title: Human Cyclin a Is Required for Mitosis until Mid Prophase

text: We have used microinjection and time-lapse video microscopy to study the role of cyclin A in mitosis. We have injected purified, active cyclin A/cyclin-dependent kinase 2 (CDK2) into synchronized cells at specific points in the cell cycle and assayed its effect on cell division. We find that cyclin A/CDK2 will drive G2 phase cells into mitosis within 30 min of microinjection, up to 4 h before control cells enter mitosis. Often this premature mitosis is abnormal; the chromosomes do not completely condense and daughter cells fuse. Remarkably, microinjecting cyclin A/CDK2 into S phase cells has no effect on progress through the following G2 phase or mitosis. In complementary experiments we have microinjected the amino terminus of p21Cip1/Waf1/Sdi1 (p21N) into cells to inhibit cyclin A/CDK2 activity. We find that p21N will prevent S phase or G2 phase cells from entering mitosis, and will cause early prophase cells to return to interphase. These results suggest that cyclin A/CDK2 is a rate-limiting component required for entry into mitosis, and for progress through mitosis until late prophase. They also suggest that cyclin A/CDK2 may be the target of the recently described prophase checkpoint.


title: Brief, four-session group CBT reduces binge eating behaviors among bariatric surgery candidates.

text:
BACKGROUND The objective of this study was to evaluate the effectiveness of a brief, 4-session cognitive behavioral, group psychotherapy for binge eating among bariatric surgery candidates at an academic medical center. Binge eating behaviors have been linked to poorer outcomes among bariatric surgery patients, and binge eating disorder have be considered a contraindication in surgery programs, some of which have mandated preoperative binge eating treatment. However, no previous studies have examined whether a preoperative binge eating intervention could successfully reduce binge eating behaviors among severely obese bariatric surgery candidates.
 METHODS A total of 243 bariatric surgery candidates completed a brief cognitive behavioral group treatment for binge eating behaviors and were administered the Binge Eating Scale and reported the number of weekly binge eating episodes at the initial psychological evaluation and again after the group sessions. The study used a pre-post intervention design.
 RESULTS The results suggested significant reductions in both binge eating behaviors and cognitions and binge eating episodes after the group intervention. The intervention's effectiveness did not differ according to gender or ethnicity (black versus white).
 CONCLUSION A brief cognitive behavioral intervention can reduce binge eating behaviors among bariatric surgery candidates. Given the potential influence of binge eating on outcomes, bariatric surgery programs could benefit by treating binge eating before surgery.


title: Influence of smoking cessation after diagnosis of early stage lung cancer on prognosis: systematic review of observational studies with meta-analysis

text:
OBJECTIVE To systematically review the evidence that smoking cessation after diagnosis of a primary lung tumour affects prognosis.
 DESIGN Systematic review with meta-analysis.
 DATA SOURCES CINAHL (from 1981), Embase (from 1980), Medline (from 1966), Web of Science (from 1966), CENTRAL (from 1977) to December 2008, and reference lists of included studies.    STUDY SELECTION Randomised controlled trials or observational longitudinal studies that measured the effect of quitting smoking after diagnosis of lung cancer on prognostic outcomes, regardless of stage at presentation or tumour histology, were included.
 DATA EXTRACTION Two researchers independently identified studies for inclusion and extracted data. Estimates were combined by using a random effects model, and the I(2) statistic was used to examine heterogeneity. Life tables were used to model five year survival for early stage non-small cell lung cancer and limited stage small cell lung cancer, using death rates for continuing smokers and quitters obtained from this review.
 RESULTS In 9/10 included studies, most patients studied were diagnosed as having an early stage lung tumour. Continued smoking was associated with a significantly increased risk of all cause mortality (hazard ratio 2.94, 95% confidence interval 1.15 to 7.54) and recurrence (1.86, 1.01 to 3.41) in early stage non-small cell lung cancer and of all cause mortality (1.86, 1.33 to 2.59), development of a second primary tumour (4.31, 1.09 to 16.98), and recurrence (1.26, 1.06 to 1.50) in limited stage small cell lung cancer. No study contained data on the effect of quitting smoking on cancer specific mortality or on development of a second primary tumour in non-small cell lung cancer. Life table modelling on the basis of these data estimated 33% five year survival in 65 year old patients with early stage non-small cell lung cancer who continued to smoke compared with 70% in those who quit smoking. In limited stage small cell lung cancer, an estimated 29% of continuing smokers would survive for five years compared with 63% of quitters on the basis of the data from this review.
 CONCLUSIONS This review provides preliminary evidence that smoking cessation after diagnosis of early stage lung cancer improves prognostic outcomes. From life table modelling, the estimated number of deaths prevented is larger than would be expected from reduction of cardiorespiratory deaths after smoking cessation, so most of the mortality gain is likely to be due to reduced cancer progression. These findings indicate that offering smoking cessation treatment to patients presenting with early stage lung cancer may be beneficial.


title: Epigenetic reprogramming and induced pluripotency.

text: The cloning of animals from adult cells has demonstrated that the developmental state of adult cells can be reprogrammed into that of embryonic cells by uncharacterized factors within the oocyte. More recently, transcription factors have been identified that can induce pluripotency in somatic cells without the use of oocytes, generating induced pluripotent stem (iPS) cells. iPS cells provide a unique platform to dissect the molecular mechanisms that underlie epigenetic reprogramming. Moreover, iPS cells can teach us about principles of normal development and disease, and might ultimately facilitate the treatment of patients by custom-tailored cell therapy.


title: Sex differences in mortality following acute coronary syndromes.

text:
CONTEXT Conflicting information exists about whether sex differences modulate short-term mortality following acute coronary syndromes (ACS).
 OBJECTIVES To investigate the relationship between sex and 30-day mortality in ACS, and to determine whether this relationship was modified by clinical syndrome or coronary anatomy using a large database across the spectrum of ACS and adjusting for potentially confounding clinical covariates.
 DESIGN, SETTING, AND PARTICIPANTS A convenience sample of patients pooled from 11 independent, international, randomized ACS clinical trials between 1993 and 2006 whose databases are maintained at the Duke Clinical Research Institute, Durham, North Carolina. Of 136 247 patients, 38 048 (28%) were women; 102 004 (26% women) with ST-segment elevation myocardial infarction (STEMI), 14 466 (29% women) with non-STEMI (NSTEMI), and 19 777 (40% women) with unstable angina.
 MAIN OUTCOME MEASURE Thirty-day mortality following ACS.
 RESULTS Thirty-day mortality was 9.6% in women and 5.3% in men (odds ratio [OR], 1.91; 95% confidence interval [CI], 1.83-2.00). After multivariable adjustment, mortality was not significantly different between women and men (adjusted OR, 1.06; 95% CI, 0.99-1.15). A significant sex by type of ACS interaction was demonstrated (P < .001). In STEMI, 30-day mortality was higher among women (adjusted OR, 1.15; 95% CI, 1.06-1.24), whereas in NSTEMI (adjusted OR, 0.77; 95% CI, 0.63-0.95) and unstable angina, mortality was lower among women (adjusted OR, 0.55; 95% CI, 0.43-0.70). In a cohort of 35 128 patients with angiographic data, women more often had nonobstructive (15% vs 8%) and less often had 2-vessel (25% vs 28%) and 3-vessel (23% vs 26%) coronary disease, regardless of ACS type. After additional adjustment for angiographic disease severity, 30-day mortality among women was not significantly different than men, regardless of ACS type. The relationship between sex and 30-day mortality was similar across the levels of angiographic disease severity (P for interaction = .70).
 CONCLUSIONS Sex-based differences existed in 30-day mortality among patients with ACS and vary depending on clinical presentation. However, these differences appear to be largely explained by clinical differences at presentation and severity of angiographically documented disease.


title: Membrane topology of the 60-kDa Oxa1p homologue from Escherichia coli.

text: We have characterized the membrane topology of a 60-kDa inner membrane protein from Escherichia coli that is homologous to the recently identified Oxa1p protein in Saccharomyces cerevisiae mitochondria implicated in the assembly of mitochondrial inner membrane proteins. Hydrophobicity and alkaline phosphatase fusion analyses suggest a membrane topology with six transmembrane segments, including an N-terminal signal-anchor sequence not present in mitochondrial Oxa1p. In contrast to partial N-terminal fusion protein constructs, the full-length protein folds into a protease-resistant conformation, suggesting that important folding determinants are present in the C-terminal part of the molecule.


title: Identifying and Characterizing Interplay between Hepatitis B Virus X Protein and Smc5/6

text: Hepatitis B X protein (HBx) plays an essential role in the hepatitis B virus (HBV) replication cycle, but the function of HBx has been elusive until recently. It was recently shown that transcription from the HBV genome (covalently-closed circular DNA, cccDNA) is inhibited by the structural maintenance of chromosome 5/6 complex (Smc5/6), and that a key function of HBx is to redirect the DNA-damage binding protein 1 (DDB1) E3 ubiquitin ligase to target this complex for degradation. By doing so, HBx alleviates transcriptional repression by Smc5/6 and stimulates HBV gene expression. In this review, we discuss in detail how the interplay between HBx and Smc5/6 was identified and characterized. We also discuss what is known regarding the repression of cccDNA transcription by Smc5/6, the timing of HBx expression, and the potential role of HBx in promoting hepatocellular carcinoma (HCC).


title: Hepatitis C Virus (HCV) Vertical Transmission in 12-Month-Old Infants Born to HCV-Infected Women and Assessment of Maternal Risk Factors

text: Background.   Hepatitis C virus (HCV) is an underappreciated cause of pediatric liver disease, most frequently acquired by vertical transmission (VT). Current guidelines that include the option of screening infants for HCV RNA at 1-2 months are based on data prior to current real-time polymerase chain reaction (PCR)-based testing. Previous studies have demonstrated VT rates of 4%-15% and an association with high maternal viral load. We evaluated HCV RNA in infants with HCV VT and assessed maternal risk factors in a prospective cohort in Cairo, Egypt. Methods.   Pregnant women were screened for HCV from December 2012 to March 2014. For those with HCV viremia, their infants were tested at 12 months for HCV RNA using real-time PCR. Maternal risk factors assessed for HCV VT association included HCV RNA levels, mode of delivery, and maternal IL28B genotype. Results.   Of 2514 women screened, a total of 54 women were viremic (2.1%) and delivered 56 infants. Of those, 51 infants of 49 women were tested at 12 months of age. Only 7 infants were viremic, with an HCV VT rate of 14.3% (7 of 49). Median HCV RNA in the infants was 2100 IU/mL. None of the maternal risk factors analyzed were associated with transmission. Conclusions.   In Egypt where HCV is highly endemic, we observed an overall 12-month HCV VT rate of 14.3%. Further studies should focus on better identification of pregnant women more likely to vertically transmit HCV and earlier testing of infants to identify those likely to develop chronicity.


title: Long-term follow-up of cervical disease in women screened by cytology and HPV testing: results from the HART study

text:
BACKGROUND Several studies have shown that testing for high-risk human papillomavirus (HPV) types results in an improved sensitivity for CIN2+, compared with cytology, although with a somewhat lower specificity.
 METHODS We obtained follow-up results, with at least one smear after participation in the HART study, which compared HPV testing (HC-II) with cytology as a primary screening modality.
 RESULTS With a median follow-up of 6 years, 42 additional cases of CIN2+ were identified; women who were HPV positive at baseline were more likely to develop CIN2+ than those who were HPV negative (hazard ratio (HR) 17.2; 95% confidence interval (CI) (9.3-31.6)) and the risk increased with increasing viral load. Compared with HPV-negative women (relative light unit (RLU) <1), the HR (95% CI) was 5.4 (1.6, 18.2) for 1-10 RLU and 25.5 (13.6, 47.9) for RLU > or = 10. Positive cytology (borderline or worse compared with negative) was also predictive of developing CIN2, although to a lesser extent (HR 8.7; 95% CI (4.5-17.1)). Only one case of CIN3 and three cases of CIN2 were found in women who showed a positive cytology result but were HPV negative at baseline.
 CONCLUSION After 5 years of follow-up, CIN2+ occurred in 0.23% of women who were HPV negative at baseline compared with 0.48% of women who showed a negative cytology result, indicating a much longer low-risk interval for CIN2+ after HPV testing.


title: The Burden of Trachoma in Ayod County of Southern Sudan

text:
BACKGROUND Blindness due to trachoma is avoidable through Surgery, Antibiotics, Facial hygiene and Environmental improvements (SAFE). Recent surveys have shown trachoma to be a serious cause of blindness in Southern Sudan. We conducted this survey in Ayod County of Jonglei State to estimate the need for intervention activities to eliminate blinding trachoma.
 METHODOLOGY AND FINDINGS A cross-sectional two-stage cluster random survey was conducted in November 2006. All residents of selected households were clinically assessed for trachoma using the World Health Organization (WHO) simplified grading scheme. A total of 2,335 people from 392 households were examined, of whom 1,107 were over 14 years of age. Prevalence of signs of active trachoma in children 1-9 years of age was: trachomatous inflammation follicular (TF) = 80.1% (95% confidence interval [CI], 73.9-86.3); trachomatous inflammation intense (TI) = 60.7% (95% CI, 54.6-66.8); and TF and/or TI (active trachoma) = 88.3% (95% CI, 83.7-92.9). Prevalence of trachomatous trichiasis (TT) was 14.6% (95% CI, 10.9-18.3) in adults over 14 years of age; 2.9% (95% CI, 0.4-5.3) in children 1-14 years of age; and 8.4% (95% CI, 5.5-11.3) overall. The prevalence of corneal opacity in persons over 14 years of age with TT was 6.4% (95% CI, 4.5-8.3). No statistically significant difference was observed in the prevalence of trachoma signs between genders. Trachoma affected almost all households surveyed: 384/392 (98.0%) had at least one person with active trachoma and 130 (33.2%) had at least one person with trichiasis.
 CONCLUSIONS Trachoma is an unnecessary public health problem in Ayod. The high prevalence of active trachoma and trichiasis confirms the severe burden of blinding trachoma found in other post-conflict areas of Southern Sudan. Based on WHO recommended thresholds, all aspects of the SAFE strategy are indicated to eliminate blinding trachoma in Ayod.


title: A Century of Cholesterol and Coronaries: From Plaques to Genes to Statins

text: One-fourth of all deaths in industrialized countries result from coronary heart disease. A century of research has revealed the essential causative agent: cholesterol-carrying low-density lipoprotein (LDL). LDL is controlled by specific receptors (LDLRs) in liver that remove it from blood. Mutations that eliminate LDLRs raise LDL and cause heart attacks in childhood, whereas mutations that raise LDLRs reduce LDL and diminish heart attacks. If we are to eliminate coronary disease, lowering LDL should be the primary goal. Effective means to achieve this goal are currently available. The key questions are: who to treat, when to treat, and how long to treat.


title: Perinatal mortality in rural China: retrospective cohort study.

text:
OBJECTIVES To explore the use of local civil registration data to assess the perinatal mortality in a typical rural county in a less developed province in China, 1999-2000.
 DESIGN Retrospective cohort study. Pregnancies in a cohort of women followed from registration of pregnancy to outcome of infant seven days after birth.
 SETTING Routine family planning records in 20 rural townships in eastern China.    SUBJECTS 3697 pregnancies registered by the local family planning system during 1999.
 MAIN OUTCOME MEASURES Abortions, stillbirths, early neonatal mortality, perinatal mortality.
 RESULTS Only three cases were lost to follow up. The average age of the women at pregnancy was 25.9 years. Three hundred and twelve pregnancies were aborted and 240 ended in miscarriage (total 552, 15%). The perinatal mortality rate was 69 per 1000 births, the rate of stillbirth was 24 per 1000 births, and the early neonatal mortality was 46 per 1000 live births. The early neonatal mortality was 29 in boys and 69 in girls per 1000 live births. The perinatal mortality rate increased notably with parity and was higher in townships having lower income per capita.
 CONCLUSIONS The family planning system at the most local level is a useful data source for studying perinatal mortality in rural China. The perinatal mortality rate in the study county was higher than previously reported for both rural and urban areas in China. The results by parity and sex of the infant raise concern over the impact of the one child policy.


title: Genetic disruption of aurora B uncovers an essential role for aurora C during early mammalian development.

text: Mitosis is controlled by multiple kinases that drive cell cycle progression and prevent chromosome mis-segregation. Aurora kinase B interacts with survivin, borealin and incenp to form the chromosomal passenger complex (CPC), which is involved in the regulation of microtubule-kinetochore attachments and cytokinesis. Whereas genetic ablation of survivin, borealin or incenp results in early lethality at the morula stage, we show here that aurora B is dispensable for CPC function during early cell divisions and aurora B-null embryos are normally implanted. This is due to a crucial function of aurora C during these early embryonic cycles. Expression of aurora C decreases during late blastocyst stages resulting in post-implantation defects in aurora B-null embryos. These defects correlate with abundant prometaphase figures and apoptotic cell death of the aurora B-deficient inner cell mass. Conditional deletion of aurora B in somatic cells that do not express aurora C results in chromosomal misalignment and lack of chromosome segregation. Re-expression of wild-type, but not kinase-dead, aurora C rescues this defect, suggesting functional overlap between these two kinases. Finally, aurora B-null cells partially arrest in the presence of nocodazole, suggesting that this kinase is not essential for the spindle assembly checkpoint.


title: NuSAP is essential for chromatin-induced spindle formation during early embryogenesis.

text: Mitotic spindle assembly is mediated by two processes: a centrosomal and a chromosomal pathway. RanGTP regulates the latter process by releasing microtubule-associated proteins from inhibitory complexes. NuSAP, a microtubule- and DNA-binding protein, is a target of RanGTP and promotes the formation of microtubules near chromosomes. However, the contribution of NuSAP to cell proliferation in vivo is unknown. Here, we demonstrate that the expression of NuSAP highly correlates with cell proliferation during embryogenesis and adult life, making it a reliable marker of proliferating cells. Additionally, we show that NuSAP deficiency in mice leads to early embryonic lethality. Spindle assembly in NuSAP-deficient cells is highly inefficient and chromosomes remain dispersed in the mitotic cytoplasm. As a result of sustained spindle checkpoint activity, the cells are unable to progress through mitosis, eventually leading to caspase activation and apoptotic cell death. Together, our findings demonstrate that NuSAP is essential for proliferation of embryonic cells and, simultaneously, they underscore the importance of chromatin-induced spindle assembly.


title: E2F-6: a novel member of the E2F family is an inhibitor of E2F-dependent transcription

text: The E2F family of transcription factors are essential for the regulation of genes required for appropriate progression through the cell cycle. Five members of the E2F family have been previously reported, namely E2F1-5. All five are key elements in transcriptional regulation of essential genes, and they can be divided into two functional groups, those that induce S-phase progression when overexpressed in quiescent cells (E2Fs 1–3), and those that do not (E2Fs 4–5). Here, we describe the identification of a novel member of this family, which we refer to as E2F-6. E2F-6 shares significant homology with E2Fs 1–5, especially within the DNA binding, heterodimerization and marked box domains. Unlike E2Fs 1–5, E2F-6 lacks a transactivation and a pocket protein binding domain, hence, forms a unique third group within the E2F family. E2F-6 is a nuclear protein that can form heterodimers with the DP proteins (both DP-1 and DP-2) in vitro and in vivo. Our results show that the complex formed between E2F-6 and the DP proteins, possesses high DNA binding activity, displaying a preference for a TTTCCCGC E2F recognition site, which is slightly different to the E2F consensus site derived from the E2 promoter (TTTCGCGC). In contrast to the other members of the E2F family, ectopic expression of E2F-6 inhibits transcription from promoters possessing E2F recognition sites rather than activating transcription. In addition, overexpression of E2F-6 suppresses the transactivational effects of co-expression of E2F-1 and DP-1. The inhibitory effect of E2F-6 is dependent on its DNA binding activity and its ability to form heterodimers with the DPs. Interestingly, ectopic expression of E2F-6 leads to accumulation of cells in S-phase. Our data suggest that E2F-6 expression delays the exit from S-phase rather than inducing S-phase, which further emphasizes the functional difference between E2F-6 and the previously known E2F family members.


title: Enhancement of hippocampal pyramidal cell excitability by the novel selective slow-afterhyperpolarization channel blocker 3-(triphenylmethylaminomethyl)pyridine (UCL2077).

text: The slow afterhyperpolarization (sAHP) in hippocampal neurons has been implicated in learning and memory. However, its precise role in cell excitability and central nervous system function has not been explicitly tested for 2 reasons: 1) there are, at present, no selective inhibitors that effectively reduce the underlying current in vivo or in intact in vitro tissue preparations, and 2) although it is known that a small conductance K(+) channel that activates after a rise in [Ca(2+)](i) underlies the sAHP, the exact molecular identity remains unknown. We show that 3-(triphenylmethylaminomethyl)pyridine (UCL2077), a novel compound, suppressed the sAHP present in hippocampal neurons in culture (IC(50) = 0.5 microM) and in the slice preparation (IC(50) approximately 10 microM). UCL2077 was selective, having minimal effects on Ca(2+) channels, action potentials, input resistance and the medium afterhyperpolarization. UCL2077 also had little effect on heterologously expressed small conductance Ca(2+)-activated K(+) (SK) channels. Moreover, UCL2077 and apamin, a selective SK channel blocker, affected spike firing in hippocampal neurons in different ways. These results provide further evidence that SK channels are unlikely to underlie the sAHP. This study also demonstrates that UCL2077, the most potent, selective sAHP blocker described so far, is a useful pharmacological tool for exploring the role of sAHP channels in the regulation of cell excitability in intact tissue preparations and, potentially, in vivo.


title: Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions.

text: This review will reconsider the current paradigm for understanding the critical, final steps in the progression of atherosclerotic lesions. That scheme, largely an outgrowth of observations of autopsy tissues by Davies and colleagues,1 2 asserts that the cause of death in atherosclerotic coronary artery disease is rupture of an advanced atherosclerotic lesion. Although this assumption may be partially true, recent autopsy studies suggest that it is incomplete.   To reconsider this paradigm, we reexamined the morphological classification scheme for lesions proposed by the American Heart Association (AHA).3 4 This scheme is difficult to use for 2 reasons. First, it uses a very long list of roman numerals modified by letter codes that are difficult to remember. Second, it implies an orderly, linear pattern of lesion progression. This tends to be ambiguous, because it is not clear whether there is a single sequence of events during the progression of all lesions. We have therefore tried to devise a simpler classification scheme that is consistent with the AHA categories but is easier to use, able to deal with a wide array of morphological variations, and not overly burdened by mechanistic implications.   The current paradigm is based on the belief that type IV lesions, or “atheromas,” described by the AHA are stable because the fatty, necrotic core is contained by a smooth muscle cell–rich fibrous cap. Virchow’s analysis5 in 1858 pointed out that historically, the term “atheroma” refers to a dermal cyst (“Grutzbalg”), a fatty …


title: Loss of allergen 1 confers a hypervirulent phenotype that resembles mucoid switch variants of Cryptococcus neoformans.

text: Microbial survival in a host is usually dependent on the ability of a pathogen to undergo changes that promote escape from host defense mechanisms. The human-pathogenic fungus Cryptococcus neoformans undergoes phenotypic switching in vivo that promotes persistence in tissue. By microarray and real-time PCR analyses, the allergen 1 gene (ALL1) was found to be downregulated in the hypervirulent mucoid switch variant, both during logarithmic growth and during intracellular growth in macrophages. The ALL1 gene encodes a small cytoplasmic protein that is involved in capsule formation. Growth of an all1Delta gene deletion mutant was normal. Similar to cells of the mucoid switch variant, all1Delta cells produced a larger polysaccharide capsule than cells of the smooth parent and the complemented strain produced, and the enlarged capsule inhibited macrophage phagocytosis. The mutant exhibited a modest defect in capsule induction compared to all of the other variants. In animal models the phenotype of the all1Delta mutant mimicked the hypervirulent phenotype of the mucoid switch variant, which is characterized by decreased host survival and elevated intracranial pressure. Decreased survival is likely the result of both an ineffective cell-mediated immune response and impaired phagocytosis by macrophages. Consequently, we concluded that, unlike loss of most virulence-associated genes, where loss of gene function results in attenuated virulence, loss of the ALL1 gene enhances virulence by altering the host-pathogen interaction and thereby impairing clearance. Our data identified the first cryptococcal gene associated with elevated intracranial pressure and support the hypothesis that an environmental opportunistic pathogen has modified its virulence in vivo by epigenetic downregulation of gene function.


title: Lessons from more than 80 structures of the GluA2 ligand-binding domain in complex with agonists, antagonists and allosteric modulators.

text: Ionotropic glutamate receptors (iGluRs) constitute a family of ligand-gated ion channels that are essential for mediating fast synaptic transmission in the central nervous system. These receptors play an important role for the development and function of the nervous system, and are essential in learning and memory. However, iGluRs are also implicated in or have causal roles for several brain disorders, e.g. epilepsy, Alzheimer's disease, Parkinson's disease and schizophrenia. Their involvement in neurological diseases has stimulated widespread interest in their structure and function. Since the first publication in 1998 of the structure of a recombinant soluble protein comprising the ligand-binding domain of GluA2 extensive studies have afforded numerous crystal structures of wildtype and mutant proteins including different ligands. The structural information obtained combined with functional data have led to models for receptor activation and desensitization by agonists, inhibition by antagonists and block of desensitization by positive allosteric modulators. Furthermore, the structural and functional studies have formed a powerful platform for the design of new selective compounds.


title: Diaphragm Atrophy and Contractile Dysfunction in a Murine Model of Pulmonary Hypertension

text: Pulmonary hypertension (PH) causes loss of body weight and inspiratory (diaphragm) muscle dysfunction. A model of PH induced by drug (monocrotaline, MCT) has been extensively used in mice to examine the etiology of PH. However, it is unclear if PH induced by MCT in mice reproduces the loss of body weight and diaphragm muscle dysfunction seen in patients. This is a pre-requisite for widespread use of mice to examine mechanisms of cachexia and diaphragm abnormalities in PH. Thus, we measured body and soleus muscle weight, food intake, and diaphragm contractile properties in mice after 6-8 weeks of saline (control) or MCT (600 mg/kg) injections. Body weight progressively decreased in PH mice, while food intake was similar in both groups. PH decreased (P<0.05) diaphragm maximal isometric specific force, maximal shortening velocity, and peak power. Protein carbonyls in whole-diaphragm lysates and the abundance of select myofibrillar proteins were unchanged by PH. Our findings show diaphragm isometric and isotonic contractile abnormalities in a murine model of PH induced by MCT. Overall, the murine model of PH elicited by MCT mimics loss of body weight and diaphragm muscle weakness reported in PH patients.


title: Identification of an anti-inflammatory protein from Faecalibacterium prausnitzii, a commensal bacterium deficient in Crohn's disease.

text:
BACKGROUND Crohn's disease (CD)-associated dysbiosis is characterised by a loss of Faecalibacterium prausnitzii, whose culture supernatant exerts an anti-inflammatory effect both in vitro and in vivo. However, the chemical nature of the anti-inflammatory compounds has not yet been determined.
 METHODS Peptidomic analysis using mass spectrometry was applied to F. prausnitzii supernatant. Anti-inflammatory effects of identified peptides were tested in vitro directly on intestinal epithelial cell lines and on cell lines transfected with a plasmid construction coding for the candidate protein encompassing these peptides. In vivo, the cDNA of the candidate protein was delivered to the gut by recombinant lactic acid bacteria to prevent dinitrobenzene sulfonic acid (DNBS)-colitis in mice.
 RESULTS The seven peptides, identified in the F. prausnitzii culture supernatants, derived from a single microbial anti-inflammatory molecule (MAM), a protein of 15 kDa, and comprising 53% of non-polar residues. This last feature prevented the direct characterisation of the putative anti-inflammatory activity of MAM-derived peptides. Transfection of MAM cDNA in epithelial cells led to a significant decrease in the activation of the nuclear factor (NF)-κB pathway with a dose-dependent effect. Finally, the use of a food-grade bacterium, Lactococcus lactis, delivering a plasmid encoding MAM was able to alleviate DNBS-induced colitis in mice.
 CONCLUSIONS A 15 kDa protein with anti-inflammatory properties is produced by F. prausnitzii, a commensal bacterium involved in CD pathogenesis. This protein is able to inhibit the NF-κB pathway in intestinal epithelial cells and to prevent colitis in an animal model.


title: Calcium store sensor stromal-interaction molecule 1-dependent signaling plays an important role in cervical cancer growth, migration, and angiogenesis.

text: Store-operated Ca(2+) entry (SOCE) is the principal Ca(2+) entry mechanism in nonexcitable cells. Stromal-interaction molecule 1 (STIM1) is an endoplasmic reticulum Ca(2+) sensor that triggers SOCE activation. However, the role of STIM1 in regulating cancer progression remains controversial and its clinical relevance is unclear. Here we show that STIM1-dependent signaling is important for cervical cancer cell proliferation, migration, and angiogenesis. STIM1 overexpression in tumor tissue is noted in 71% cases of early-stage cervical cancer. In tumor tissues, the level of STIM1 expression is significantly associated with the risk of metastasis and survival. EGF-stimulated cancer cell migration requires STIM1 expression and EGF increases the interaction between STIM1 and Orai1 in juxta-membrane areas, and thus induces Ca(2+) influx. STIM1 involves the activation of Ca(2+)-regulated protease calpain, as well as Ca(2+)-regulated cytoplasmic kinase Pyk2, which regulate the focal-adhesion dynamics of migratory cervical cancer cells. Because of an increase of p21 protein levels and a decrease of Cdc25C protein levels, STIM1-silencing in cervical cancer cells significantly inhibits cell proliferation by arresting the cell cycle at the S and G2/M phases. STIM1 also regulates the production of VEGF in cervical cancer cells. Interference with STIM1 expression or blockade of SOCE activity inhibits tumor angiogenesis and growth in animal models, confirming the crucial role of STIM1-mediated Ca(2+) influx in aggravating tumor development in vivo. These results make STIM1-dependent signaling an attractive target for therapeutic intervention.


title: A feasibility study for a randomised controlled trial of the Positive Reappraisal Coping Intervention, a novel supportive technique for recurrent miscarriage

text:
INTRODUCTION Recurrent miscarriage (RM) is diagnosed when a woman has had three or more miscarriages. Increased levels of distress and anxiety are common during the waiting period of any subsequent pregnancies, posing a significant threat to psychological well-being. However, only limited support and therapy are available for these women, and many are left to cope alone. The Positive Reappraisal Coping Intervention (PRCI) is a novel self-administered supportive technique which has been shown to be effective in patients awaiting the outcome of in vitro fertilisation treatment. The primary objective of this study is to assess the feasibility and effectiveness of the PRCI in improving quality of life in the difficult waiting period which women with previous RM endure before an ongoing pregnancy can be confirmed.
 METHODS AND ANALYSIS A randomised controlled trial (RCT) feasibility study will establish the viability of conducting a multicentre RCT to definitively test the effects of the PRCI on the psychological well-being of women who have experienced RM during the initial waiting period of a subsequent pregnancy. A second component consists of a qualitative process evaluation exploring the initial experience of pregnancy following repeated miscarriages. Participants (n=50) will be randomised into one of two groups. The PRCI intervention group will receive the PRCI card and weekly questionnaires to assess their psychological well-being during the waiting period of their new pregnancy. The non-intervention group will be asked to complete the same weekly questionnaires. The qualitative process analysis will employ semistructured interviews (n=20) to address relevant aspects of the study objectives.    ETHICS AND DISSEMINATION Ethics approval has been obtained from the National Research Ethics Service Committee South Central-Hampshire A. Participating centres have given National Health Service R&D approval. Study findings will be disseminated through peer reviewed journals, national and international conferences and lay user groups.
 TRIAL REGISTRATION NUMBER ISRCTN43571276. This study was registered with the ISRCTN 18/02/2014 following adoption onto the United Kingdom Clinical Research Network (UKCRN) portfolio. Recruitment of the first participant occurred 04/02/2014.


title: Report of the National Heart, Lung, and Blood Institute Workshop on Lipoprotein(a) and Cardiovascular Disease: recent advances and future directions.

text: It has been estimated that approximately 37% of the US population judged to be at high risk for developing coronary artery disease (CAD), based on the National Cholesterol Education Program guidelines, have increased plasma lipoprotein(a) [Lp(a)], whereas Lp(a) is increased in only 14% of those judged to be at low risk. Therefore, the importance of establishing a better understanding of the relative contribution of Lp(a) to the risk burden for CAD and other forms of vascular disease, as well as the underlying mechanisms, is clearly evident. However, the structural complexity and size heterogeneity of Lp(a) have hindered the development of immunoassays to accurately measure Lp(a) concentrations in plasma. The large intermethod variation in Lp(a) values has made it difficult to compare data from different clinical studies and to achieve a uniform interpretation of clinical data. A workshop was recently convened by the National Heart, Lung, and Blood Institute (NHLBI) to evaluate our current understanding of Lp(a) as a risk factor for atherosclerotic disorders; to determine how future studies could be designed to more clearly define the extent to which, and mechanisms by which, Lp(a) participates in these processes; and to present the results of the NHLBI-supported program for the evaluation and standardization of Lp(a) immunoassays. This report includes the most recent data presented by the workshop participants and the resulting practical and research recommendations.


title: Classification, functions, and clinical relevance of extracellular vesicles.

text: Both eukaryotic and prokaryotic cells release small, phospholipid-enclosed vesicles into their environment. Why do cells release vesicles? Initial studies showed that eukaryotic vesicles are used to remove obsolete cellular molecules. Although this release of vesicles is beneficial to the cell, the vesicles can also be a danger to their environment, for instance in blood, where vesicles can provide a surface supporting coagulation. Evidence is accumulating that vesicles are cargo containers used by eukaryotic cells to exchange biomolecules as transmembrane receptors and genetic information. Because also bacteria communicate to each other via extracellular vesicles, the intercellular communication via extracellular cargo carriers seems to be conserved throughout evolution, and therefore vesicles are likely to be a highly efficient, robust, and economic manner of exchanging information between cells. Furthermore, vesicles protect cells from accumulation of waste or drugs, they contribute to physiology and pathology, and they have a myriad of potential clinical applications, ranging from biomarkers to anticancer therapy. Because vesicles may pass the blood-brain barrier, they can perhaps even be considered naturally occurring liposomes. Unfortunately, pathways of vesicle release and vesicles themselves are also being used by tumors and infectious diseases to facilitate spreading, and to escape from immune surveillance. In this review, the different types, nomenclature, functions, and clinical relevance of vesicles will be discussed.


title: Estimating the sample mean and standard deviation from the sample size, median, range and/or interquartile range

text: In systematic reviews and meta-analysis, researchers often pool the results of the sample mean and standard deviation from a set of similar clinical trials. A number of the trials, however, reported the study using the median, the minimum and maximum values, and/or the first and third quartiles. Hence, in order to combine results, one may have to estimate the sample mean and standard deviation for such trials. In this paper, we propose to improve the existing literature in several directions. First, we show that the sample standard deviation estimation in Hozo et al. (2005) has some serious limitations and is always less satisfactory in practice. Inspired by this, we propose a new estimation method by incorporating the sample size. Second, we systematically study the sample mean and standard deviation estimation problem under more general settings where the first and third quartiles are also available for the trials. Through simulation studies, we demonstrate that the proposed methods greatly improve the existing methods and enrich the literature. We conclude our work with a summary table that serves as a comprehensive guidance for performing meta-analysis in different situations.


title: Fascin protein is critical for transforming growth factor β protein-induced invasion and filopodia formation in spindle-shaped tumor cells.

text: Fascin, an actin-bundling protein overexpressed in all carcinomas, has been associated with poor prognosis, shorter survival, and more metastatic diseases. It is believed that fascin facilitates tumor metastasis by promoting the formation of invasive membrane protrusions. However, the mechanisms by which fascin is overexpressed in tumors are not clear. TGFβ is a cytokine secreted by tumor and mesenchymal cells and promotes metastasis in many late stage tumors. The pro-metastasis mechanisms of TGFβ remain to be fully elucidated. Here we demonstrated that TGFβ induced fascin expression in spindle-shaped tumor cells through the canonical Smad-dependent pathway. Fascin was critical for TGFβ-promoted filopodia formation, migration, and invasion in spindle tumor cells. More importantly, fascin expression significantly correlates with TGFβ1 and TGFβ receptor I levels in a cohort of primary breast tumor samples. Our results indicate that elevated TGFβ level in the tumor microenvironment may be responsible for fascin overexpression in some of the metastatic tumors. Our data also suggest that fascin could play a central role in TGFβ-promoted tumor metastasis.


title: DNA methylation age of human tissues and cell types

text:
BACKGROUND It is not yet known whether DNA methylation levels can be used to accurately predict age across a broad spectrum of human tissues and cell types, nor whether the resulting age prediction is a biologically meaningful measure.
 RESULTS I developed a multi-tissue predictor of age that allows one to estimate the DNA methylation age of most tissues and cell types. The predictor, which is freely available, was developed using 8,000 samples from 82 Illumina DNA methylation array datasets, encompassing 51 healthy tissues and cell types. I found that DNA methylation age has the following properties: first, it is close to zero for embryonic and induced pluripotent stem cells; second, it correlates with cell passage number; third, it gives rise to a highly heritable measure of age acceleration; and, fourth, it is applicable to chimpanzee tissues. Analysis of 6,000 cancer samples from 32 datasets showed that all of the considered 20 cancer types exhibit significant age acceleration, with an average of 36 years. Low age-acceleration of cancer tissue is associated with a high number of somatic mutations and TP53 mutations, while mutations in steroid receptors greatly accelerate DNA methylation age in breast cancer. Finally, I characterize the 353 CpG sites that together form an aging clock in terms of chromatin states and tissue variance.
 CONCLUSIONS I propose that DNA methylation age measures the cumulative effect of an epigenetic maintenance system. This novel epigenetic clock can be used to address a host of questions in developmental biology, cancer and aging research.


title: Whole-body MR vascular screening detects unsuspected concomitant vascular disease in coronary heart disease patients

text: Coronary heart disease (CHD) patients often show atherosclerotic vascular disease in other vascular territories. We evaluated how often whole-body MR imaging detects concomitant arterial pathologies in CHD patients, and how often these pathologies were not known to the patients previously. Of 4,814 participants in the population-based Heinz Nixdorf Recall Study, 327 reported CHD (i.e., previous coronary bypass surgery, angioplasty); of those, 160 patients (mean age 66.4 years) were examined using MR of the brain, the heart (excluding the coronary arteries), and whole-body MR angiography. The prevalence of each vascular pathology was assessed, correlated to the others and compared to patients’ histories. Of the 160 CHD patients, 16 (10%) showed MR signs of stroke, and 77 (48.1%) had a stenosis >50% in at least one extracerebral peripheral artery (other than the coronaries), including 28 (17.5%) with relevant renal artery stenoses, and 20 (12.5%) with relevant extracerebral internal carotid artery stenoses. False negative histories were reported in 12 of 81 cases with myocardial infarctions, and in 11 of 16 cases with cerebrovascular infarctions. This whole-body atherosclerosis MR screening program allows previously unknown concomitant vascular disease to be detected in coronary heart disease patients. Its prospective value should be assessed in further studies.


title: Key role of Ubc5 and lysine-63 polyubiquitination in viral activation of IRF3.

text: The mitochondrial antiviral signaling protein (MAVS; also known as IPS-1, VISA, and CARDIF) is essential for innate immune response against RNA viruses. MAVS transduces signals from the cytosolic RIG-I-like receptors, which bind to viral RNAs. But how MAVS activates downstream transcription factors such as IRF3 to induce type-I interferons is not well understood. We have established a cell-free system in which mitochondria derived from virus-infected cells activate IRF3 in the cytosol. Fractionation of the cytosol led to the identification of Ubc5 as a ubiquitin-conjugating enzyme (E2) required for IRF3 activation. Using an inducible RNAi strategy, we demonstrate that catalytically active Ubc5 is required for IRF3 activation by viral infection. The activation of IRF3 also requires two ubiquitin-binding domains of NEMO. Furthermore, we show that replacement of endogenous ubiquitin with its K63R mutant abolishes viral activation of IRF3, demonstrating that K63 polyubiquitination plays a key role in IRF3 activation.


title: Profiling of residual breast cancers after neoadjuvant chemotherapy identifies DUSP4 deficiency as a mechanism of drug resistance

text: Neoadjuvant chemotherapy (NAC) induces a pathological complete response (pCR) in ∼30% of patients with breast cancer. However, many patients have residual cancer after chemotherapy, which correlates with a higher risk of metastatic recurrence and poorer outcome than those who achieve a pCR. We hypothesized that molecular profiling of tumors after NAC would identify genes associated with drug resistance. Digital transcript counting was used to profile surgically resected breast cancers after NAC. Low concentrations of dual specificity protein phosphatase 4 (DUSP4), an ERK phosphatase, correlated with high post-NAC tumor cell proliferation and with basal-like breast cancer (BLBC) status. BLBC had higher DUSP4 promoter methylation and gene expression patterns of Ras-ERK pathway activation relative to other breast cancer subtypes. DUSP4 overexpression increased chemotherapy-induced apoptosis, whereas DUSP4 depletion dampened the response to chemotherapy. Reduced DUSP4 expression in primary tumors after NAC was associated with treatment-refractory high Ki-67 scores and shorter recurrence-free survival. Finally, inhibition of mitogen-activated protein kinase kinase (MEK) synergized with docetaxel treatment in BLBC xenografts. Thus, DUSP4 downregulation activates the Ras-ERK pathway in BLBC, resulting in an attenuated response to anti-cancer chemotherapy.


title: Age-related behaviors have distinct transcriptional profiles in Caenorhabditis elegans.

text: There has been a great deal of interest in identifying potential biomarkers of aging. Biomarkers of aging would be useful to predict potential vulnerabilities in an individual that may arise well before they are chronologically expected, due to idiosyncratic aging rates that occur between individuals. Prior attempts to identify biomarkers of aging have often relied on the comparisons of long-lived animals to a wild-type control. However, the effect of interventions in model systems that prolong lifespan (such as single gene mutations or caloric restriction) can sometimes be difficult to interpret due to the manipulation itself having multiple unforeseen consequences on physiology, unrelated to aging itself. The search for predictive biomarkers of aging therefore is problematic, and the identification of metrics that can be used to predict either physiological or chronological age would be of great value. One methodology that has been used to identify biomarkers for numerous pathologies is gene expression profiling. Here, we report whole-genome expression profiles of individual wild-type Caenorhabditis elegans covering the entire wild-type nematode lifespan. Individual nematodes were scored for either age-related behavioral phenotypes, or survival, and then subsequently associated with their respective gene expression profiles. This facilitated the identification of transcriptional profiles that were highly associated with either physiological or chronological age. Overall, our approach serves as a paradigm for identifying potential biomarkers of aging in higher organisms that can be repeatedly sampled throughout their lifespan.


title: Synchronization of Diffusively-Connected Nonlinear Systems: Results Based on Contractions with Respect to General Norms

text: Contraction theory provides an elegant way to analyze the behavior of certain nonlinear dynamical systems. In this paper, we discuss the application of contraction to synchronization of diffusively interconnected components described by nonlinear differential equations. We provide estimates of convergence of the difference in states between components, in the cases of line, complete, and star graphs, and Cartesian products of such graphs. We base our approach on contraction theory, using matrix measures derived from norms that are not induced by inner products. Such norms are the most appropriate in many applications, but proofs cannot rely upon Lyapunov-like linear matrix inequalities, and different techniques, such as the use of the Perron-Frobenious Theorem in the cases of L1 or L∞ norms, must be introduced.


title: GeneTrack--a genomic data processing and visualization framework.

text: MOTIVATION High-throughput 'ChIP-chip' and 'ChIP-seq' methodologies generate sufficiently large data sets that analysis poses significant informatics challenges, particularly for research groups with modest computational support. To address this challenge, we devised a software platform for storing, analyzing and visualizing high resolution genome-wide binding data. GeneTrack automates several steps of a typical data processing pipeline, including smoothing and peak detection, and facilitates dissemination of the results via the web. Our software is freely available via the Google Project Hosting environment at http://genetrack.googlecode.com


title: Multisite optical recording of excitability in the enteric nervous system.

text: A multisite optical recording technique consisting of an array of 464 photodiodes was used to measure dynamic changes in transmembrane potentials (Vm) of guinea-pig and mouse enteric neurones stained with the voltage-sensitive dye Di-8-ANEPPS. Optical recordings of Vm changes in enteric neurones which were evoked by depolarizing current pulses or synaptic activation mirrored the Vm changes measured intracellularly in the same neurone. Action potentials had fractional change in fluorescence of -0.09 +/- 0.06% and their peak to peak noise level was 20 +/- 14% of the action potential amplitude. Optical recordings after electrical stimulation of interganglionic nerve strands revealed slow EPSPs, nicotinergic supra- and subthreshold fast EPSPs as well as propagation of action potentials along interganglionic strands. Local application of acetylcholine onto a single ganglion induced reproducibly and dose dependently action potential discharge demonstrating the feasibility of neuropharmacological studies. The optical mapping made it possible to record action potentials simultaneously in a large number of neurones with high spatiotemporal resolution that is unattainable by conventional techniques. This technique presents a powerful tool to study excitability spread within enteric circuits and to assess differential activation of enteric populations in response to a number of stimuli which modulate neuronal activity directly or through synaptic mechanisms.


title: Telomere-bound TRF1 and TRF2 stall the replication fork at telomeric repeats.

text: Vertebrate telomeres consist of tandem repeats of T2AG3 and associated proteins including the telomeric DNA-binding proteins, TRF1 and TRF2. It has been proposed that telomeres assume two interswitchable states, the open state that is accessible to various trans-acting factors and the closed state that excludes those factors. TRF1 and TRF2 are believed to promote the formation of the closed state. However, little is known about how those two states influence DNA replication. We analyzed the effects of TRF1 and TRF2 on telomeric replication both in vitro and in vivo. By exploiting the in vitro replication system of linear SV40 DNA, we found that telomeric repeats are a poor replication template. Moreover, the addition of recombinant TRF1 and TRF2 significantly stalled the replication fork progression at telomeric repeats. When TRF1 was overexpressed in HeLa cells, cells with 4N DNA content were accumulated. Furthermore, cytological analyses revealed that the replication focus overlapped with telomere signals at a significantly higher frequency in TRF1-overexpressing cells than in control cells. The results suggest that TRF1 and TRF2 exert inhibitory effects on replication fork progression.


title: Margination of white blood cells in microcapillary flow.

text: Margination of white blood cells (WBCs) towards vessel walls is an essential precondition for their efficient adhesion to the vascular endothelium. We perform numerical simulations with a two-dimensional blood flow model to investigate the dependence of WBC margination on hydrodynamic interactions of blood cells with the vessel walls, as well as on their collective behavior and deformability. We find WBC margination to be optimal in intermediate ranges of red blood cell (RBC) volume fractions and flow rates, while, beyond these ranges, it is substantially attenuated. RBC aggregation enhances WBC margination, while WBC deformability reduces it. These results are combined in state diagrams, which identify WBC margination for a wide range of flow and cell suspension conditions.


title: Lung cancer treatment waiting times and tumour growth.

text: We report a single-centre prospective audit of 29 lung cancer patients who were awaiting radical (potentially curative) radiotherapy. This was the total number assessed as suitable for radical treatment by one consultant during 1999. At the time of assessment they had been newly diagnosed and staged with a computed tomographic (CT) scan of chest. They had a subsequent CT scan prior to starting treatment for the purpose of planning the radiation fields. We have now measured tumour size on the diagnostic scans and compared this with the size on the planning scans. We have documented the delay between diagnostic and planning CT scanning and the total time between first hospital visit and starting treatment. Two patients had progression of symptoms while on the waiting list, making them unfit for radical treatment, and another four had tumour progression on planning CT such that the tumour volume was too large for radical treatment. Therefore, 21% of potentially curable patients became incurable on the waiting list. The delay between diagnostic and planning CT scans ranged from 18 to 131 days (median 54), with increases in the cross-sectional tumour size over that period ranging zero to 373%. The delay between the first hospital visit and starting treatment was 35-187 days (median 94); between the date of the radiotherapy request and the starting date for treatment it was 23-61 days (median 44). Limited access to specialists is the reason most often advanced for the poor performance of the UK in treating lung cancer. This study demonstrates that, even for the select minority of patients who have specialist referral and are deemed suitable for potentially curative treatment, the outcome is prejudiced by waiting times that allow tumour progression.


title: Functional coupling of RNAP II transcription to spliceosome assembly.

text: The pathway of gene expression in higher eukaryotes involves a highly complex network of physical and functional interactions among the different machines involved in each step of the pathway. Here we established an efficient in vitro system to determine how RNA polymerase II (RNAP II) transcription is functionally coupled to pre-mRNA splicing. Strikingly, our data show that nascent pre-messenger RNA (pre-mRNA) synthesized by RNAP II is immediately and quantitatively directed into the spliceosome assembly pathway. In contrast, nascent pre-mRNA synthesized by T7 RNA polymerase is quantitatively assembled into the nonspecific H complex, which consists of heterogeneous nuclear ribonucleoprotein (hnRNP) proteins and is inhibitory for spliceosome assembly. Consequently, RNAP II transcription results in a dramatic increase in both the kinetics of splicing and overall yield of spliced mRNA relative to that observed for T7 transcription. We conclude that RNAP II mediates the functional coupling of transcription to splicing by directing the nascent pre-mRNA into spliceosome assembly, thereby bypassing interaction of the pre-mRNA with the inhibitory hnRNP proteins.


title: Calicum microdomains form within neutrophils at the neutrophil–tumor cell synapse: role in antibody-dependent target cell apoptosis

text: Ca2+ messages are broadly important in cellular signal transduction. In immune cells, Ca2+ signaling is an essential step in many forms of activation. Neutrophil-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) is one form of leukocyte activation that plays an important role in tumor cell killing in vitro and in patient care. Using fluorescence methodologies, we found that neutrophils exhibit Ca2+ signals during ADCC directed against breast fibrosarcoma cells. Importantly, these signals were localized to Ca2+ microdomains at the neutrophil-to-tumor cell interface where they display dynamic features such as movement, fusion, and fission. These signals were blocked by the intracellular Ca2+ buffer BAPTA. At the neutrophil–tumor cell synapse, the neutrophil’s cytoplasm was enriched in STIM1, a crucial mediator of Ca2+ signaling, whereas the Ca2+-binding proteins calbindin and parvalbumin were not affected. Our findings suggest that Ca2+ microdomains are due to an active signaling process. As Ca2+ signals within neutrophils were necessary for specific tumor cell apoptosis, a central role of microdomains in leukocyte-mediated tumor cell destruction is indicated.


title: Platelet glycoprotein IIb/IIIa inhibitors reduce mortality in diabetic patients with non-ST-segment-elevation acute coronary syndromes.

text:
BACKGROUND Diabetes mellitus is a major risk factor for adverse outcomes after acute coronary syndromes (ACS). Because this disease may be associated with increased platelet aggregation, we investigated whether diabetic patients with ACS derive particular benefit from platelet glycoprotein (GP) IIb/IIIa receptor inhibition.
 METHODS AND RESULTS We performed a meta-analysis of the diabetic populations enrolled in the 6 large-scale platelet GP IIb/IIIa inhibitor ACS trials: PRISM, PRISM-PLUS, PARAGON A, PARAGON B, PURSUIT, and GUSTO IV. Among 6458 diabetic patients, platelet GP IIb/IIIa inhibition was associated with a significant mortality reduction at 30 days, from 6.2% to 4.6% (OR 0.74; 95% CI 0.59 to 0.92; P=0.007). Conversely, 23 072 nondiabetic patients had no survival benefit (3.0% versus 3.0%). The interaction between platelet GP IIb/IIIa inhibition and diabetic status was statistically significant (P=0.036). Among 1279 diabetic patients undergoing percutaneous coronary intervention (PCI) during index hospitalization, the use of these agents was associated with a mortality reduction at 30 days from 4.0% to 1.2% (OR 0.30; 95% CI 0.14 to 0.69; P=0.002).
 CONCLUSIONS This meta-analysis, including the entire large-scale trial experience of intravenous platelet GP IIb/IIIa inhibitors for the medical management of non-ST-segment-elevation ACS, shows that these agents may significantly reduce mortality at 30 days in diabetic patients. Although not based on a randomized assessment, the survival benefit appears to be of greater magnitude in patients undergoing PCI. Therefore, the use of platelet GP IIb/IIIa inhibitors should be strongly considered in diabetic patients with ACS.


title: Glucose deprivation-induced cytotoxicity and alterations in mitogen-activated protein kinase activation are mediated by oxidative stress in multidrug-resistant human breast carcinoma cells.

text: We previously observed that glucose deprivation induces cell death in multidrug-resistant human breast carcinoma cells (MCF-7/ADR). As a follow up we wished to test the hypothesis that metabolic oxidative stress was the causative process or at least the link between causative processes behind the cytotoxicity. In the studies described here, we demonstrate that mitogen-activated protein kinase (MAPK) was activated within 3 min of being in glucose-free medium and remained activated for 3 h. Glucose deprivation for 2-4 h also caused oxidative stress as evidenced by a 3-fold greater steady state concentration of oxidized glutathione and a 3-fold increase in pro-oxidant production. Glucose and glutamate treatment rapidly suppressed MAPK activation and rescued cells from cytotoxicity. Glutamate and the peroxide scavenger, pyruvate, rescued the cells from cell killing as well as suppressed pro-oxidant production. In addition the thiol antioxidant, N-acetyl-L-cysteine, rescued cells from glucose deprivation-induced cytotoxicity and suppressed MAPK activation. These results suggest that glucose deprivation-induced cytotoxicity and alterations in MAPK signal transduction are mediated by oxidative stress in MCF-7/ADR. These results also support the speculation that a common mechanism of glucose deprivation-induced cytotoxicity in mammalian cells may involve metabolic oxidative stress.


title: A structural basis for kinetochore recruitment of the Ndc80 complex via two distinct centromere receptors.

text: The Ndc80 complex is the key microtubule-binding element of the kinetochore. In contrast to the well-characterized interaction of Ndc80-Nuf2 heads with microtubules, little is known about how the Spc24-25 heterodimer connects to centromeric chromatin. Here, we present molecular details of Spc24-25 in complex with the histone-fold protein Cnn1/CENP-T illustrating how this connection ultimately links microtubules to chromosomes. The conserved Ndc80 receptor motif of Cnn1 is bound as an α helix in a hydrophobic cleft at the interface between Spc24 and Spc25. Point mutations that disrupt the Ndc80-Cnn1 interaction also abrogate binding to the Mtw1 complex and are lethal in yeast. We identify a Cnn1-related motif in the Dsn1 subunit of the Mtw1 complex, necessary for Ndc80 binding and essential for yeast growth. Replacing this region with the Cnn1 peptide restores viability demonstrating functionality of the Ndc80-binding module in different molecular contexts. Finally, phosphorylation of the Cnn1 N-terminus coordinates the binding of the two competing Ndc80 interaction partners. Together, our data provide structural insights into the modular binding mechanism of the Ndc80 complex to its centromere recruiters.


title: Feedback Circuit among INK4 Tumor Suppressors Constrains Human Glioblastoma Development

text: We have developed a nonheuristic genome topography scan (GTS) algorithm to characterize the patterns of genomic alterations in human glioblastoma (GBM), identifying frequent p18(INK4C) and p16(INK4A) codeletion. Functional reconstitution of p18(INK4C) in GBM cells null for both p16(INK4A) and p18(INK4C) resulted in impaired cell-cycle progression and tumorigenic potential. Conversely, RNAi-mediated depletion of p18(INK4C) in p16(INK4A)-deficient primary astrocytes or established GBM cells enhanced tumorigenicity in vitro and in vivo. Furthermore, acute suppression of p16(INK4A) in primary astrocytes induced a concomitant increase in p18(INK4C). Together, these findings uncover a feedback regulatory circuit in the astrocytic lineage and demonstrate a bona fide tumor suppressor role for p18(INK4C) in human GBM wherein it functions cooperatively with other INK4 family members to constrain inappropriate proliferation.


title: The SMC5/6 complex maintains telomere length in ALT cancer cells through SUMOylation of telomere-binding proteins

text: Most cancer cells activate telomerase to elongate telomeres and achieve unlimited replicative potential. Some cancer cells cannot activate telomerase and use telomere homologous recombination (HR) to elongate telomeres, a mechanism termed alternative lengthening of telomeres (ALT). A hallmark of ALT cells is the recruitment of telomeres to PML bodies (termed APBs). Here, we show that the SMC5/6 complex localizes to APBs in ALT cells and is required for targeting telomeres to APBs. The MMS21 SUMO ligase of the SMC5/6 complex SUMOylates multiple telomere-binding proteins, including TRF1 and TRF2. Inhibition of TRF1 or TRF2 SUMOylation prevents APB formation. Depletion of SMC5/6 subunits by RNA interference inhibits telomere HR, causing telomere shortening and senescence in ALT cells. Thus, the SMC5/6 complex facilitates telomere HR and elongation in ALT cells by promoting APB formation through SUMOylation of telomere-binding proteins.


title: Dynein pulls microtubules without rotating its stalk.

text: Dynein is a microtubule motor that powers motility of cilia and flagella. There is evidence that the relative sliding of the doublet microtubules is due to a conformational change in the motor domain that moves a microtubule bound to the end of an extension known as the stalk. A predominant model for the movement involves a rotation of the head domain, with its stalk, toward the microtubule plus end. However, stalks bound to microtubules have been difficult to observe. Here, we present the clearest views so far of stalks in action, by observing sea urchin, outer arm dynein molecules bound to microtubules, with a new method, "cryo-positive stain" electron microscopy. The dynein molecules in the complex were shown to be active in in vitro motility assays. Analysis of the electron micrographs shows that the stalk angles relative to microtubules do not change significantly between the ADP.vanadate and no-nucleotide states, but the heads, together with their stalks, shift with respect to their A-tubule attachments. Our results disagree with models in which the stalk acts as a lever arm to amplify structural changes. The observed movement of the head and stalk relative to the tail indicates a new plausible mechanism, in which dynein uses its stalk as a grappling hook, catching a tubulin subunit 8 nm ahead and pulling on it by retracting a part of the tail (linker).


title: The INO80 chromatin remodeling complex in transcription, replication and repair.

text: The Ino80 ATPase is a member of the SNF2 family of ATPases and functions as an integral component of a multisubunit ATP-dependent chromatin remodeling complex. Although INO80 complexes from yeast and higher eukaryotes share a common core of conserved subunits, the complexes have diverged substantially during evolution and have acquired new subunits with apparently species-specific functions. Recent studies have shown that the INO80 complex contributes to a wide variety of chromatin-dependent nuclear transactions, including transcription, DNA repair and DNA replication.


title: VEX1 controls the allelic exclusion required for antigenic variation in trypanosomes.

text: Allelic exclusion underpins antigenic variation and immune evasion in African trypanosomes. These bloodstream parasites use RNA polymerase-I (pol-I) to transcribe just one telomeric variant surface glycoprotein (VSG) gene at a time, producing superabundant and switchable VSG coats. We identified trypanosome VSG exclusion-1 (VEX1) using a genetic screen for defects in telomere-exclusive expression. VEX1 was sequestered by the active VSG and silencing of other VSGs failed when VEX1 was either ectopically expressed or depleted, indicating positive and negative regulation, respectively. Positive regulation affected VSGs and nontelomeric pol-I-transcribed genes, whereas negative regulation primarily affected VSGs. Negative regulation by VEX1 also affected telomeric pol-I-transcribed reporter constructs, but only when they contained blocks of sequence sharing homology with a pol-I-transcribed locus. We conclude that restricted positive regulation due to VEX1 sequestration, combined with VEX1-dependent, possibly homology-dependent silencing, drives a "winner-takes-all" mechanism of allelic exclusion.


title: Kruppel-like factor 2 (KLF2) regulates monocyte differentiation and functions in mBSA and IL-1β-induced arthritis.

text: Kruppel-like factor 2 (KLF2) plays an important role in the regulation of a variety of immune cells, including monocytes. We have previously shown that KLF2 inhibits proinflammatory activation of monocytes. However, the role of KLF2 in arthritis is yet to be investigated. In the current study, we show that recruitment of significantly greater numbers of inflammatory subset of CD11b(+)F4/80(+)Ly6C+ monocytes to the inflammatory sites in KLF2 hemizygous mice compared to the wild type littermate controls. In parallel, inflammatory mediators, MCP-1, Cox-2 and PAI-1 were significantly up-regulated in bone marrow-derived monocytes isolated from KLF2 hemizygous mice, in comparison to wild-type controls. Methylated-BSA and IL-1β-induced arthritis was more severe in KLF2 hemizygous mice as compared to the littermate wild type controls. Consistent with this observation, monocytes isolated from KLF2 hemizygous mice showed an increased number of cells matured and differentiated towards osteoclastic lineage, potentially contributing to the severity of cartilage and bone damage in induced arthritic mice. The severity of arthritis was associated with the higher expression of proteins such as HSP60, HSP90 and MMP13 and attenuated levels of pPTEN, p21, p38 and HSP25/27 molecules in bone marrow cells of arthritic KLF2 hemizygous mice compared to littermate wild type controls. The data provide new insights and evidences of KLF2-mediated transcriptional regulation of arthritis via modulation of monocyte differentiation and function.


title: Relationship of collegiate football experience and concussion with hippocampal volume and cognitive outcomes.

text:
IMPORTANCE Concussion and subconcussive impacts have been associated with short-term disrupted cognitive performance in collegiate athletes, but there are limited data on their long-term neuroanatomic and cognitive consequences.
 OBJECTIVE To assess the relationships of concussion history and years of football experience with hippocampal volume and cognitive performance in collegiate football athletes.
 DESIGN, SETTING, AND PARTICIPANTS Cross-sectional study conducted between June 2011 and August 2013 at a US psychiatric research institute specializing in neuroimaging among collegiate football players with a history of clinician-diagnosed concussion (n = 25), collegiate football players without a history of concussion (n = 25), and non-football-playing, age-, sex-, and education-matched healthy controls (n = 25).    EXPOSURES History of clinician-diagnosed concussion and years of football experience.
 MAIN OUTCOMES AND MEASURES High-resolution anatomical magnetic resonance imaging was used to quantify brain volumes. Baseline scores on a computerized concussion-related cognitive battery were used for cognitive assessment in athletes.
 RESULTS Players with and without a history of concussion had smaller hippocampal volumes relative to healthy control participants (with concussion: t48 = 7.58; P < .001; mean difference, 1788 μL; 95% CI, 1317-2258 μL; without concussion: t48 = 4.35; P < .001, mean difference, 1027 μL; 95% CI, 556-1498 μL). Players with a history of concussion had smaller hippocampal volumes than players without concussion (t48 = 3.15; P < .001; mean difference, 761 μL; 95% CI, 280-1242 μL). In both athlete groups, there was a statistically significant inverse relationship between left hippocampal volume and number of years of football played (t46 = -3.62; P < .001; coefficient = -43.54; 95% CI, -67.66 to -19.41). Behavioral testing demonstrated no differences between athletes with and without a concussion history on 5 cognitive measures but did show an inverse correlation between years of playing football and reaction time (ρ42 = -0.43; 95% CI, -0.46 to -0.40; P = .005).
 CONCLUSIONS AND RELEVANCE Among a group of collegiate football athletes, there was a significant inverse relationship of concussion and years of football played with hippocampal volume. Years of football experience also correlated with slower reaction time. Further research is needed to determine the temporal relationships of these findings.


title: STING and the innate immune response to nucleic acids in the cytosol

text: Cytosolic detection of pathogen-derived nucleic acids is critical for the initiation of innate immune defense against diverse bacterial, viral and eukaryotic pathogens. Conversely, inappropriate responses to cytosolic nucleic acids can produce severe autoimmune pathology. The host protein STING has been identified as a central signaling molecule in the innate immune response to cytosolic nucleic acids. STING seems to be especially critical for responses to cytosolic DNA and the unique bacterial nucleic acids called 'cyclic dinucleotides'. Here we discuss advances in the understanding of STING and highlight the many unresolved issues in the field.


title: Global Assessment of Genomic Regions Required for Growth in Mycobacterium tuberculosis

text: Identifying genomic elements required for viability is central to our understanding of the basic physiology of bacterial pathogens. Recently, the combination of high-density mutagenesis and deep sequencing has allowed for the identification of required and conditionally required genes in many bacteria. Genes, however, make up only a part of the complex genomes of important bacterial pathogens. Here, we use an unbiased analysis to comprehensively identify genomic regions, including genes, domains, and intergenic elements, required for the optimal growth of Mycobacterium tuberculosis, a major global health pathogen. We found that several proteins jointly contain both domains required for optimal growth and domains that are dispensable. In addition, many non-coding regions, including regulatory elements and non-coding RNAs, are critical for mycobacterial growth. Our analysis shows that the genetic requirements for growth are more complex than can be appreciated using gene-centric analysis.


title: Cyclooxygenase-Dependent Tumor Growth through Evasion of Immunity

text: The mechanisms by which melanoma and other cancer cells evade anti-tumor immunity remain incompletely understood. Here, we show that the growth of tumors formed by mutant Braf(V600E) mouse melanoma cells in an immunocompetent host requires their production of prostaglandin E2, which suppresses immunity and fuels tumor-promoting inflammation. Genetic ablation of cyclooxygenases (COX) or prostaglandin E synthases in Braf(V600E) mouse melanoma cells, as well as in Nras(G12D) melanoma or in breast or colorectal cancer cells, renders them susceptible to immune control and provokes a shift in the tumor inflammatory profile toward classic anti-cancer immune pathways. This mouse COX-dependent inflammatory signature is remarkably conserved in human cutaneous melanoma biopsies, arguing for COX activity as a driver of immune suppression across species. Pre-clinical data demonstrate that inhibition of COX synergizes with anti-PD-1 blockade in inducing eradication of tumors, implying that COX inhibitors could be useful adjuvants for immune-based therapies in cancer patients.


title: Anti-alphav integrin monoclonal antibody intetumumab enhances the efficacy of radiation therapy and reduces metastasis of human cancer xenografts in nude rats.

text: We previously reported that intetumumab (CNTO 95), a fully human anti-αv integrin monoclonal antibody, is a radiosensitizer in mice with xenograft tumors. Because intetumumab does not cross-react with mouse integrins, but has cross-reactivity with rat integrins, we next studied the potential combined use of radiation therapy and intetumumab in human cancer xenograft models in nude rats to assess effects on both tumor cells and the tumor microenvironment. Nude rats bearing human head and neck cancer and non-small cell lung cancer (NSCLC) xenografts were treated with intetumumab and fractionated local tumor radiotherapy. Effects on tumor growth and metastasis, blood perfusion, oxygenation, and gastrointestinal toxicity were studied. Intetumumab alone had a moderate effect on tumor growth. When combined with fractionated radiation therapy, intetumumab significantly inhibited tumor growth and produced a tumor response rate that was significantly better than with radiation therapy alone. Treatment with intetumumab also significantly reduced lung metastasis in the A549 NSCLC xenograft model. The oxygenation and blood perfusion in xenograft tumors measured by microbubble-enhanced ultrasound imaging were substantially increased after treatment with intetumumab. The combined use of intetumumab and radiation therapy reduced the microvessel density and increased apoptosis in tumor cells and the tumor microenvironment. Toxicity studies showed that treatment with intetumumab did not cause the histopathologic changes in the lungs and did not sensitize the sensitive gastrointestinal epithelium to the effect of radiation therapy. Intetumumab can potentiate the efficacy of fractionated radiation therapy in human cancer xenograft tumors in nude rats without increased toxicity.


title: Methylomic trajectories across human fetal brain development.

text: Epigenetic processes play a key role in orchestrating transcriptional regulation during development. The importance of DNA methylation in fetal brain development is highlighted by the dynamic expression of de novo DNA methyltransferases during the perinatal period and neurodevelopmental deficits associated with mutations in the methyl-CpG binding protein 2 (MECP2) gene. However, our knowledge about the temporal changes to the epigenome during fetal brain development has, to date, been limited. We quantified genome-wide patterns of DNA methylation at ∼ 400,000 sites in 179 human fetal brain samples (100 male, 79 female) spanning 23 to 184 d post-conception. We identified highly significant changes in DNA methylation across fetal brain development at >7% of sites, with an enrichment of loci becoming hypomethylated with fetal age. Sites associated with developmental changes in DNA methylation during fetal brain development were significantly underrepresented in promoter regulatory regions but significantly overrepresented in regions flanking CpG islands (shores and shelves) and gene bodies. Highly significant differences in DNA methylation were observed between males and females at a number of autosomal sites, with a small number of regions showing sex-specific DNA methylation trajectories across brain development. Weighted gene comethylation network analysis (WGCNA) revealed discrete modules of comethylated loci associated with fetal age that are significantly enriched for genes involved in neurodevelopmental processes. This is, to our knowledge, the most extensive study of DNA methylation across human fetal brain development to date, confirming the prenatal period as a time of considerable epigenomic plasticity.


title: Synaptic Determinants of Rett Syndrome

text: There is mounting evidence showing that the structural and molecular organization of synaptic connections is affected both in human patients and in animal models of neurological and psychiatric diseases. As a consequence of these experimental observations, it has been introduced the concept of synapsopathies, a notion describing brain disorders of synaptic function and plasticity. A close correlation between neurological diseases and synaptic abnormalities is especially relevant for those syndromes including also mental retardation in their symptomatology, such as Rett syndrome (RS). RS (MIM312750) is an X-linked dominant neurological disorder that is caused in the majority of cases by mutations in methyl-CpG-binding protein 2 (MeCP2). This review will focus on the current knowledge of the synaptic alterations produced by mutations of the gene MeCP2 in mouse models of RS and will highlight prospects experimental therapies currently in use. Different experimental approaches have revealed that RS could be the consequence of an impairment in the homeostasis of synaptic transmission in specific brain regions. Indeed, several forms of experience-induced neuronal plasticity are impaired in the absence of MeCP2. Based on the results presented in this review, it is reasonable to propose that understanding how the brain is affected by diseases such as RS is at reach. This effort will bring us closer to identify the neurobiological bases of human cognition.


title: Depressive symptoms and physical decline in community-dwelling older persons.

text:
CONTEXT Significant symptoms of depression are common in the older community-dwelling population. Although depressive symptoms and disability may commonly occur in the same person, whether depressive symptoms contribute to subsequent functional decline has not been elucidated.
 OBJECTIVE To determine whether depressive symptoms in older persons increase the risk of subsequent decline in physical function as measured by objective performance-based tests.
 DESIGN A 4-year prospective cohort study.
 SETTING The communities of Iowa and Washington counties, Iowa.
 PARTICIPANTS A total of 1286 persons aged 71 years and older who completed a short battery of physical performance tests in 1988 and again 4 years later.
 MAIN OUTCOME MEASURES Baseline depressive symptoms were assessed by the Center for Epidemiological Studies Depression Scale. Physical performance tests included an assessment of standing balance, a timed 2.4-m (8-ft) walk, and a timed test of 5 repetitions of rising from a chair and sitting down.
 RESULTS After adjustment for baseline performance score, health status, and sociodemographic factors, increasing levels of depressive symptoms were predictive of greater decline in physical performance over 4 years (odds ratio for decline in those with depressed mood vs those without, 1.55; 95% confidence interval [CI], 1.02-2.34). Even among those at the high end of the functional spectrum, who reported no disability, the severity of depressive symptoms predicted subsequent decline in physical performance (odds ratio for decline, 1.03; 95% CI, 1.00-1.08).
 CONCLUSIONS This study provides evidence that older persons who report depressive symptoms are at higher risk of subsequent physical decline. These results suggest that prevention or reduction of depressed mood could play a role in reducing functional decline in older persons.


title: Foxk proteins repress the initiation of starvation-induced atrophy and autophagy programs

text: Autophagy is the primary catabolic process triggered in response to starvation. Although autophagic regulation within the cytosolic compartment is well established, it is becoming clear that nuclear events also regulate the induction or repression of autophagy. Nevertheless, a thorough understanding of the mechanisms by which sequence-specific transcription factors modulate expression of genes required for autophagy is lacking. Here, we identify Foxk proteins (Foxk1 and Foxk2) as transcriptional repressors of autophagy in muscle cells and fibroblasts. Interestingly, Foxk1/2 serve to counter-balance another forkhead transcription factor, Foxo3, which induces an overlapping set of autophagic and atrophic targets in muscle. Foxk1/2 specifically recruits Sin3A-HDAC complexes to restrict acetylation of histone H4 and expression of critical autophagy genes. Remarkably, mTOR promotes the transcriptional activity of Foxk1 by facilitating nuclear entry to specifically limit basal levels of autophagy in nutrient-rich conditions. Our study highlights an ancient, conserved mechanism whereby nutritional status is interpreted by mTOR to restrict autophagy by repressing essential autophagy genes through Foxk-Sin3-mediated transcriptional control.


title: PRRSV receptors and their roles in virus infection

text: Porcine reproductive and respiratory syndrome virus (PRRSV) has a restricted cell tropism and prefers to invade well-differentiated cells of the monocyte/macrophage lineage, such as pulmonary alveolar macrophages and African green monkey kidney cell line MA-104 and its derivatives, such as Marc-145, Vero and CL-2621. PRRSV infection of the host cells actually is a receptor-mediated endocytosis and replication process. The presence and absence of the cellular receptors decide whether the cell lines are permissive or non-permissive to PRRSV infection. Several PRRSV non-permissive cell lines, such as BHK-21, PK-15 and CHO-K1, have been shown to become sensitive to the virus infection upon expression of the recombinant receptor proteins. Up to now, heparin sulfate, sialoadhesin, CD163, CD151 and vimentin have been identified as the important PRRSV receptors via their involvement in virus attachment, internalization or uncoating. Each receptor is characterized by the distribution in different cells, the function in virus different infection stages and the interaction model with the viral proteins or genes. Joint forces of the receptors recently attract attentions due to the specific function. PRRSV receptors have become the targets for designing the new anti-viral reagents or the recombinant cell lines used for isolating the viruses or developing more effective vaccines due to their more conserved sequences compared with the genetic variation of the virus. In this paper, the role of PRRSV receptors and the molecular mechanism of the interaction between the virus and the receptors are reviewed.


title: Kinetic Analysis Reveals the Fate of a MicroRNA following Target Regulation in Mammalian Cells

text: Considerable details about microRNA (miRNA) biogenesis and regulation have been uncovered, but little is known about the fate of the miRNA subsequent to target regulation. To gain insight into this process, we carried out kinetic analysis of a miRNA's turnover following termination of its biogenesis and during regulation of a target that is not subject to Ago2-mediated catalytic cleavage. By quantitating the number of molecules of the miRNA and its target in steady state and in the course of its decay, we found that each miRNA molecule was able to regulate at least two target transcripts, providing in vivo evidence that the miRNA is not irreversibly sequestered with its target and that the nonslicing pathway of miRNA regulation is multiple-turnover. Using deep sequencing, we further show that miRNA recycling is limited by target regulation, which promotes posttranscriptional modifications to the 3' end of the miRNA and accelerates the miRNA's rate of decay. These studies provide new insight into the efficiency of miRNA regulation that help to explain how a miRNA can regulate a vast number of transcripts and that identify one of the mechanisms that impart specificity to miRNA decay in mammalian cells.


title: Estimation of nuclear DNA content in plants using flow cytometry

text: Flow cytometry (FCM) using DNA-selective fluorochromes is now the prevailing method for the measurement of nuclear DNA content in plants. Ease of sample preparation and high sample throughput make it generally better suited than other methods such as Feulgen densitometry to estimate genome size, level of generative polyploidy, nuclear replication state and endopolyploidy (polysomaty). Here we present four protocols for sample preparation (suspensions of intact cell nuclei) and describe the analysis of nuclear DNA amounts using FCM. We consider the chemicals and equipment necessary, the measurement process, data analysis, and describe the most frequent problems encountered with plant material such as the interference of secondary metabolites. The purpose and requirement of internal and external standardization are discussed. The importance of using a correct terminology for DNA amounts and genome size is underlined, and its basic principles are explained.


title: Predictive value of bone resorption and formation markers in cancer patients with bone metastases receiving the bisphosphonate zoledronic acid.

text:
PURPOSE Three large, randomized trials of patients with bone metastases recently demonstrated that zoledronic acid reduces the risk of skeletal-related events. These trials provide an opportunity for investigating the correlation between bone metabolism and clinical outcome during bisphosphonate therapy.
 PATIENTS AND METHODS Urinary measurements of N-telopeptide (Ntx) and serum bone alkaline phosphatase (BAP) were obtained in 1,824 bisphosphonate-treated patients-1,462 with zoledronic acid (breast, 490; prostate, 411; myeloma, 210; non-small-cell lung, 183; other, 168) and 362 with pamidronate (breast, 254; myeloma, 108). This exploratory cohort analysis grouped patients by baseline and most recent levels of Ntx as low (< 50 nmol/mmol creatinine), moderate (50 to 99 nmol/mmol creatinine), or high (> or = 100 nmol/mmol creatinine), and BAP as low (< 146 U/L) or high (> or = 146 U/L). The relative risks for negative clinical outcomes were estimated for each group using multiple-event and Cox regression models with time-varying covariates.
 RESULTS Patients with high and moderate Ntx levels had 2-fold increases in their risk of skeletal complications and disease progression compared with patients with low Ntx levels (P < .001 for all). High Ntx levels in each solid tumor category were associated with a 4- to 6-fold increased risk of death on study, and moderate Ntx levels a 2- to 4-fold increased risk compared with low Ntx levels (P < .001 for all). Bone alkaline phosphatase also showed some correlation with risk of negative clinical outcomes.
 CONCLUSION The bone resorption marker Ntx provides valuable prognostic information in patients with bone metastases receiving bisphosphonates.


title: Role of CD28-B7 interactions in generation and maintenance of CD8 T cell memory.

text: Although the role of CD28-B7 interaction in the activation of naive T cells is well established, its importance in the generation and maintenance of T cell memory is not well understood. In this study, we examined the requirement for CD28-B7 interactions in primary T cell activation and immune memory. Ag-specific CD8 T cell responses were compared between wild-type (+/+) and CD28-deficient (CD28(-/-)) mice following an acute infection with lymphocytic choriomeningitis virus (LCMV). During the primary response, there was a substantial activation and expansion of LCMV-specific CD8 T cells in both +/+ and CD28(-/-) mice. However, the magnitude of the primary CD8 T cell response to both dominant and subdominant LCMV CTL epitopes was approximately 2- to 3-fold lower in CD28(-/-) mice compared with +/+ mice; the lack of CD28-mediated costimulation did not lead to preferential suppression of CD8 T cell responses to the weaker subdominant epitopes. As seen in CD28(-/-) mice, blockade of B7-mediated costimulation by CTLA4-Ig treatment of +/+ mice also resulted in a 2-fold reduction in the anti-LCMV CD8 T cell responses. Loss of CD28/B7 interactions did not significantly affect the generation and maintenance of CD8 T cell memory; the magnitude of CD8 T cell memory was approximately 2-fold lower in CD28(-/-) mice as compared with +/+ mice. Further, in CD28(-/-) mice, LCMV-specific memory CD8 T cells showed normal homeostatic proliferation in vivo and also conferred protective immunity. Therefore, CD28 signaling is not necessary for the proliferative renewal and maintenance of memory CD8 T cells.


title: Systematic reviews from astronomy to zoology: myths and misconceptions.

text: Systematic literature reviews are widely used as an aid to evidence based decision making. For example, reviews of randomised controlled trials are regularly used to answer questions about the effectiveness of healthcare interventions. The high profile of systematic reviews as a cornerstone of evidence based medicine, however, has led to several misconceptions about their purpose and methods. Among these is the belief that systematic reviews are applicable only to randomised controlled trials and that they are incapable of dealing with other forms of evidence, such as from non-randomised studies or qualitative research.   The systematic literature review is a method of locating, appraising, and synthesising evidence. The value of regularly updated systematic reviews in the assessment of effectiveness of healthcare interventions was dramatically illustrated by Antman and colleagues, who showed that review articles failed to mention advances in treatment identified by an updated systematic review.1  It is nearly a quarter of a century since Gene Glass coined the term “meta-analysis” to refer to the quantitative synthesis of the results of primary studies.2 The importance of making explicit efforts to limit bias in the review of literature, however, has been emphasised by social scientists at least since the 1960s.3 In recent years systematic reviews have found an important role in health services research, and the growing interest in evidence based approaches to decision making makes it likely that their use will increase. Not everybody accepts that systematic reviews are necessary or desirable, and as one moves further away from the clinical applications of systematic reviews cynicism about their utility grows. Several arguments are commonly used to reject a wider role for systematic reviews, and these arguments are often based on major misconceptions about the history, purpose, methods, and uses of systematic reviews. I have examined eight common myths about …


title: Acellular pertussis vaccines protect against disease but fail to prevent infection and transmission in a nonhuman primate model.

text: Pertussis is a highly contagious respiratory illness caused by the bacterial pathogen Bordetella pertussis. Pertussis rates in the United States have been rising and reached a 50-y high of 42,000 cases in 2012. Although pertussis resurgence is not completely understood, we hypothesize that current acellular pertussis (aP) vaccines fail to prevent colonization and transmission. To test our hypothesis, infant baboons were vaccinated at 2, 4, and 6 mo of age with aP or whole-cell pertussis (wP) vaccines and challenged with B. pertussis at 7 mo. Infection was followed by quantifying colonization in nasopharyngeal washes and monitoring leukocytosis and symptoms. Baboons vaccinated with aP were protected from severe pertussis-associated symptoms but not from colonization, did not clear the infection faster than naïve animals, and readily transmitted B. pertussis to unvaccinated contacts. Vaccination with wP induced a more rapid clearance compared with naïve and aP-vaccinated animals. By comparison, previously infected animals were not colonized upon secondary infection. Although all vaccinated and previously infected animals had robust serum antibody responses, we found key differences in T-cell immunity. Previously infected animals and wP-vaccinated animals possess strong B. pertussis-specific T helper 17 (Th17) memory and Th1 memory, whereas aP vaccination induced a Th1/Th2 response instead. The observation that aP, which induces an immune response mismatched to that induced by natural infection, fails to prevent colonization or transmission provides a plausible explanation for the resurgence of pertussis and suggests that optimal control of pertussis will require the development of improved vaccines.


title: Specific and cooperative binding of E. coli single-stranded DNA binding protein to mRNA.

text: Fluorometric titration of E. coli single-stranded DNA binding protein with various RNAs showed that the protein specifically and cooperatively binds to its own mRNA. The binding inhibited in vitro expression of ssb and bla but not nusA. This inhibition takes place at a physiological concentration of SSB. The function of the protein in gene regulation is discussed.