour initiation in NOD-SCID (non-obese diabetic, severe combined immunodeficient) mouse brains. Injection of as few as 100 CD133+ cells produced a tumour that could be serially transplanted and was a phenocopy of the patient's original tumour, whereas injection of 10(5) CD133- cells engrafted but did not cause a tumour. Thus, the identification of brain tumour initiating cells provides insights into human brain tumour pathogenesis, giving strong support for the CSC hypothesis as the basis for many solid tumours, and establishes a previously unidentified cellular target for more effective cancer therapies.",
"The idea that conversion of glucose to ATP is an attractive target for cancer therapy has been supported in part by the observation that glucose deprivation induces apoptosis in rodent cells transduced with the proto-oncogene MYC, but not in the parental line. Here, we found that depletion of glucose killed normal human cells irrespective of induced MYC activity and by a mechanism different from apoptosis. However, depletion of glutamine, another major nutrient consumed by cancer cells, induced apoptosis depending on MYC activity. This apoptosis was preceded by depletion of the Krebs cycle intermediates, was prevented by two Krebs cycle substrates, but was unrelated to ATP synthesis or several other reported consequences of glutamine starvation. Our results suggest that the fate of normal human cells should be considered in evaluating nutrient deprivation as a strategy for cancer therapy, and that understanding how glutamine metabolism is linked to cell viability might provide new approaches for treatment of cancer.",
"The mitochondrial electron transport chain (ETC) enables many metabolic processes, but why its inhibition suppresses cell proliferation is unclear. It is also not well understood why pyruvate supplementation allows cells lacking ETC function to proliferate. We used a CRISPR-based genetic screen to identify genes whose loss sensitizes human cells to phenformin, a complex I inhibitor. The screen yielded GOT1, the cytosolic aspartate aminotransferase, loss of which kills cells upon ETC inhibition. GOT1 normally consumes aspartate to transfer electrons into mitochondria, but, upon ETC inhibition, it reverses to generate aspartate in the cytosol, which partially compensates for the loss of mitochondrial aspartate synthesis. Pyruvate stimulates aspartate synthesis in a GOT1-dependent fashion, which is required for pyruvate to rescue proliferation of cells with ETC dysfunction. Aspartate supplementation or overexpression of an aspartate transporter allows cells without ETC activity to proliferate. Thus, enabling aspartate synthesis is an essential role of the ETC in cell proliferation.",
"Cancer cells show increased glycolysis and take advantage of this metabolic pathway to generate ATP. The TP53-induced glycolysis and apoptosis regulator (TIGAR) inhibits aerobic glycolysis and protects tumor cells from intracellular reactive oxygen species (ROS)-associated apoptosis. However, the function of TIGAR in glycolysis and survival of acute myeloid leukemia cells remains unclear. We analyzed TIGAR expression in cytogenetically normal (CN-) AML patients and the correlations with clinical and biological parameters. In vivo and in vitro, we tested whether glycolysis may induce TIGAR expression and evaluated the combination effect of glycolysis inhibitor and TIGAR knockdown on human leukemia cell proliferation. High TIGAR expression was an independent predictor of poor survival and high incidence of relapse in adult patients with CN-AML. TIGAR also showed high expression in multiple human leukemia cell lines and knockdown of TIGAR activated glycolysis through PFKFB3 upregulation in human leukemia cells. Knockdown of TIGAR inhibited the proliferation of human leukemia cells and sensitized leukemia cells to glycolysis inhibitor both in vitro and in vivo. Furthermore, TIGAR knockdown in combination with glycolysis inhibitor 2-DG led leukemia cells to apoptosis. In addition, the p53 activator Nutlin-3α showed a significant combinational effect with TIGAR knockdown in leukemia cells. However, TIGAR expression and its anti-apoptotic effects were uncoupled from overexpression of exogenous p53 in leukemia cells. TIGAR might be a predictor of poor survival and high incidence of relapse in AML patients, and the combination of TIGAR inhibitors with anti-glycolytic agents may be novel therapies for the future clinical use in AML patients."
] |
Sequence alignment-free identification of recent and chronic hepatitis C virus infections using next-generation sequencing data
|
Identification of acute or recent hepatitis C virus (HCV) infections is important for detecting outbreaks and devising timely public health interventions for interruption of transmission. Epidemiological investigations and chemistry-based laboratory tests are 2 main approaches that are available for identification of acute HCV infection. However, owing to complexity, both approaches are not efficient. Here, we describe a new sequence alignment-free method to discriminate between recent (R) and chronic (C) HCV infection using next-generation sequencing (NGS) data derived from the HCV hypervariable region 1 (HVR1).
|
[
"DiProDB (http://diprodb.fli-leibniz.de) is a database of conformational and thermodynamic dinucleotide properties. It includes datasets both for DNA and RNA, as well as for single and double strands. The data have been shown to be important for understanding different aspects of nucleic acid structure and function, and they can also be used for encoding nucleic acid sequences. The database is intended to facilitate further applications of dinucleotide properties. A number of property datasets is highly correlated. Therefore, the database comes with a correlation analysis facility. Authors having determined new sets of dinucleotide property values are invited to submit these data to DiProDB.",
"More and more genomes are being sequenced, and to keep up with the pace of sequencing projects, automated annotation techniques are required. One of the most challenging problems in genome annotation is the identification of the core promoter. Because the identification of the transcription initiation region is such a challenging problem, it is not yet a common practice to integrate transcription start site prediction in genome annotation projects. Nevertheless, better core promoter prediction can improve genome annotation and can be used to guide experimental work. Comparing the average structural profile based on base stacking energy of transcribed, promoter and intergenic sequences demonstrates that the core promoter has unique features that cannot be found in other sequences. We show that unsupervised clustering by using self-organizing maps can clearly distinguish between the structural profiles of promoter sequences and other genomic sequences. An implementation of this promoter prediction program, called ProSOM, is available and has been compared with the state-of-the-art. We propose an objective, accurate and biologically sound validation scheme for core promoter predictors. ProSOM performs at least as well as the software currently available, but our technique is more balanced in terms of the number of predicted sites and the number of false predictions, resulting in a better all-round performance. Additional tests on the ENCODE regions of the human genome show that 98% of all predictions made by ProSOM can be associated with transcriptionally active regions, which demonstrates the high precision. Predictions for the human genome, the validation datasets and the program (ProSOM) are available upon request.",
"A new method for the prediction of promoter regions based on atomic molecular dynamics simulations of small oligonucleotides has been developed. The method works independently of gene structure conservation and orthology and of the presence of detectable sequence features. Results obtained with our method confirm the existence of a hidden physical code that modulates genome expression.",
"RNA secondary structure is often predicted from sequence by free energy minimization. Over the past two years, advances have been made in the estimation of folding free energy change, the mapping of secondary structure and the implementation of computer programs for structure prediction. The trends in computer program development are: efficient use of experimental mapping of structures to constrain structure prediction; use of statistical mechanics to improve the fidelity of structure prediction; inclusion of pseudoknots in secondary structure prediction; and use of two or more homologous sequences to find a common structure."
] |
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.