Monitoring spatiotemporal biogenesis of macromolecular assemblies by pulse-chase epitope labeling.

Abstract: Many cellular proteins perform their roles within macromolecular assemblies. Hence, an understanding of how these multiprotein complexes form is a fundamental question in cell biology. We developed a translation-controlled pulse-chase system that allows time-resolved isolation of newly forming multiprotein complexes in chemical quantities suitable for biochemical and cell biological analysis. The "pulse" is triggered by an unnatural amino acid, which induces immediate translation of an amber stop codon repressed mRNA encoding the protein of interest with a built-in tag for detection and purification. The "chase" is elicited by stopping translation of this bait via a riboswitch in the respective mRNA. Over the course of validating our method, we discovered a distinct time-resolved assembly step during NPC biogenesis and could directly monitor the spatiotemporal maturation of preribosomes via immunofluorescence detection and purification of a pulse-labeled ribosomal protein. Thus, we provide an innovative strategy to study dynamic protein assembly within cellular networks.


A beginning of the end: new insights into the functional organization of telomeres

Abstract: Ever since the first demonstration of their repetitive sequence and unique replication pathway, telomeres have beguiled researchers with how they function in protecting chromosome ends. Of course much has been learned over the years, and we now appreciate that telomeres are comprised of the multimeric protein/DNA shelterin complex and that the formation of t-loops provides protection from DNA damage machinery. Deriving their name from D-loops, t-loops are generated by the insertion of the 3' overhang into telomeric repeats facilitated by the binding of TRF2. Recent studies have uncovered novel forms of chromosome end-structure that may implicate telomere organization in cellular processes beyond its essential role in telomere protection and homeostasis. In particular, we have recently described that t-loops form in a TRF2-dependent manner at interstitial telomere repeat sequences, which we termed interstitial telomere loops (ITLs). These structures are also dependent on association of lamin A/C, a canonical component of the nucleoskeleton that is mutated in myriad human diseases, including human segmental progeroid syndromes. Since ITLs are associated with telomere stability and require functional lamin A/C, our study suggests a mechanistic link between cellular aging (replicative senescence induced by telomere shortening) and organismal aging (modeled by Hutchinson Gilford Progeria Syndrome). Here we speculate on other potential ramifications of ITL formation, from gene expression to genome stability to chromosome structure.


Involvement of ER Stress in Dysmyelination of Pelizaeus-Merzbacher Disease with PLP1 Missense Mutations Shown by iPSC-Derived Oligodendrocytes

Abstract: Pelizaeus-Merzbacher disease (PMD) is a form of X-linked leukodystrophy caused by mutations in the proteolipid protein 1 (PLP1) gene. Although PLP1 proteins with missense mutations have been shown to accumulate in the rough endoplasmic reticulum (ER) in disease model animals and cell lines transfected with mutant PLP1 genes, the exact pathogenetic mechanism of PMD has not previously been clarified. In this study, we established induced pluripotent stem cells (iPSCs) from two PMD patients carrying missense mutation and differentiated them into oligodendrocytes in vitro. In the PMD iPSC-derived oligodendrocytes, mislocalization of mutant PLP1 proteins to the ER and an association between increased susceptibility to ER stress and increased numbers of apoptotic oligodendrocytes were observed. Moreover, electron microscopic analysis demonstrated drastically reduced myelin formation accompanied by abnormal ER morphology. Thus, this study demonstrates the involvement of ER stress in pathogenic dysmyelination in the oligodendrocytes of PMD patients with the PLP1 missense mutation.


Genomic Nucleosome Organization Reconstituted with Pure Proteins

Abstract: Chromatin remodelers regulate genes by organizing nucleosomes around promoters, but their individual contributions are obfuscated by the complex in vivo milieu of factor redundancy and indirect effects. Genome-wide reconstitution of promoter nucleosome organization with purified proteins resolves this problem and is therefore a critical goal. Here, we reconstitute four stages of nucleosome architecture using purified components: yeast genomic DNA, histones, sequence-specific Abf1/Reb1, and remodelers RSC, ISW2, INO80, and ISW1a. We identify direct, specific, and sufficient contributions that in vivo observations validate. First, RSC clears promoters by translating poly(dA:dT) into directional nucleosome removal. Second, partial redundancy is recapitulated where INO80 alone, or ISW2 at Abf1/Reb1sites, positions +1 nucleosomes. Third, INO80 and ISW2 each align downstream nucleosomal arrays. Fourth, ISW1a tightens the spacing to canonical repeat lengths. Such a minimal set of rules and proteins establishes core mechanisms by which promoter chromatin architecture arises through a blend of redundancy and specialization.


Identification of splenic reservoir monocytes and their deployment to inflammatory sites.

Abstract: A current paradigm states that monocytes circulate freely and patrol blood vessels but differentiate irreversibly into dendritic cells (DCs) or macrophages upon tissue entry. Here we show that bona fide undifferentiated monocytes reside in the spleen and outnumber their equivalents in circulation. The reservoir monocytes assemble in clusters in the cords of the subcapsular red pulp and are distinct from macrophages and DCs. In response to ischemic myocardial injury, splenic monocytes increase their motility, exit the spleen en masse, accumulate in injured tissue, and participate in wound healing. These observations uncover a role for the spleen as a site for storage and rapid deployment of monocytes and identify splenic monocytes as a resource that the body exploits to regulate inflammation.


Prox1 Is Required for Granule Cell Maturation and Intermediate Progenitor Maintenance During Brain Neurogenesis

Abstract: The dentate gyrus has an important role in learning and memory, and adult neurogenesis in the subgranular zone of the dentate gyrus may play a role in the acquisition of new memories. The homeobox gene Prox1 is expressed in the dentate gyrus during embryonic development and adult neurogenesis. Here we show that Prox1 is necessary for the maturation of granule cells in the dentate gyrus during development and for the maintenance of intermediate progenitors during adult neurogenesis. We also demonstrate that Prox1-expressing intermediate progenitors are required for adult neural stem cell self-maintenance in the subgranular zone; thus, we have identified a previously unknown non-cell autonomous regulatory feedback mechanism that controls adult neurogenesis in this region of the mammalian brain. Finally, we show that the ectopic expression of Prox1 induces premature differentiation of neural stem cells.


Senescent growth arrest in mesenchymal stem cells is bypassed by Wip1-mediated downregulation of intrinsic stress signaling pathways.

Abstract: Human mesenchymal stem cells (hMSCs) have been widely studied as a source of primary adult stem cells for cell therapy because of their multidifferentiation potential; however, the growth arrest (also known as "premature senescence") often found in hMSCs cultured in vitro has been a major obstacle to the in-depth characterization of these cells. In addition, the inability to maintain constant cell growth hampers the development of additional genetic modifications aimed at achieving desired levels of differentiation to specific tissues; however, the molecular mechanisms that govern this phenomenon remain unclear, with the exception of a few studies demonstrating that induction of p16INK4a is responsible for this senescence-like event. Here, we observed that the premature growth arrest in hMSCs occurs in parallel with the induction of p16INK4a, following abrogation of inhibitory phosphorylation of retinoblastoma protein. These stress responses were concurrent with increased formation of reactive oxygen species (ROSs) from mitochondria and increased p38 mitogen-activated protein kinase (MAPK) activity. The introduction of Wip1 (wild-type p53 inducible phosphatase-1), a well-studied stress modulator, significantly lowered p16INK4a expression and led to p38 MAPK inactivation, although it failed to affect the levels of ROSs. Moreover, the suppression of stress responses by Wip1 apparently extended the life span of hMSCs, compared with control conditions, while maintaining their multilineage differentiation potential. Based on these results, we suggest that senescent growth arrest in hMSCs may result from activation of stress signaling pathways and consequent onset of stress responses, due in part to ROS production during prolonged in vitro culture.


Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis.

Abstract: Mononuclear phagocytes, including monocytes, macrophages, and dendritic cells, contribute to tissue integrity as well as to innate and adaptive immune defense. Emerging evidence for labor division indicates that manipulation of these cells could bear therapeutic potential. However, specific ontogenies of individual populations and the overall functional organization of this cellular network are not well defined. Here we report a fate-mapping study of the murine monocyte and macrophage compartment taking advantage of constitutive and conditional CX(3)CR1 promoter-driven Cre recombinase expression. We have demonstrated that major tissue-resident macrophage populations, including liver Kupffer cells and lung alveolar, splenic, and peritoneal macrophages, are established prior to birth and maintain themselves subsequently during adulthood independent of replenishment by blood monocytes. Furthermore, we have established that short-lived Ly6C(+) monocytes constitute obligatory steady-state precursors of blood-resident Ly6C(-) cells and that the abundance of Ly6C(+) blood monocytes dynamically controls the circulation lifespan of their progeny.


The centrosome cycle: Centriole biogenesis, duplication and inherent asymmetries

Abstract: Centrosomes are microtubule-organizing centres of animal cells. They influence the morphology of the microtubule cytoskeleton, function as the base for the primary cilium and serve as a nexus for important signalling pathways. At the core of a typical centrosome are two cylindrical microtubule-based structures termed centrioles, which recruit a matrix of associated pericentriolar material. Cells begin the cell cycle with exactly one centrosome, and the duplication of centrioles is constrained such that it occurs only once per cell cycle and at a specific site in the cell. As a result of this duplication mechanism, the two centrioles differ in age and maturity, and thus have different functions; for example, the older of the two centrioles can initiate the formation of a ciliary axoneme. We discuss spatial aspects of the centrosome duplication cycle, the mechanism of centriole assembly and the possible consequences of the inherent asymmetry of centrioles and centrosomes.


Osteoclasts control reactivation of dormant myeloma cells by remodelling the endosteal niche

Abstract: Multiple myeloma is largely incurable, despite development of therapies that target myeloma cell-intrinsic pathways. Disease relapse is thought to originate from dormant myeloma cells, localized in specialized niches, which resist therapy and repopulate the tumour. However, little is known about the niche, and how it exerts cell-extrinsic control over myeloma cell dormancy and reactivation. In this study, we track individual myeloma cells by intravital imaging as they colonize the endosteal niche, enter a dormant state and subsequently become activated to form colonies. We demonstrate that dormancy is a reversible state that is switched 'on' by engagement with bone-lining cells or osteoblasts, and switched 'off' by osteoclasts remodelling the endosteal niche. Dormant myeloma cells are resistant to chemotherapy that targets dividing cells. The demonstration that the endosteal niche is pivotal in controlling myeloma cell dormancy highlights the potential for targeting cell-extrinsic mechanisms to overcome cell-intrinsic drug resistance and prevent disease relapse.


IDEAL in meshes for prolapse, urinary incontinence, and hernia repair.

Abstract:
PURPOSE Mesh surgeries are counted among the most frequently applied surgical procedures. Despite global spread of mesh applying surgeries, there is no current systematic analysis of incidence and possible prevention of adverse events after mesh implantation.    MATERIALS AND METHODS Based on the recommendations of IDEAL an in vitro test system for biocompatibility of surgical meshes has been generated (Innovation). Coating strategies for biocompatibility optimization have been developed (Development). The native and modified alloplastic materials have been tested in an animal model over 2 years (Exploration and Assessment and Long-term study).
 RESULTS In 3 meshes, implanted in sheep and explanted at 4 different time points (a, 3 months; b, 6 months; c, 12 months; and d, 24 months) over 24 months, thickness of inflammatory tissue (TVT a, 35 µm; b, 32 µm; c, 33 µm; d, 28 µm; UltraPro, a, 25 µm; b, 24 µm; c, 21 µm; d, 22 µm; PVDF a, 20 µm; b, 21 µm; c, 14 µm; d, 15µm), connective tissue (TVT a, 37 µm; b, 36 µm; c, 43 µm; d, 41 µm; UltraPro a, 33 µm; b, 32 µm; c, 40 µm; d, 38 µm; PVDF a, 25 µm; b, 22 µm; c, 22 µm; d, 24 µm), and macrophage infiltration (TVT a, 36%; b, 33%; c, 23%; d, 20%; UltraPro a, 34%; b, 28%; c, 25%; d, 22%; PVDF a, 24%; b, 18%; c, 18%; d, 16%) revealed comparable ranking characteristics at every time point after explantation. The in vivo performance of these meshes in a sheep model was predictable with a previously developed in vitro test system. Coating of meshes with autologous plasma prior to implantation seems to have a positive effect on the meshes biocompatibility.
 CONCLUSION We have applied IDEAL criteria on a new innovation for surgical meshes. The results permit the generation of a ranking of currently available meshes with potential to optimize future meshes.


Aspirin in the chemoprevention of colorectal neoplasia: an overview.

Abstract: Considerable evidence supports the effectiveness of aspirin for chemoprevention of colorectal cancer (CRC) in addition to its well-established benefits in the prevention of vascular disease. Epidemiologic studies have consistently observed an inverse association between aspirin use and risk of CRC. A recent pooled analysis of a long-term posttrial follow-up of nearly 14,000 patients from four randomized, cardiovascular disease prevention trials showed that daily aspirin treatment for about five years was associated with a 34% reduction in 20-year CRC mortality. A separate metaanalysis of nearly 3,000 patients with a history of colorectal adenoma or cancer in four randomized adenoma prevention trials showed that aspirin reduced the occurrence of advanced adenomas by 28% and any adenoma by 17%. Aspirin has also been shown to be beneficial in a clinical trial of patients with Lynch syndrome, a hereditary CRC syndrome; in those treated with aspirin for at least two years, there was a 50% or more reduction in the risk of CRC commencing five years after randomization and after aspirin had been discontinued. A few observational studies have shown an increase in survival among patients with CRC who use aspirin. Taken together, these findings strengthen the case for consideration of long-term aspirin use in CRC prevention. Despite these compelling data, there is a lack of consensus about the balance of risks and benefits associated with long-term aspirin use, particularly in low-risk populations. The optimal dose to use for cancer prevention and the precise mechanism underlying aspirin's anticancer effect require further investigation.


Chemokine receptor trio: CXCR3, CXCR4 and CXCR7 crosstalk via CXCL11 and CXCL12.

Abstract: Although chemokines are well established to function in immunity and endothelial cell activation and proliferation, a rapidly growing literature suggests that CXC Chemokine receptors CXCR3, CXCR4 and CXCR7 are critical in the development and progression of solid tumors. The effect of these chemokine receptors in tumorigenesis is mediated via interactions with shared ligands I-TAC (CXCL11) and SDF-1 (CXCL12). Over the last decade, CXCR4 has been extensively reported to be overexpressed in most human solid tumors and has earned considerable attention toward elucidating its role in cancer metastasis. To enrich the existing armamentarium of anti-cancerous agents, many inhibitors of CXCL12-CXCR4 axis have emerged as additional or alternative agents for neo-adjuvant treatments and even many of them are in preclinical and clinical stages of their development. However, the discovery of CXCR7 as another receptor for CXCL12 with rather high binding affinity and recent reports about its involvement in cancer progression, has questioned the potential of "selective blockade" of CXCR4 as cancer chemotherapeutics. Interestingly, CXCR7 can also bind another chemokine CXCL11, which is an established ligand for CXCR3. Recent reports have documented that CXCR3 and their ligands are overexpressed in different solid tumors and regulate tumor growth and metastasis. Therefore, it is important to consider the interactions and crosstalk between these three chemokine receptors and their ligand mediated signaling cascades for the development of effective anti-cancer therapies. Emerging evidence also indicates that these receptors are differentially expressed in tumor endothelial cells as well as in cancer stem cells, suggesting their direct role in regulating tumor angiogenesis and metastasis. In this review, we will focus on the signals mediated by this receptor trio via their shared ligands and their role in tumor growth and progression.


Human Cyclin a Is Required for Mitosis until Mid Prophase

Abstract: 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.


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

Abstract:
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.


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

Abstract: 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.


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

Abstract: 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).


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

Abstract: 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.


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

Abstract:
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.


The Burden of Trachoma in Ayod County of Southern Sudan

Abstract:
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.


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

Abstract: 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.


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

Abstract: 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.


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

Abstract: 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.


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

Abstract: 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.


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

Abstract: 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.


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

Abstract: 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.


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

Abstract:
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.


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

Abstract: 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.


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

Abstract:
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.


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

Abstract: 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.


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

Abstract: 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.


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

Abstract: 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.


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

Abstract: 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.


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

Abstract: 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.


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

Abstract: 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.


GeneTrack--a genomic data processing and visualization framework.

Abstract: 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


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

Abstract: 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.


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

Abstract: 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.


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

Abstract: 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.


Feedback Circuit among INK4 Tumor Suppressors Constrains Human Glioblastoma Development

Abstract: 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.


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

Abstract: 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.


Dynein pulls microtubules without rotating its stalk.

Abstract: 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).


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

Abstract: 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.


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

Abstract: 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.


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

Abstract:
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.


Global Assessment of Genomic Regions Required for Growth in Mycobacterium tuberculosis

Abstract: 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.


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

Abstract: 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.


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

Abstract: 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.


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

Abstract: 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.


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

Abstract:
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.


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

Abstract: 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.


Systematic reviews from astronomy to zoology: myths and misconceptions.

Abstract: 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 …


Rps14 haploinsufficiency causes a block in erythroid differentiation mediated by S100A8 and S100A9

Abstract: Impaired erythropoiesis in the deletion 5q (del(5q)) subtype of myelodysplastic syndrome (MDS) has been linked to heterozygous deletion of RPS14, which encodes the ribosomal protein small subunit 14. We generated mice with conditional inactivation of Rps14 and demonstrated an erythroid differentiation defect that is dependent on the tumor suppressor protein p53 (encoded by Trp53 in mice) and is characterized by apoptosis at the transition from polychromatic to orthochromatic erythroblasts. This defect resulted in age-dependent progressive anemia, megakaryocyte dysplasia and loss of hematopoietic stem cell (HSC) quiescence. As assessed by quantitative proteomics, mutant erythroblasts expressed higher levels of proteins involved in innate immune signaling, notably the heterodimeric S100 calcium-binding proteins S100a8 and S100a9. S100a8—whose expression was increased in mutant erythroblasts, monocytes and macrophages—is functionally involved in the erythroid defect caused by the Rps14 deletion, as addition of recombinant S100a8 was sufficient to induce a differentiation defect in wild-type erythroid cells, and genetic inactivation of S100a8 expression rescued the erythroid differentiation defect of Rps14-haploinsufficient HSCs. Our data link Rps14 haploinsufficiency in del(5q) MDS to activation of the innate immune system and induction of S100A8-S100A9 expression, leading to a p53-dependent erythroid differentiation defect.


Rational Extension of the Ribosome Biogenesis Pathway Using Network-Guided Genetics

Abstract: Biogenesis of ribosomes is an essential cellular process conserved across all eukaryotes and is known to require >170 genes for the assembly, modification, and trafficking of ribosome components through multiple cellular compartments. Despite intensive study, this pathway likely involves many additional genes. Here, we employ network-guided genetics-an approach for associating candidate genes with biological processes that capitalizes on recent advances in functional genomic and proteomic studies-to computationally identify additional ribosomal biogenesis genes. We experimentally evaluated >100 candidate yeast genes in a battery of assays, confirming involvement of at least 15 new genes, including previously uncharacterized genes (YDL063C, YIL091C, YOR287C, YOR006C/TSR3, YOL022C/TSR4). We associate the new genes with specific aspects of ribosomal subunit maturation, ribosomal particle association, and ribosomal subunit nuclear export, and we identify genes specifically required for the processing of 5S, 7S, 20S, 27S, and 35S rRNAs. These results reveal new connections between ribosome biogenesis and mRNA splicing and add >10% new genes-most with human orthologs-to the biogenesis pathway, significantly extending our understanding of a universally conserved eukaryotic process.


Roles of induced expression of MAPK phosphatase-2 in tumor development in RET-MEN2A transgenic mice

Abstract: Germline mutations in the RET tyrosine kinase gene are responsible for the development of multiple endocrine neoplasia 2A and 2B (MEN2A and MEN2B). However, knowledge of the fundamental principles that determine the mutant RET-mediated signaling remains elusive. Here, we report increased expression of mitogen-activated protein kinase phosphatase-2 (MKP-2) in carcinomas developed in transgenic mice carrying RET with the MEN2A mutation (RET-MEN2A). The expression of MKP-2 was not only induced by RET-MEN2A or RET-MEN2B mutant proteins but also by the activation of endogenous RET by its ligand, glial cell line-derived neurotrophic factor (GDNF). MKP-2 expression was also evident in the MKK-f cell line, which was established from a mammary tumor developed in a RET-MEN2A transgenic mouse. Inhibition of MKP-2 attenuated the in vitro and in vivo proliferation of MKK-f cells, which was mediated by the suppression of cyclin B1 expression. Furthermore, we found that MKP-2 is highly expressed in medullary thyroid carcinomas derived from MEN2A patients. These findings suggest that the increased expression of MKP-2 may play a crucial role in oncogenic signaling downstream of mutant RET, leading to deregulation of cell cycle.


Effects of repeated social stress on leukocyte distribution in bone marrow, peripheral blood and spleen

Abstract: Leukocyte trafficking between the various body compartments has an important surveillance function that ensures the detection of antigen and enables the immune system to initiate a rapid and effective response. Repeated social defeat of group-housed male mice induced by daily, acute encounters with an aggressive conspecific substantially altered leukocyte trafficking and led to a gradual redistribution of immune cells in bone marrow, peripheral blood and spleen. Recurrent exposure to the stressor over a period of 2, 4 or 6 consecutive days was associated with cell mobilization and increased myelopoiesis in the bone marrow that was paralleled by an accumulation of neutrophils and monocytes in circulation and spleen. Substantial depletion of B cells in bone marrow and blood was associated with an increase in splenic B cells indicating a redirection of this cell type to the spleen. In contrast, T cells were markedly reduced in these immune compartments. The recruitment of CD11b+ leukocytes (i.e., monocytes/macrophages and neutrophils) from the bone marrow to the spleen might play a critical role in the development of functional glucocorticoid resistance in the murine spleen that was reported in context with repeated social defeat.


Lentivirally transduced dendritic cells as a tool for cancer immunotherapy.

Abstract:
BACKGROUND Dendritic cells (DC) are the professional antigen-presenting cells of the immune system, fully equipped to prime naive T cells and thus essential components for cancer immunotherapy.
 METHODS We tested the influence of several elements (cPPT, trip, WPRE, SIN) on the transduction efficiency of human DC. Human and murine DC were transduced with tNGFR-encoding lentiviruses to assess the effect of transduction on phenotype and function. Human DC were transduced with lentiviruses encoding huIi80MAGE-A3 and murine DC with huIi80tOVA to test antigen presentation.
 RESULTS A self-inactivating (SIN) lentiviral vector containing the trip element was most efficient in transducing human DC. The transduction of DC with trip/SIN tNGFR encoding lentiviral vectors at MOI 15 resulted in stable gene expression in up to 94.6% (murine) and 88.2% (human) of the mature DC, without perturbing viability, phenotype and function. Human huIi80MAGE-A3-transduced DC were able to stimulate MAGE-A3-specific CD4(+) and CD8(+) T cell clones and could prime both MAGE-A3-specific CD4(+) and CD8(+) T cells in vitro. Murine huIi80tOVA-transduced DC were able to present OVA peptides in the context of MHC class I and class II in vitro and induced a strong OVA-specific cytotoxic T lymphocyte response in vivo, that was protective against subsequent challenge with OVA-expressing tumor cells.
 CONCLUSIONS We show that, using lentiviral vectors, efficient gene transfer in human and murine DC can be obtained and that these DC can elicit antigen-specific immune responses in vitro and in vivo. The composition of the transfer vector has a major impact on the transduction efficiency.


Molecular characterization of the yeast meiotic regulatory gene RIM1.

Abstract: In the yeast Saccharomyces cerevisiae, genetic studies suggest that the RIM1 gene encodes a positive regulator of meiosis. rim1 mutations cause reduced expression of IME1, which is required for expression of many meiotic genes, and thus lead to a partial defect in meiosis and spore formation. We report the sequence of RIM1 and functional analysis of its coding region. The RIM1 gene product (RIM1) contains three regions similar to C2H2 zinc fingers. Serine substitutions for cysteine in each of the putative zinc fingers abolish RIM1 function. The carboxyl-terminus of RIM1 is enriched in acidic amino acids and is required for full RIM1 activity. RIM1 also contains two putative cAMP-dependent protein kinase (cAPK) phosphorylation sites. At one site, substitution of alanine for serine does not affect RIM1 activity; at the other site, this substitution impairs activity. This analysis of RIM1 suggests that the protein may function as a transcriptional activator. We have used the cloned RIM1 gene to create a complete rim1 deletion. This null allele, like previously isolated rim1 mutations, causes a partial meiotic defect. In addition to RIM1, maximum IME1 expression requires the MCK1 and IME4 gene products. Defects associated with rim1, mck1, and ime4 mutations in expression of a meiotic reporter gene (ime2-lacZ) and in sporulation are additive. These findings suggest that RIM1 acts independently of MCK1 and IME4 to stimulate IME1 expression.


Butyrate greatly enhances derivation of human induced pluripotent stem cells by promoting epigenetic remodeling and the expression of pluripotency-associated genes.

Abstract: We report here that butyrate, a naturally occurring fatty acid commonly used as a nutritional supplement and differentiation agent, greatly enhances the efficiency of induced pluripotent stem (iPS) cell derivation from human adult or fetal fibroblasts. After transient butyrate treatment, the iPS cell derivation efficiency is enhanced by 15- to 51-fold using either retroviral or piggyBac transposon vectors expressing 4 to 5 reprogramming genes. Butyrate stimulation is more remarkable (>100- to 200-fold) on reprogramming in the absence of either KLF4 or MYC transgene. Butyrate treatment did not negatively affect properties of iPS cell lines established by either 3 or 4 retroviral vectors or a single piggyBac DNA transposon vector. These characterized iPS cell lines, including those derived from an adult patient with sickle cell disease by either the piggyBac or retroviral vectors, show normal karyotypes and pluripotency. To gain insights into the underlying mechanisms of butyrate stimulation, we conducted genome-wide gene expression and promoter DNA methylation microarrays and other epigenetic analyses on established iPS cells and cells from intermediate stages of the reprogramming process. By days 6 to 12 during reprogramming, butyrate treatment enhanced histone H3 acetylation, promoter DNA demethylation, and the expression of endogenous pluripotency-associated genes, including DPPA2, whose overexpression partially substitutes for butyrate stimulation. Thus, butyrate as a cell permeable small molecule provides a simple tool to further investigate molecular mechanisms of cellular reprogramming. Moreover, butyrate stimulation provides an efficient method for reprogramming various human adult somatic cells, including cells from patients that are more refractory to reprogramming.


Nanoparticle conjugation of CpG enhances adjuvancy for cellular immunity and memory recall at low dose.

Abstract: In subunit vaccines, strong CD8(+) T-cell responses are desired, yet they are elusive at reasonable adjuvant doses. We show that targeting adjuvant to the lymph node (LN) via ultrasmall polymeric nanoparticles (NPs), which rapidly drain to the LN after intradermal injection, greatly enhances adjuvant efficacy at low doses. Coupling CpG-B or CpG-C oligonucleotides to NPs led to better dual-targeting of adjuvant and antigen (codelivered on separate NPs) in cross-presenting dendritic cells compared with free adjuvant. This led to enhanced dendritic cell maturation and T helper 1 (Th1)-cytokine secretion, in turn driving stronger effector CD8(+) T-cell activation with enhanced cytolytic profiles and, importantly, more powerful memory recall. With only 4 μg CpG, NP-CpG-B could substantially protect mice from syngeneic tumor challenge, even after 4 mo of vaccination, compared with free CpG-B. Together, these results show that nanocarriers can enhance vaccine efficacy at a low adjuvant dose for inducing potent and long-lived cellular immunity.


Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds

Abstract: Reprogramming of mouse and human somatic cells can be achieved by ectopic expression of transcription factors, but with low efficiencies. We report that DNA methyltransferase and histone deacetylase (HDAC) inhibitors improve reprogramming efficiency. In particular, valproic acid (VPA), an HDAC inhibitor, improves reprogramming efficiency by more than 100-fold, using Oct4-GFP as a reporter. VPA also enables efficient induction of pluripotent stem cells without introduction of the oncogene c-Myc.


Noninvasive ventilation for treatment of acute respiratory failure in patients undergoing solid organ transplantation: a randomized trial.

Abstract:
CONTEXT Noninvasive ventilation (NIV) has been associated with lower rates of endotracheal intubation in populations of patients with acute respiratory failure.
 OBJECTIVE To compare NIV with standard treatment using supplemental oxygen administration to avoid endotracheal intubation in recipients of solid organ transplantation with acute hypoxemic respiratory failure.
 DESIGN AND SETTING Prospective randomized study conducted at a 14-bed, general intensive care unit of a university hospital.
 PATIENTS Of 238 patients who underwent solid organ transplantation from December 1995 to October 1997, 51 were treated for acute respiratory failure. Of these, 40 were eligible and 20 were randomized to each group.
 INTERVENTION Noninvasive ventilation vs standard treatment with supplemental oxygen administration.
 MAIN OUTCOME MEASURES The need for endotracheal intubation and mechanical ventilation at any time during the study, complications not present on admission, duration of ventilatory assistance, length of hospital stay, and intensive care unit mortality.
 RESULTS The 2 groups were similar at study entry. Within the first hour of treatment, 14 patients (70%) in the NIV group, and 5 patients (25%) in the standard treatment group improved their ratio of the PaO2 to the fraction of inspired oxygen (FIO2). Over time, a sustained improvement in PaO2 to FIO2 was noted in 12 patients (60%) in the NIV group, and in 5 patients (25%) randomized to standard treatment (P = .03). The use of NIV was associated with a significant reduction in the rate of endotracheal intubation (20% vs 70%; P = .002), rate of fatal complications (20% vs 50%; P = .05), length of stay in the intensive care unit by survivors (mean [SD] days, 5.5 [3] vs 9 [4]; P = .03), and intensive care unit mortality (20% vs 50%; P = .05). Hospital mortality did not differ.
 CONCLUSIONS These results indicate that transplantation programs should consider NIV in the treatment of selected recipients of transplantation with acute respiratory failure.


Use of synthetic peptides to locate novel integrin alpha2beta1-binding motifs in human collagen III.

Abstract: A set of 57 synthetic peptides encompassing the entire triplehelical domain of human collagen III was used to locate binding sites for the collagen-binding integrin alpha(2)beta(1). The capacity of the peptides to support Mg(2+)-dependent binding of several integrin preparations was examined. Wild-type integrins (recombinant alpha(2) I-domain, alpha(2)beta(1) purified from platelet membranes, and recombinant soluble alpha(2)beta(1) expressed as an alpha(2)-Fos/beta(1)-Jun heterodimer) bound well to only three peptides, two containing GXX'GER motifs (GROGER and GMOGER, where O is hydroxyproline) and one containing two adjacent GXX'GEN motifs (GLKGEN and GLOGEN). Two mutant alpha(2) I-domains were tested: the inactive T221A mutant, which recognized no peptides, and the constitutively active E318W mutant, which bound a larger subset of peptides. Adhesion of activated human platelets to GER-containing peptides was greater than that of resting platelets, and HT1080 cells bound well to more of the peptides compared with platelets. Binding of cells and recombinant proteins was abolished by anti-alpha(2) monoclonal antibody 6F1 and by chelation of Mg(2+). We describe two novel high affinity integrin-binding motifs in human collagen III (GROGER and GLOGEN) and a third motif (GLKGEN) that displays intermediate activity. Each motif was verified using shorter synthetic peptides.


The Viral and Cellular MicroRNA Targetome in Lymphoblastoid Cell Lines

Abstract: Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus linked to a number of B cell cancers and lymphoproliferative disorders. During latent infection, EBV expresses 25 viral pre-microRNAs (miRNAs) and induces the expression of specific host miRNAs, such as miR-155 and miR-21, which potentially play a role in viral oncogenesis. To date, only a limited number of EBV miRNA targets have been identified; thus, the role of EBV miRNAs in viral pathogenesis and/or lymphomagenesis is not well defined. Here, we used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) combined with deep sequencing and computational analysis to comprehensively examine the viral and cellular miRNA targetome in EBV strain B95-8-infected lymphoblastoid cell lines (LCLs). We identified 7,827 miRNA-interaction sites in 3,492 cellular 3'UTRs. 531 of these sites contained seed matches to viral miRNAs. 24 PAR-CLIP-identified miRNA:3'UTR interactions were confirmed by reporter assays. Our results reveal that EBV miRNAs predominantly target cellular transcripts during latent infection, thereby manipulating the host environment. Furthermore, targets of EBV miRNAs are involved in multiple cellular processes that are directly relevant to viral infection, including innate immunity, cell survival, and cell proliferation. Finally, we present evidence that myc-regulated host miRNAs from the miR-17/92 cluster can regulate latent viral gene expression. This comprehensive survey of the miRNA targetome in EBV-infected B cells represents a key step towards defining the functions of EBV-encoded miRNAs, and potentially, identifying novel therapeutic targets for EBV-associated malignancies.


Mitochondrial Dysfunction Leads to Nuclear Genome Instability via an Iron-Sulfur Cluster Defect

Abstract: Mutations and deletions in the mitochondrial genome (mtDNA), as well as instability of the nuclear genome, are involved in multiple human diseases. Here, we report that in Saccharomyces cerevisiae, loss of mtDNA leads to nuclear genome instability, through a process of cell-cycle arrest and selection we define as a cellular crisis. This crisis is not mediated by the absence of respiration, but instead correlates with a reduction in the mitochondrial membrane potential. Analysis of cells undergoing this crisis identified a defect in iron-sulfur cluster (ISC) biogenesis, which requires normal mitochondrial function. We found that downregulation of nonmitochondrial ISC protein biogenesis was sufficient to cause increased genomic instability in cells with intact mitochondrial function. These results suggest mitochondrial dysfunction stimulates nuclear genome instability by inhibiting the production of ISC-containing protein(s), which are required for maintenance of nuclear genome integrity. For a video summary of this article, see the PaperFlick file available with the online Supplemental Data.


Replisome stall events have shaped the distribution of replication origins in the genomes of yeasts

Abstract: During S phase, the entire genome must be precisely duplicated, with no sections of DNA left unreplicated. Here, we develop a simple mathematical model to describe the probability of replication failing due to the irreversible stalling of replication forks. We show that the probability of complete genome replication is maximized if replication origins are evenly spaced, the largest inter-origin distances are minimized, and the end-most origins are positioned close to chromosome ends. We show that origin positions in the yeast Saccharomyces cerevisiae genome conform to all three predictions thereby maximizing the probability of complete replication if replication forks stall. Origin positions in four other yeasts-Kluyveromyces lactis, Lachancea kluyveri, Lachancea waltii and Schizosaccharomyces pombe-also conform to these predictions. Equating failure rates at chromosome ends with those in chromosome interiors gives a mean per nucleotide fork stall rate of ∼5 × 10(-8), which is consistent with experimental estimates. Using this value in our theoretical predictions gives replication failure rates that are consistent with data from replication origin knockout experiments. Our theory also predicts that significantly larger genomes, such as those of mammals, will experience a much greater probability of replication failure genome-wide, and therefore will likely require additional compensatory mechanisms.