Engineered RNA viral synthesis of microRNAs.

Abstract: MicroRNAs (miRNAs) are short noncoding RNAs that exert posttranscriptional gene silencing and regulate gene expression. In addition to the hundreds of conserved cellular miRNAs that have been identified, miRNAs of viral origin have been isolated and found to modulate both the viral life cycle and the cellular transcriptome. Thus far, detection of virus-derived miRNAs has been largely limited to DNA viruses, suggesting that RNA viruses may be unable to exploit this aspect of transcriptional regulation. Lack of RNA virus-produced miRNAs has been attributed to the replicative constraints that would incur following RNase III processing of a genomic hairpin. To ascertain whether the generation of viral miRNAs is limited to DNA viruses, we investigated whether influenza virus could be designed to deliver functional miRNAs without affecting replication. Here, we describe a modified influenza A virus that expresses cellular microRNA-124 (miR-124). Insertion of the miR-124 hairpin into an intron of the nuclear export protein transcript resulted in endogenous processing and functional miR-124. We demonstrate that a viral RNA genome incorporating a hairpin does not result in segment instability or miRNA-mediated genomic targeting, thereby permitting the virus to produce a miRNA without having a negative impact on viral replication. This work demonstrates that RNA viruses can produce functional miRNAs and suggests that this level of transcriptional regulation may extend beyond DNA viruses.


Aluminum fluoride stimulates surface protrusions in cells overexpressing the ARF6 GTPase

Abstract: To study the effector function of the ADP- ribosylation factor (ARF) 6 GTP-binding protein, we transfected HeLa cells with wild-type, epitope-tagged ARF6. Previously shown to indirectly activate the ARF1 GTPase, aluminum fluoride (AIF) treatment of ARF6-transfected cells resulted in a redistribution of both ARF6 and actin to discrete sites on the plasma membrane, which became increasingly protrusive over time. The effects of AIF were reversible, specific to cells transfected with wild-type ARF6, and resembled the cellular protrusions observed in cells expressing the GTPase defective mutant of ARF6. Importantly, the protrusions observed in cells transfected with ARF6 were distinct from the enhanced stress fibers and membrane ruffles observed in cells transfected with RhoA and Rac1, respectively. In cells forming protrusions, there was an apparent stimulation of macropinocytosis and membrane recycling within the protrusive structures. In contrast, no block in transferrin uptake or alteration of the distribution of clathrin AP-2 complexes was detected in these cells. The AIF-induced, ARF6- dependent formation of protrusive structures was blocked by cytochalasin D and inhibitors of the lipoxygenase pathway. These observations support a novel role for the ARF6 GTPase in modeling the plasma membrane and underlying cytoskeleton.


Novel proteinaceous infectious particles cause scrapie.

Abstract: After infection and a prolonged incubation period, the scrapie agent causes a degenerative disease of the central nervous system in sheep and goats. Six lines of evidence including sensitivity to proteases demonstrate that this agent contains a protein that is required for infectivity. Although the scrapie agent is irreversibly inactivated by alkali, five procedures with more specificity for modifying nucleic acids failed to cause inactivation. The agent shows heterogeneity with respect to size, apparently a result of its hydrophobicity; the smallest form may have a molecular weight of 50,000 or less. Because the novel properties of the scrapie agent distinguish it from viruses, plasmids, and viroids, a new term "prion" is proposed to denote a small proteinaceous infectious particle which is resistant to inactivation by most procedures that modify nucleic acids. Knowledge of the scrapie agent structure may have significance for understanding the causes of several degenerative diseases.


Long term pharmacotherapy for obesity and overweight: updated meta-analysis.

Abstract:
OBJECTIVE To summarise the long term efficacy of anti-obesity drugs in reducing weight and improving health status.
 DESIGN Updated meta-analysis of randomised trials.
 DATA SOURCES Medline, Embase, the Cochrane controlled trials register, the Current Science meta-register of controlled trials, and reference lists of identified articles. All data sources were searched from December 2002 (end date of last search) to December 2006.    STUDIES REVIEWED Double blind randomised placebo controlled trials of approved anti-obesity drugs used in adults (age over 18) for one year or longer.
 RESULTS 30 trials of one to four years' duration met the inclusion criteria: 16 orlistat (n=10 631 participants), 10 sibutramine (n=2623), and four rimonabant (n=6365). Of these, 14 trials were new and 16 had previously been identified. Attrition rates averaged 30-40%. Compared with placebo, orlistat reduced weight by 2.9 kg (95% confidence interval 2.5 kg to 3.2 kg), sibutramine by 4.2 kg (3.6 kg to 4.7 kg), and rimonabant by 4.7 kg (4.1 kg to 5.3 kg). Patients receiving active drug treatment were significantly more likely to achieve 5% and 10% weight loss thresholds. Orlistat reduced the incidence of diabetes and improved concentrations of total cholesterol and low density lipoprotein cholesterol, blood pressure, and glycaemic control in patients with diabetes but increased rates of gastrointestinal side effects and slightly lowered concentrations of high density lipoprotein. Sibutramine improved [corrected] concentrations of high density lipoprotein cholesterol and triglycerides [corrected] Rimonabant improved concentrations of high density lipoprotein cholesterol and triglycerides, blood pressure, and glycaemic control in patients with diabetes but increased the risk of mood disorders.
 CONCLUSIONS Orlistat, sibutramine, and rimonabant modestly reduce weight, have differing effects on cardiovascular risk profiles, and have specific adverse effects.


TRPM7 is required for breast tumor cell metastasis.

Abstract: TRPM7 encodes a Ca2+-permeable nonselective cation channel with kinase activity. TRPM7 has been implicated in control of cell adhesion and migration, but whether TRPM7 activity contributes to cancer progression has not been established. Here we report that high levels of TRPM7 expression independently predict poor outcome in breast cancer patients and that it is functionally required for metastasis formation in a mouse xenograft model of human breast cancer. Mechanistic investigation revealed that TRPM7 regulated myosin II-based cellular tension, thereby modifying focal adhesion number, cell-cell adhesion and polarized cell movement. Our findings therefore suggest that TRPM7 is part of a mechanosensory complex adopted by cancer cells to drive metastasis formation.


Frailty Syndrome: A Transitional State in a Dynamic Process

Abstract: Frailty has long been considered synonymous with disability and comorbidity, to be highly prevalent in old age and to confer a high risk for falls, hospitalization and mortality. However, it is becoming recognized that frailty may be a distinct clinical syndrome with a biological basis. The frailty process appears to be a transitional state in the dynamic progression from robustness to functional decline. During this process, total physiological reserves decrease and become less likely to be sufficient for the maintenance and repair of the ageing body. Central to the clinical concept of frailty is that no single altered system alone defines it, but that multiple systems are involved. Clinical consensus regarding the phenotype which constitutes frailty, drawing upon the opinions of numerous authors, shows the characteristics to include wasting (loss of both muscle mass and strength and weight loss), loss of endurance, decreased balance and mobility, slowed performance, relative inactivity and, potentially, decreased cognitive function. Frailty is a distinct entity easily recognized by clinicians, with multiple manifestations and with no single symptom being sufficient or essential in its presentation. Manifestations include appearance (consistent or not with age), nutritional status (thin, weight loss), subjective health rating (health perception), performance (cognition, fatigue), sensory/physical impairments (vision, hearing, strength) and current care (medication, hospital). Although the early stages of the frailty process may be clinically silent, when depleted reserves reach an aggregate threshold leading to serious vulnerability, the syndrome may become detectable by looking at clinical, functional, behavioral and biological markers. Thus, a better understanding of these clinical changes and their underlying mechanisms, beginning in the pre-frail state, may confirm the impression held by many geriatricians that increasing frailty is distinguishable from ageing and in consequence is potentially reversible. We therefore provide an update of the physiopathology and clinical and biological characteristics of the frailty process and speculate on possible preventative approaches.


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

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


Metabolic Reprograming in Macrophage Polarization

Abstract: Studying the metabolism of immune cells in recent years has emphasized the tight link existing between the metabolic state and the phenotype of these cells. Macrophages in particular are a good example of this phenomenon. Whether the macrophage obtains its energy through glycolysis or through oxidative metabolism can give rise to different phenotypes. Classically activated or M1 macrophages are key players of the first line of defense against bacterial infections and are known to obtain energy through glycolysis. Alternatively activated or M2 macrophages on the other hand are involved in tissue repair and wound healing and use oxidative metabolism to fuel their longer-term functions. Metabolic intermediates, however, are not just a source of energy but can be directly implicated in a particular macrophage phenotype. In M1 macrophages, the Krebs cycle intermediate succinate regulates HIF1α, which is responsible for driving the sustained production of the pro-inflammatory cytokine IL1β. In M2 macrophages, the sedoheptulose kinase carbohydrate kinase-like protein is critical for regulating the pentose phosphate pathway. The potential to target these events and impact on disease is an exciting prospect.


Maternal and child undernutrition: consequences for adult health and human capital

Abstract: In this paper we review the associations between maternal and child undernutrition with human capital and risk of adult diseases in low-income and middle-income countries. We analysed data from five long-standing prospective cohort studies from Brazil, Guatemala, India, the Philippines, and South Africa and noted that indices of maternal and child undernutrition (maternal height, birthweight, intrauterine growth restriction, and weight, height, and body-mass index at 2 years according to the new WHO growth standards) were related to adult outcomes (height, schooling, income or assets, offspring birthweight, body-mass index, glucose concentrations, blood pressure). We undertook systematic reviews of studies from low-income and middle-income countries for these outcomes and for indicators related to blood lipids, cardiovascular disease, lung and immune function, cancers, osteoporosis, and mental illness. Undernutrition was strongly associated, both in the review of published work and in new analyses, with shorter adult height, less schooling, reduced economic productivity, and--for women--lower offspring birthweight. Associations with adult disease indicators were not so clear-cut. Increased size at birth and in childhood were positively associated with adult body-mass index and to a lesser extent with blood pressure values, but not with blood glucose concentrations. In our new analyses and in published work, lower birthweight and undernutrition in childhood were risk factors for high glucose concentrations, blood pressure, and harmful lipid profiles once adult body-mass index and height were adjusted for, suggesting that rapid postnatal weight gain--especially after infancy--is linked to these conditions. The review of published works indicates that there is insufficient information about long-term changes in immune function, blood lipids, or osteoporosis indicators. Birthweight is positively associated with lung function and with the incidence of some cancers, and undernutrition could be associated with mental illness. We noted that height-for-age at 2 years was the best predictor of human capital and that undernutrition is associated with lower human capital. We conclude that damage suffered in early life leads to permanent impairment, and might also affect future generations. Its prevention will probably bring about important health, educational, and economic benefits. Chronic diseases are especially common in undernourished children who experience rapid weight gain after infancy.


The Cytosolic Chaperonin CCT/TRiC and Cancer Cell Proliferation

Abstract: The molecular chaperone CCT/TRiC plays a central role in maintaining cellular proteostasis as it mediates the folding of the major cytoskeletal proteins tubulins and actins. CCT/TRiC is also involved in the oncoprotein cyclin E, the Von Hippel-Lindau tumour suppressor protein, cyclin B and p21(ras) folding which strongly suggests that it is involved in cell proliferation and tumor genesis. To assess the involvement of CCT/TRiC in tumor genesis, we quantified its expression levels and activity in 18 cancer, one non-cancer human cell lines and a non-cancer human liver. We show that the expression levels of CCT/TRiC in cancer cell lines are higher than that in normal cells. However, CCT/TRiC activity does not always correlate with its expression levels. We therefore documented the expression levels of CCT/TRiC modulators and partners PhLP3, Hop/P60, prefoldin and Hsc/Hsp70. Our analysis reveals a functional interplay between molecular chaperones that might account for a precise modulation of CCT/TRiC activity in cell proliferation through changes in the cellular levels of prefoldin and/or Hsc/p70 and CCT/TRiC client protein availability. Our observation and approaches bring novel insights in the role of CCT/TRiC-mediated protein folding machinery in cancer cell development.


Persistence of HIV-1 Receptor-Positive Cells after HSV-2 Reactivation: A Potential Mechanism for Increased HIV-1 Acquisition

Abstract: To explore the mechanism by which herpes simplex virus (HSV)-2 infection is related to HIV-1 acquisition, we conducted in situ analysis of the cellular infiltrate from sequential biopsies of HSV-2 lesions from patients on and off antiviral therapy. CD4(+) and CD8(+) T cells and a mixed population of plasmacytoid and myeloid dendritic cells (DCs), including cells expressing the C-type lectin receptor DC-SIGN, persisted at sites of HSV-2 reactivation for months after healing, even with daily antiviral therapy. The CD4(+) T cells that persisted reacted to HSV-2 antigen, were enriched for expression of the chemokine receptor CCR5, and were contiguous to DCs expressing the interleukin-3 receptor CD123 or DC-SIGN. Ex vivo infection with a CCR5-tropic strain of HIV-1 revealed greater concentrations of integrated HIV-1 DNA in cells derived from healed genital lesion biopsies than in cells from control skin biopsies. The persistence and enrichment of HIV receptor-positive inflammatory cells in the genitalia help explain the inability of anti-HSV-2 therapy to reduce HIV acquisition.


Mechanisms of progression and regression of renal lesions of chronic nephropathies and diabetes.

Abstract: The incidence of chronic kidney diseases is increasing worldwide, and these conditions are emerging as a major public health problem. While genetic factors contribute to susceptibility and progression of renal disease, proteinuria has been claimed as an independent predictor of outcome. Reduction of urinary protein levels by various medications and a low-protein diet limits renal function decline in individuals with nondiabetic and diabetic nephropathies to the point that remission of the disease and regression of renal lesions have been observed in experimental animals and even in humans. In animal models, regression of glomerular structural changes is associated with remodeling of the glomerular architecture. Instrumental to this discovery were 3D reconstruction studies of the glomerular capillary tuft, which allowed the quantification of sclerosis volume reduction and capillary regeneration upon treatment. Regeneration of capillary segments might result from the contribution of resident cells, but progenitor cells of renal or extrarenal origin may also have a role. This review describes recent advances in our understanding of the mechanisms and mediators underlying renal tissue repair ultimately responsible for regression of renal injury.


Molecular Mechanisms of Vitamin D Action

Abstract: The hormonal metabolite of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25D), initiates biological responses via binding to the vitamin D receptor (VDR). When occupied by 1,25D, VDR interacts with the retinoid X receptor (RXR) to form a heterodimer that binds to vitamin D responsive elements in the region of genes directly controlled by 1,25D. By recruiting complexes of either coactivators or corepressors, ligand-activated VDR-RXR modulates the transcription of genes encoding proteins that promulgate the traditional functions of vitamin D, including signaling intestinal calcium and phosphate absorption to effect skeletal and calcium homeostasis. Thus, vitamin D action in a particular cell depends upon the metabolic production or delivery of sufficient concentrations of the 1,25D ligand, expression of adequate VDR and RXR coreceptor proteins, and cell-specific programming of transcriptional responses to regulate select genes that encode proteins that function in mediating the effects of vitamin D. For example, 1,25D induces RANKL, SPP1 (osteopontin), and BGP (osteocalcin) to govern bone mineral remodeling; TRPV6, CaBP9k, and claudin 2 to promote intestinal calcium absorption; and TRPV5, klotho, and Npt2c to regulate renal calcium and phosphate reabsorption. VDR appears to function unliganded by 1,25D in keratinocytes to drive mammalian hair cycling via regulation of genes such as CASP14, S100A8, SOSTDC1, and others affecting Wnt signaling. Finally, alternative, low-affinity, non-vitamin D VDR ligands, e.g., lithocholic acid, docosahexaenoic acid, and curcumin, have been reported. Combined alternative VDR ligand(s) and 1,25D/VDR control of gene expression may delay chronic disorders of aging such as osteoporosis, type 2 diabetes, cardiovascular disease, and cancer.


Vitamin d deficiency in postmenopausal women - biological correlates.

Abstract:
INTRODUCTION Low vitamin D (VD) is associated with secondary hyperparathyroidism and both contribute to deleterious consequences (reduced bone mineral density (BMD), risk of fractures and falls).
 OBJECTIVE To study the VD status and biological correlates in a group of postmenopausal women.    MATERIAL AND METHODS We studied 123 postmenopausal women evaluated in the C.I.Parhon National Institute of Endocrinology, the Pituitary and Neuroendocrine Diseases department. All cases had been reffered for the evaluation of BMD by the general practitioner. The evaluation included serum measurements of total and ionised calcium, phosphorus, alkaline phosphatase (ALP), 25 hydroxi vitaminD (25OHD), parathyroid hormone (PTH), osteocalcin, betacrosslaps. Central DXA osteodensitometry was performed.
 RESULTS 91.9% of cases had 25OHD serum levels below 30 ng/ml (74.8% had VD deficiency, 17.1% VD insufficiency). Only 8.1% had sufficient VD levels. A history of fragility fractures was present in 45.83% of the osteoporotic patients, 27.27% of the osteopenic ones and 15.15% of the women with normal BMD. 32 women (26%) were on VD supplementation at the time of evaluation. Among these subjects, the 25OHD level was significantly higher in those with prior fragility fractures (p=0.018) and osteoporosis (p=0.008). 25OHD concentration negatively correlated with PTH, alkaline phosphatase (ALP) and osteocalcin. The bone markers evaluated had a significant inverse correlation with the radius BMD, T and Z scores (p=0.004). 27.17% of the cases with VD deficiency had secondary hyperparathyroidism. The 25OHD concentration was significantly lower in these cases (p=0.000).
 CONCLUSIONS VD insufficiency is widely prevalent but still under-recognized and under-treated, possibly leading to secondary hyperparathyroidism. The compliance to VD supplementation is lower in subjects without osteoporosis or fragility fractures. Primary prevention measures should be more actively implemented.


Advances in high-resolution imaging--techniques for three-dimensional imaging of cellular structures.

Abstract: A fundamental goal in biology is to determine how cellular organization is coupled to function. To achieve this goal, a better understanding of organelle composition and structure is needed. Although visualization of cellular organelles using fluorescence or electron microscopy (EM) has become a common tool for the cell biologist, recent advances are providing a clearer picture of the cell than ever before. In particular, advanced light-microscopy techniques are achieving resolutions below the diffraction limit and EM tomography provides high-resolution three-dimensional (3D) images of cellular structures. The ability to perform both fluorescence and electron microscopy on the same sample (correlative light and electron microscopy, CLEM) makes it possible to identify where a fluorescently labeled protein is located with respect to organelle structures visualized by EM. Here, we review the current state of the art in 3D biological imaging techniques with a focus on recent advances in electron microscopy and fluorescence super-resolution techniques.


Induction of ectopic eyes by targeted expression of the eyeless gene in Drosophila.

Abstract: The Drosophila gene eyeless (ey) encodes a transcription factor with both a paired domain and a homeodomain. It is homologous to the mouse Small eye (Pax-6) gene and to the Aniridia gene in humans. These genes share extensive sequence identity, the position of three intron splice sites is conserved, and these genes are expressed similarly in the developing nervous system and in the eye during morphogenesis. Loss-of-function mutations in both the insect and in the mammalian genes have been shown to lead to a reduction or absence of eye structures, which suggests that ey functions in eye morphogenesis. By targeted expression of the ey complementary DNA in various imaginal disc primordia of Drosophila, ectopic eye structures were induced on the wings, the legs, and on the antennae. The ectopic eyes appeared morphologically normal and consisted of groups of fully differentiated ommatidia with a complete set of photoreceptor cells. These results support the proposition that ey is the master control gene for eye morphogenesis. Because homologous genes are present in vertebrates, ascidians, insects, cephalopods, and nemerteans, ey may function as a master control gene throughout the metazoa.


Niche-Independent Symmetrical Self-Renewal of a Mammalian Tissue Stem Cell

Abstract: Pluripotent mouse embryonic stem (ES) cells multiply in simple monoculture by symmetrical divisions. In vivo, however, stem cells are generally thought to depend on specialised cellular microenvironments and to undergo predominantly asymmetric divisions. Ex vivo expansion of pure populations of tissue stem cells has proven elusive. Neural progenitor cells are propagated in combination with differentiating progeny in floating clusters called neurospheres. The proportion of stem cells in neurospheres is low, however, and they cannot be directly observed or interrogated. Here we demonstrate that the complex neurosphere environment is dispensable for stem cell maintenance, and that the combination of fibroblast growth factor 2 (FGF-2) and epidermal growth factor (EGF) is sufficient for derivation and continuous expansion by symmetrical division of pure cultures of neural stem (NS) cells. NS cells were derived first from mouse ES cells. Neural lineage induction was followed by growth factor addition in basal culture media. In the presence of only EGF and FGF-2, resulting NS cells proliferate continuously, are diploid, and clonogenic. After prolonged expansion, they remain able to differentiate efficiently into neurons and astrocytes in vitro and upon transplantation into the adult brain. Colonies generated from single NS cells all produce neurons upon growth factor withdrawal. NS cells uniformly express morphological, cell biological, and molecular features of radial glia, developmental precursors of neurons and glia. Consistent with this profile, adherent NS cell lines can readily be established from foetal mouse brain. Similar NS cells can be generated from human ES cells and human foetal brain. The extrinsic factors EGF plus FGF-2 are sufficient to sustain pure symmetrical self-renewing divisions of NS cells. The resultant cultures constitute the first known example of tissue-specific stem cells that can be propagated without accompanying differentiation. These homogenous cultures will enable delineation of molecular mechanisms that define a tissue-specific stem cell and allow direct comparison with pluripotent ES cells.


Molecular mechanisms of ribosomal protein gene coregulation.

Abstract: The 137 ribosomal protein genes (RPGs) of Saccharomyces provide a model for gene coregulation. We examined the positional and functional organization of their regulators (Rap1 [repressor activator protein 1], Fhl1, Ifh1, Sfp1, and Hmo1), the transcription machinery (TFIIB, TFIID, and RNA polymerase II), and chromatin at near-base-pair resolution using ChIP-exo, as RPGs are coordinately reprogrammed. Where Hmo1 is enriched, Fhl1, Ifh1, Sfp1, and Hmo1 cross-linked broadly to promoter DNA in an RPG-specific manner and demarcated by general minor groove widening. Importantly, Hmo1 extended 20-50 base pairs (bp) downstream from Fhl1. Upon RPG repression, Fhl1 remained in place. Hmo1 dissociated, which was coupled to an upstream shift of the +1 nucleosome, as reflected by the Hmo1 extension and core promoter region. Fhl1 and Hmo1 may create two regulatable and positionally distinct barriers, against which chromatin remodelers position the +1 nucleosome into either an activating or a repressive state. Consistent with in vitro studies, we found that specific TFIID subunits, in addition to cross-linking at the core promoter, made precise cross-links at Rap1 sites, which we interpret to reflect native Rap1-TFIID interactions. Our findings suggest how sequence-specific DNA binding regulates nucleosome positioning and transcription complex assembly >300 bp away and how coregulation coevolved with coding sequences.


Probing the phenomenon of trained immunity in invertebrates during a transgenerational study, using brine shrimp Artemia as a model system

Abstract: The invertebrate's innate immune system was reported to show some form of adaptive features, termed trained immunity. However, the memory characteristics of innate immune system and the mechanisms behind such phenomena remain unclear. Using the invertebrate model Artemia, we verified the possibility or impossibility of trained immunity, examining the presence or absence of enduring memory against homologous and heterologous antigens (Vibrio spp.) during a transgenerational study. We also determined the mechanisms behind such phenomenon. Our results showed the occurrence of memory and partial discrimination in Artemia's immune system, as manifested by increased resistance, for three successive generations, of the progenies of Vibrio-exposed ancestors towards a homologous bacterial strain, rather than to a heterologous strain. This increased resistance phenotype was associated with elevated levels of hsp70 and hmgb1 signaling molecules and alteration in the expression of key innate immunity-related genes. Our results also showed stochastic pattern in the acetylation and methylation levels of H4 and H3K4me3 histones, respectively, in the progenies whose ancestors were challenged. Overall results suggest that innate immune responses in invertebrates have the capacity to be trained, and epigenetic reprogramming of (selected) innate immune effectors is likely to have central place in the mechanisms leading to trained immunity.


Fresh meat and further processing characteristics of ham muscles from finishing pigs fed ractopamine hydrochloride.

Abstract: Ractopamine hydrochloride (RAC) has consistently led to an advantage in carcass cutting yields of finishing pigs and remains a common feed additive in US finishing pig diets. Less is known about the effect of RAC on further processing characteristics. Some researchers have reported advantages in ultimate pH of the LM in pigs fed RAC. If a greater ultimate pH was also observed in hams, the increased pH could affect further processing characteristics and lead to better protein interaction and improved textural properties. The objective of this experiment was to determine if RAC-fed pigs yielded hams with a greater ultimate pH, and if so, whether or not that advantage improves textural properties and water retention of further processed hams. Two hundred hams from barrows and gilts fed RAC or control diets were selected based on HCW. Hams were fabricated into 5 separate pieces to determine cutting yields, and 6 muscles were evaluated for ultimate pH. Hams were processed to make cured and smoked hams. Ractopamine increased cutting yields of the whole ham (P < 0.0001), inside (P < 0.01), outside (P < 0.01), and knuckle (P < 0.01) when expressed as a percentage of chilled side weight. Ultimate pH of the rectus femoris, vastus lateralis, and semitendinosus were all 0.06 pH units greater (P < 0.05), the biceps femoris was 0.04 pH units greater (P = 0.02), and the semimembranosus and adductor muscles were 0.03 pH units greater in pigs fed 7.4 mg/kg of RAC when compared with control pigs. Cured hams from RAC-fed pigs were heavier at all stages of production. No differences were detected in binding strengths (P = 0.88) or protein fat-free values (P = 0.13) between RAC (9.06 kg and 20.37) and control hams (9.01 kg and 20.13). Ractopamine increased cutting yields, total weight of cured hams, and ultimate muscle pH. Ractopamine can be fed to pigs to achieve the desired growth characteristic advantages and cutting yields without affecting further processed ham characteristics.


Mechanism and regulation of DNA end resection in eukaryotes.

Abstract: The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is initiated by nucleolytic degradation of the 5'-terminated strands in a process termed end resection. End resection generates 3'-single-stranded DNA tails, substrates for Rad51 to catalyze homologous pairing and DNA strand exchange, and for activation of the DNA damage checkpoint. The commonly accepted view is that end resection occurs by a two-step mechanism. In the first step, Sae2/CtIP activates the Mre11-Rad50-Xrs2/Nbs1 (MRX/N) complex to endonucleolytically cleave the 5'-terminated DNA strands close to break ends, and in the second step Exo1 and/or Dna2 nucleases extend the resected tracts to produce long 3'-ssDNA-tailed intermediates. Initiation of resection commits a cell to repair a DSB by HR because long ssDNA overhangs are poor substrates for non-homologous end joining (NHEJ). Thus, the initiation of end resection has emerged as a critical control point for repair pathway choice. Here, I review recent studies on the mechanism of end resection and how this process is regulated to ensure the most appropriate repair outcome.


Folate and vitamin B6 from diet and supplements in relation to risk of coronary heart disease among women.

Abstract:
CONTEXT Hyperhomocysteinemia is caused by genetic and lifestyle influences, including low intakes of folate and vitamin B6. However, prospective data relating intake of these vitamins to risk of coronary heart disease (CHD) are not available.
 OBJECTIVE To examine intakes of folate and vitamin B6 in relation to the incidence of nonfatal myocardial infarction (MI) and fatal CHD.
 DESIGN Prospective cohort study.
 SETTING AND PATIENTS In 1980, a total of 80082 women from the Nurses' Health Study with no previous history of cardiovascular disease, cancer, hypercholesterolemia, or diabetes completed a detailed food frequency questionnaire from which we derived usual intake of folate and vitamin B6.
 MAIN OUTCOME MEASURE Nonfatal MI and fatal CHD confirmed by World Health Organization criteria.
 RESULTS During 14 years of follow-up, we documented 658 incident cases of nonfatal MI and 281 cases of fatal CHD. After controlling for cardiovascular risk factors, including smoking and hypertension and intake of alcohol, fiber, vitamin E, and saturated, polyunsaturated, and trans fat, the relative risks (RRs) of CHD between extreme quintiles were 0.69 (95% confidence interval [CI], 0.55-0.87) for folate (median intake, 696 microg/d vs 158 microg/d) and 0.67 (95% CI, 0.53-0.85) for vitamin B6 (median intake, 4.6 mg/d vs 1.1 mg/d). Controlling for the same variables, the RR was 0.55 (95% CI, 0.41-0.74) among women in the highest quintile of both folate and vitamin B6 intake compared with the opposite extreme. Risk of CHD was reduced among women who regularly used multiple vitamins (RR=0.76; 95% CI, 0.65-0.90), the major source of folate and vitamin B6, and after excluding multiple vitamin users, among those with higher dietary intakes of folate and vitamin B6. In a subgroup analysis, compared with nondrinkers, the inverse association between a high-folate diet and CHD was strongest among women who consumed up to 1 alcoholic beverage per day (RR =0.69; 95% CI, 0.49-0.97) or more than 1 drink per day (RR=0.27; 95% CI, 0.13-0.58).
 CONCLUSION These results suggest that intake of folate and vitamin B6 above the current recommended dietary allowance may be important in the primary prevention of CHD among women.


Prevalence and cardiovascular disease correlates of low cardiorespiratory fitness in adolescents and adults.

Abstract:
CONTEXT Population surveys indicate that physical activity levels are low in the United States. One consequence of inactivity, low cardiorespiratory fitness, is an established risk factor for cardiovascular disease (CVD) morbidity and mortality, but the prevalence of cardiorespiratory fitness has not been quantified in representative US population samples.
 OBJECTIVES To describe the prevalence of low fitness in the US population aged 12 through 49 years and to relate low fitness to CVD risk factors in this population.
 DESIGN, SETTING, AND PARTICIPANTS Inception cohort study using data from the cross-sectional nationally representative National Health and Nutrition Examination Survey 1999-2002. Participants were adolescents (aged 12-19 years; n = 3110) and adults (aged 20-49 years; n = 2205) free from previously diagnosed CVD who underwent submaximal graded exercise treadmill testing to achieve at least 75% to 90% of their age-predicted maximum heart rate. Maximal oxygen consumption (VO2max) was estimated by measuring the heart rate response to reference levels of submaximal work.
 MAIN OUTCOME MEASURES Low fitness defined using percentile cut points of estimated VO2max from existing external referent populations; anthropometric and other CVD risk factors measured according to standard methods.
 RESULTS Low fitness was identified in 33.6% of adolescents (approximately 7.5 million US adolescents) and 13.9% of adults (approximately 8.5 million US adults); the prevalence was similar in adolescent females (34.4%) and males (32.9%) (P = .40) but was higher in adult females (16.2%) than in males (11.8%) (P = .03). Non-Hispanic blacks and Mexican Americans were less fit than non-Hispanic whites. In all age-sex groups, body mass index and waist circumference were inversely associated with fitness; age- and race-adjusted odds ratios of overweight or obesity (body mass index > or =25) ranged from 2.1 to 3.7 (P<.01 for all), comparing persons with low fitness with those with moderate or high fitness. Total cholesterol levels and systolic blood pressure were higher and levels of high-density lipoprotein cholesterol were lower among participants with low vs high fitness.
 CONCLUSION Low fitness in adolescents and adults is common in the US population and is associated with an increased prevalence of CVD risk factors.


Hypoglycemic episodes and risk of dementia in older patients with type 2 diabetes mellitus.

Abstract:
CONTEXT Although acute hypoglycemia may be associated with cognitive impairment in children with type 1 diabetes, no studies to date have evaluated whether hypoglycemia is a risk factor for dementia in older patients with type 2 diabetes.
 OBJECTIVE To determine if hypoglycemic episodes severe enough to require hospitalization are associated with an increased risk of dementia in a population of older patients with type 2 diabetes followed up for 27 years.
 DESIGN, SETTING, AND PATIENTS A longitudinal cohort study from 1980-2007 of 16,667 patients with a mean age of 65 years and type 2 diabetes who are members of an integrated health care delivery system in northern California.
 MAIN OUTCOME MEASURE Hypoglycemic events from 1980-2002 were collected and reviewed using hospital discharge and emergency department diagnoses. Cohort members with no prior diagnoses of dementia, mild cognitive impairment, or general memory complaints as of January 1, 2003, were followed up for a dementia diagnosis through January 15, 2007. Dementia risk was examined using Cox proportional hazard regression models, adjusted for age, sex, race/ethnicity, education, body mass index, duration of diabetes, 7-year mean glycated hemoglobin, diabetes treatment, duration of insulin use, hyperlipidemia, hypertension, cardiovascular disease, stroke, transient cerebral ischemia, and end-stage renal disease.
 RESULTS At least 1 episode of hypoglycemia was diagnosed in 1465 patients (8.8%) and dementia was diagnosed in 1822 patients (11%) during follow-up; 250 patients had both dementia and at least 1 episode of hypoglycemia (16.95%). Compared with patients with no hypoglycemia, patients with single or multiple episodes had a graded increase in risk with fully adjusted hazard ratios (HRs): for 1 episode (HR, 1.26; 95% confidence interval [CI], 1.10-1.49); 2 episodes (HR, 1.80; 95% CI, 1.37-2.36); and 3 or more episodes (HR, 1.94; 95% CI, 1.42-2.64). The attributable risk of dementia between individuals with and without a history of hypoglycemia was 2.39% per year (95% CI, 1.72%-3.01%). Results were not attenuated when medical utilization rates, length of health plan membership, or time since initial diabetes diagnosis were added to the model. When examining emergency department admissions for hypoglycemia for association with risk of dementia (535 episodes), results were similar (compared with patients with 0 episodes) with fully adjusted HRs: for 1 episode (HR, 1.42; 95% CI, 1.12-1.78) and for 2 or more episodes (HR, 2.36; 95% CI, 1.57-3.55).
 CONCLUSIONS Among older patients with type 2 diabetes, a history of severe hypoglycemic episodes was associated with a greater risk of dementia. Whether minor hypoglycemic episodes increase risk of dementia is unknown.


Novel syndromes associated with JC virus infection of neurons and meningeal cells: no longer a gray area.

Abstract: PURPOSE OF REVIEW The availability of a growing number of immunomodulatory medications over the past few years has been associated with various JC virus (JCV)-associated brain syndromes in patients with autoimmune diseases, including multiple sclerosis, Crohn's disease, and psoriasis that had not been previously recognized as predisposing factors for progressive multifocal leukoencephalopathy. This review covers the three novel syndromes discovered in the last decade that are caused by JCV infection of neurons and meningeal cells.    RECENT FINDINGS For more than 30 years, JCV was thought to exclusively infect oligodendrocytes and astrocytes in the white matter of the brain of immunosuppressed individuals. We now recognize that JCV-infected glial cells are frequently located at the gray-white matter junction or exclusively within the gray matter causing demyelination in the cortex. Mutations in JCV can trigger a change in tropism leading to involvement of other cell types, such as neurons and meningeal cells, causing clinically distinct entities. These new features of JCV infection provide challenges for clinicians taking care of affected patients and investigators studying the biology of this polyomavirus, its pathogenesis, and tropism.    SUMMARY We hope that increasing awareness of these syndromes will lead to early diagnosis, and pave the way for new avenues of research to better understand all aspects of JCV pathogenesis and develop efficient therapies for our patients. However, we need to remain vigilant and open to the possibility that additional JC variants or yet unknown polyomaviruses may also be associated with neurological diseases.


Preventing obesity in children and adolescents.

Abstract: In this review, we address the natural history of obesity in children, the most promising family- and school-based approaches to the prevention of obesity, and the barriers and opportunities associated with secondary prevention. In childhood, the most important periods of risk appear to be the periods of adiposity rebound and adolescence. Caution regarding the period of adiposity rebound is still warranted, because it is not yet clear that early rebound is attributable to changes in body fat. Families and schools represent the most important foci for preventive efforts in children and adolescents. One productive approach is to proceed from an examination of factors that affect energy balance to the identification of more proximal influences on those factors. This approach may help to narrow the strategies necessary to prevent or treat childhood obesity. For example, television viewing affects both energy intake and energy expenditure, and therefore represents a logical target for interventions. Anticipatory guidance by pediatricians may offer an effective mechanism by which to change parental attitudes and practices regarding television viewing. A similar process is used to emphasize the potential influence of school-based interventions directed at changes in food choices and sedentary behavior.


Culture of Preimplantation Mouse Embryos Affects Fetal Development and the Expression of Imprinted Genes1

Abstract: Abstract Culture of preimplantation mammalian embryos and cells can influence their subsequent growth and differentiation. Previously, we reported that culture of mouse embryonic stem cells is associated with deregulation of genomic imprinting and affects the potential for these cells to develop into normal fetuses. The purpose of our current study was to determine whether culture of preimplantation mouse embryos in a chemically defined medium (M16) with or without fetal calf serum (FCS) can affect their subsequent development and imprinted gene expression. Only one third of the blastocysts that had been cultured from two-cell embryos in M16 medium complemented with FCS developed into viable Day 14 fetuses after transfer into recipients. These M16 + FCS fetuses were reduced in weight as compared with controls and M16 fetuses and had decreased expression of the imprinted H19 and insulin-like growth factor 2 genes associated with a gain of DNA methylation at an imprinting control region upstream of H19. They also displayed increased expression of the imprinted gene Grb10. The growth factor receptor binding gene Grb7, in contrast, was strongly reduced in its expression in most of the M16 + FCS fetuses. No alterations were detected for the imprinted gene Mest. Preimplantation culture in the presence of serum can influence the regulation of multiple growth-related imprinted genes, thus leading to aberrant fetal growth and development.


Grip strength in healthy caucasian adults: reference values.

Abstract:
PURPOSE The aim of this study was to update reference data of handgrip strength for healthy adults of both genders spanning a wide age range and to analyze possible factors of influence.
 METHODS Intraindividual and interindividual variations of grip strength and their relation to several anthropometric factors were analyzed in a standardized manner for 769 healthy adults (women, n = 403; men, n = 366) aged between 20 years and 95 years. Measurements were done in neutral position of arm, forearm, and wrist on setting II of a Baseline digital hydraulic dynamometer (NexGen Ergonomics Inc. Quebec, Canada).
 RESULTS Mean strength was about 41% less in women (right 29 kg; left 27 kg) than in men (right 49 kg; left 47 kg) resulting in a ratio of left to right hand slightly above .95 in both genders. During the course of life, hand strength develops comparably in both genders peaking at 35 years of age and decreasing continuously further on. Anthropometric variables such as forearm circumference and length, hand size, or body mass showed a positive correlation with grip strength. Body mass index, type of work, and hand dominance showed only a partial positive correlation or no correlation with grip strength. Gender and age, followed by parameters representing body length and obesity, were observed to have the highest predictive value for handgrip strength and were therefore entered into the generation of prediction equations.
 CONCLUSIONS We recommend side adjustment of measured values for intraindividual comparison and inclusion of information regarding anthropometric characteristics, as well as using gender- and age-adjusted reference values, whereas hand dominance can be neglected. The regression equations we generated might prove to be useful for clinicians or for those who use normative values within software to provide more accurate predictions of strength scores for specific applications.


Chemokine-like receptor 1 (CMKLR1) and chemokine (C-C motif) receptor-like 2 (CCRL2); two multifunctional receptors with unusual properties.

Abstract: Chemokine-like receptor 1 (CMKLR1), also known as ChemR23, and chemokine (C-C motif) receptor-like 2 (CCRL2) are 7-transmembrane receptors that were cloned in the late 1990s based on their homology to known G-protein-coupled receptors. They were previously orphan receptors without any known biological roles; however, recent studies identified ligands for these receptors and their functions have begun to be unveiled. The plasma protein-derived chemoattractant chemerin is a ligand for CMKLR1 and activation of CMKLR1 with chemerin induces the migration of macrophages and dendritic cells (DCs) in vitro, suggesting a proinflammatory role. However, in vivo studies using CMKLR-deficient mice suggest an anti-inflammatory role for this receptor, possibly due to the recruitment of tolerogenic plasmacytoid DCs. Chemerin/CMKLR1 interaction also promotes adipogenesis and angiogenesis. The anti-inflammatory lipid mediator, resolving E1, is another CMKLR1 ligand and it inhibits leukocyte infiltration and proinflammatory gene expression. These divergent results suggest that CMKLR1 is a multifunctional receptor. The chemokine CCL5 and CCL19 are reported to bind to CCRL2. Like Duffy antigen for chemokine receptor (DARC), D6 and CCX-CKR, CCRL2 does not signal, but it constitutively recycles, potentially reducing local concentration of CCL5 and CCL19 and subsequent immune responses. Surprisingly, chemerin, a ligand for CMKLR1, is a ligand for CCRL2. CCRL2 binds chemerin and increases local chemerin concentration to efficiently present it to CMKLR1 on nearby cells, providing a link between CCRL2 and CMKLR1. Although these findings suggest an anti-inflammatory role, a recent study using CCRL2-deficient mice indicates a proinflammatory role; thus, CCRL2 may also be multifunctional. Further studies using CMKLR1- or CCRL2-deficient mice are needed to further define the role of these receptors in immune responses and other cellular processes.


Prophylactic and therapeutic efficacy of the epitope vaccine CTB-UA against Helicobacter pylori infection in a BALB/c mice model

Abstract: Epitope vaccine based on the enzyme urease of Helicobacter pylori is a promising option for prophylactic and therapeutic vaccination against H. pylori infection. In our previous study, the epitope vaccine CTB-UA, which was composed of the mucosal adjuvant cholera toxin B subunit (CTB) and an epitope (UreA183–203) from the H. pylori urease A subunit (UreA) was constructed. This particular vaccine was shown to have good immunogenicity and immunoreactivity and could induce specific neutralizing antibodies, which exhibited effectively inhibitory effects on the enzymatic activity of H. pylori urease. In this study, the prophylactic and therapeutic efficacy of the epitope vaccine CTB-UA was evaluated in a BALB/c mice model. The experimental results indicated that oral prophylactic or therapeutic immunization with CTB-UA significantly decreased H. pylori colonization compared with oral immunization with PBS. The results also revealed that the protection was correlated with antigen-specific IgG, IgA, and mucosal secretory IgA antibody responses. CTB-UA may be a promising vaccine candidate for the control of H. pylori infection.


Directed Actin Polymerization Is the Driving Force for Epithelial Cell–Cell Adhesion

Abstract: We have found that epithelial cells engage in a process of cadherin-mediated intercellular adhesion that utilizes calcium and actin polymerization in unexpected ways. Calcium stimulates filopodia, which penetrate and embed into neighboring cells. E-cadherin complexes cluster at filopodia tips, generating a two-rowed zipper of embedded puncta. Opposing cell surfaces are clamped by desmosomes, while vinculin, zyxin, VASP, and Mena are recruited to adhesion zippers by a mechanism that requires alpha-catenin. Actin reorganizes and polymerizes to merge puncta into a single row and seal cell borders. In keratinocytes either null for alpha-catenin or blocked in VASP/Mena function, filopodia embed, but actin reorganization/polymerization is prevented, and membranes cannot seal. Taken together, a dynamic mechanism for intercellular adhesion is unveiled involving calcium-activated filopodia penetration and VASP/Mena-dependent actin reorganization/polymerization.


Mammalian RAD52 Functions in Break-Induced Replication Repair of Collapsed DNA Replication Forks

Abstract: Human cancers are characterized by the presence of oncogene-induced DNA replication stress (DRS), making them dependent on repair pathways such as break-induced replication (BIR) for damaged DNA replication forks. To better understand BIR, we performed a targeted siRNA screen for genes whose depletion inhibited G1 to S phase progression when oncogenic cyclin E was overexpressed. RAD52, a gene dispensable for normal development in mice, was among the top hits. In cells in which fork collapse was induced by oncogenes or chemicals, the Rad52 protein localized to DRS foci. Depletion of Rad52 by siRNA or knockout of the gene by CRISPR/Cas9 compromised restart of collapsed forks and led to DNA damage in cells experiencing DRS. Furthermore, in cancer-prone, heterozygous APC mutant mice, homozygous deletion of the Rad52 gene suppressed tumor growth and prolonged lifespan. We therefore propose that mammalian RAD52 facilitates repair of collapsed DNA replication forks in cancer cells.


Reprogramming factor expression initiates widespread targeted chromatin remodeling.

Abstract: Despite rapid progress in characterizing transcription factor-driven reprogramming of somatic cells to an induced pluripotent stem cell (iPSC) state, many mechanistic questions still remain. To gain insight into the earliest events in the reprogramming process, we systematically analyzed the transcriptional and epigenetic changes that occur during early factor induction after discrete numbers of divisions. We observed rapid, genome-wide changes in the euchromatic histone modification, H3K4me2, at more than a thousand loci including large subsets of pluripotency-related or developmentally regulated gene promoters and enhancers. In contrast, patterns of the repressive H3K27me3 modification remained largely unchanged except for focused depletion specifically at positions where H3K4 methylation is gained. These chromatin regulatory events precede transcriptional changes within the corresponding loci. Our data provide evidence for an early, organized, and population-wide epigenetic response to ectopic reprogramming factors that clarify the temporal order through which somatic identity is reset during reprogramming.


Enhancer Evolution across 20 Mammalian Species

Abstract: The mammalian radiation has corresponded with rapid changes in noncoding regions of the genome, but we lack a comprehensive understanding of regulatory evolution in mammals. Here, we track the evolution of promoters and enhancers active in liver across 20 mammalian species from six diverse orders by profiling genomic enrichment of H3K27 acetylation and H3K4 trimethylation. We report that rapid evolution of enhancers is a universal feature of mammalian genomes. Most of the recently evolved enhancers arise from ancestral DNA exaptation, rather than lineage-specific expansions of repeat elements. In contrast, almost all liver promoters are partially or fully conserved across these species. Our data further reveal that recently evolved enhancers can be associated with genes under positive selection, demonstrating the power of this approach for annotating regulatory adaptations in genomic sequences. These results provide important insight into the functional genetics underpinning mammalian regulatory evolution.


Plasma lipids and prolactin in patients with breast cancer.

Abstract: In a comparative study of pre- and postmenopausal women with benign and malignant breast disease, a number of differences were observed in circulating plasma prolactin and lipid concentrations. Plasma lipids, phospholipids, triglycerides, cholesterol and free fatty acids were all higher in blood obtained from breast cancer patients prior to surgery. HDL-Cholesterol levels were significantly lower in these patients. These differences remained when the patient groups were sub-divided according to menopausal status. Plasma prolactin concentrations were also found to be higher in cancer compared with non-cancer patients, this effect being more marked in premenopausal than in postmenopausal patients. Premenopausal patients with invasive or poorly differentiated disease had significantly higher prolactin levels than those with non-invasive disease. No correlations were found between plasma prolactin and any of the lipid fractions.


Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference.

Abstract: In the present study, the relationship between short interfering RNA (siRNA) sequence and RNA interference (RNAi) effect was extensively analyzed using 62 targets of four exogenous and two endogenous genes and three mammalian and Drosophila cells. We present the rules that may govern siRNA sequence preference and in accordance with which highly effective siRNAs essential for systematic mammalian functional genomics can be readily designed. These rules indicate that siRNAs which simultaneously satisfy all four of the following sequence conditions are capable of inducing highly effective gene silencing in mammalian cells: (i) A/U at the 5' end of the antisense strand; (ii) G/C at the 5' end of the sense strand; (iii) at least five A/U residues in the 5' terminal one-third of the antisense strand; and (iv) the absence of any GC stretch of more than 9 nt in length. siRNAs opposite in features with respect to the first three conditions give rise to little or no gene silencing in mammalian cells. Essentially the same rules for siRNA sequence preference were found applicable to DNA-based RNAi in mammalian cells and in ovo RNAi using chick embryos. In contrast to mammalian and chick cells, little siRNA sequence preference could be detected in Drosophila in vivo RNAi.


Reprogramming of human fibroblasts to pluripotency with lineage specifiers.

Abstract: Since the initial discovery that OCT4, SOX2, KLF4, and c-MYC overexpression sufficed for the induction of pluripotency in somatic cells, methodologies replacing the original factors have enhanced our understanding of the reprogramming process. However, unlike in mouse, OCT4 has not been replaced successfully during reprogramming of human cells. Here we report on a strategy to accomplish this replacement. Through a combination of transcriptome and bioinformatic analysis we have identified factors previously characterized as being lineage specifiers that are able to replace OCT4 and SOX2 in the reprogramming of human fibroblasts. Our results show that it is possible to replace OCT4 and SOX2 simultaneously with alternative lineage specifiers in the reprogramming of human cells. At a broader level, they also support a model in which counteracting lineage specification networks underlies the induction of pluripotency.


PHENIX: a comprehensive Python-based system for macromolecular structure solution

Abstract: Macromolecular X-ray crystallography is routinely applied to understand biological processes at a molecular level. However, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromolecular crystallographic structure solution with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms.


Telomeric 3′ Overhangs Derive from Resection by Exo1 and Apollo and Fill-In by POT1b-Associated CST

Abstract: A 3' overhang is critical for the protection and maintenance of mammalian telomeres, but its synthesis must be regulated to avoid excessive resection of the 5' end, which could cause telomere shortening. How this balance is achieved in mammals has not been resolved. Here, we determine the mechanism for 3' overhang synthesis in mouse cells by evaluating changes in telomeric overhangs throughout the cell cycle and at leading- and lagging-end telomeres. Apollo, a nuclease bound to the shelterin subunit TRF2, initiates formation of the 3' overhang at leading-, but not lagging-end telomeres. Hyperresection by Apollo is blocked at both ends by the shelterin protein POT1b. Exo1 extensively resects both telomere ends, generating transient long 3' overhangs in S/G2. CST/AAF, a DNA polα.primase accessory factor, binds POT1b and shortens the extended overhangs produced by Exo1, likely through fill-in synthesis. 3' overhang formation is thus a multistep, shelterin-controlled process, ensuring functional telomeric overhangs at chromosome ends.


Notch signaling: cell fate control and signal integration in development.

Abstract: Notch signaling defines an evolutionarily ancient cell interaction mechanism, which plays a fundamental role in metazoan development. Signals exchanged between neighboring cells through the Notch receptor can amplify and consolidate molecular differences, which eventually dictate cell fates. Thus, Notch signals control how cells respond to intrinsic or extrinsic developmental cues that are necessary to unfold specific developmental programs. Notch activity affects the implementation of differentiation, proliferation, and apoptotic programs, providing a general developmental tool to influence organ formation and morphogenesis.


Common BRCA1 variants and susceptibility to breast and ovarian cancer in the general population.

Abstract: Most multiple case families of young onset breast cancer and ovarian cancer are thought to be due to highly penetrant mutations in the predisposing genes BRCA1 and BRCA2. However, these mutations are uncommon in the population and they probably account for only a few percent of all breast cancer incidence. A much larger fraction of breast cancer might, in principle, be due to common variants which confer more modest individual risks. There are several common polymorphisms in the BRCA1 gene which generate amino acid substitutions. We have examined the frequency of four of these polymorphisms: Gln356Arg, Pro871Leu, Glu1038Gly and Ser1613Gly in large series of breast and ovarian cancer cases and matched controls. Due to strong linkage disequilibrium, these four sites generate only three haplotypes with a frequency > 1.3%. The most common haplotypes, defined by the alleles Gln356Pro871Glu1038Ser1613 and Gln356Leu871Gly1038Gly1613, have frequencies of 0.57 and 0.32 respectively, and these frequencies do not differ significantly between patient and control groups. Thus the most common polymorphisms of the BRCA1 gene do not make a significant contribution to breast or ovarian cancer risk. However, our data suggest that the Arg356 allele may have a different genotype distribution in breast cancer patients from that in controls (Arg356 homozygotes are more frequent in the control groups, P = 0.01), indicating that it may be protective against breast cancer. If this finding can be confirmed, it may provide an insight into the structural features of the BRCA1 protein that are important for its function.


Clonal analysis of epiblast fate during germ layer formation in the mouse embryo.

Abstract: The fate of cells in the epiblast at prestreak and early primitive streak stages has been studied by injecting horseradish peroxidase (HRP) into single cells in situ of 6.7-day mouse embryos and identifying the labelled descendants at midstreak to neural plate stages after one day of culture. Ectoderm was composed of descendants of epiblast progenitors that had been located in the embryonic axis anterior to the primitive streak. Embryonic mesoderm was derived from all areas of the epiblast except the distal tip and the adjacent region anterior to it: the most anterior mesoderm cells originated posteriorly, traversing the primitive streak early; labelled cells in the posterior part of the streak at the neural plate stage were derived from extreme anterior axial and paraxial epiblast progenitors; head process cells were derived from epiblast at or near the anterior end of the primitive streak. Endoderm descendants were most frequently derived from a region that included, but extended beyond, the region producing the head process: descendants of epiblast were present in endoderm by the midstreak stage, as well as at later stages. Yolk sac and amnion mesoderm developed from posterolateral and posterior epiblast. The resulting fate map is essentially the same as those of the chick and urodele and indicates that, despite geometrical differences, topological fate relationships are conserved among these vertebrates. Clonal descendants were not necessarily confined to a single germ layer or to extraembryonic mesoderm, indicating that these lineages are not separated at the beginning of gastrulation. The embryonic axis lengthened up to the neural plate stage by (1) elongation of the primitive streak through progressive incorporation of the expanding lateral and initially more anterior regions of epiblast and, (2) expansion of the region of epiblast immediately cranial to the anterior end of the primitive streak. The population doubling time of labelled cells was 7.5 h; a calculated 43% were in, or had completed, a 4th cell cycle, and no statistically significant regional differences in the number of descendants were found. This clonal analysis also showed that (1) growth in the epiblast was noncoherent and in most regions anisotropic and directed towards the primitive streak and (2) the midline did not act as a barrier to clonal spread, either in the epiblast in the anterior half of the axis or in the primitive streak. These results taken together with the fate map indicate that, while individual cells in the epiblast sheet behave independently with respect to their neighbours, morphogenetic movement during germ layer formation is coordinated in the population as a whole.


Immunological mechanisms for desensitization and tolerance in food allergy

Abstract: Food allergy is a major public health concern in westernized countries, estimated to affect 5 % of children and 3–4 % of adults. Allergen-specific immunotherapy for food allergy is currently being actively evaluated, but is still experimental. The optimal protocol, in terms of the route of administration of the food, target maintenance dose, and duration of maintenance therapy, and the optimal patient for these procedures are still being worked out. The mechanisms underlying successful food desensitization are also unclear, in part, because there is no standard immunotherapy protocol. The mechanisms involved, however, may include mast cell and basophil suppression, development of food-specific IgG4 antibodies, reduction in the food-specific IgE/IgG4 ratio, up-regulation and expansion of natural or inducible regulatory T cells, a skewing from a Th2 to a Th1 profile, and the development of anergy and/or deletion in antigen-specific cells. Additional studies are required to elucidate and understand these mechanisms by which desensitization and tolerance are achieved, which may reveal valuable biomarkers for evaluating and following food allergic patients on immunotherapy.


Susceptibility to exacerbation in chronic obstructive pulmonary disease.

Abstract:
BACKGROUND Although we know that exacerbations are key events in chronic obstructive pulmonary disease (COPD), our understanding of their frequency, determinants, and effects is incomplete. In a large observational cohort, we tested the hypothesis that there is a frequent-exacerbation phenotype of COPD that is independent of disease severity.
 METHODS We analyzed the frequency and associations of exacerbation in 2138 patients enrolled in the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE) study. Exacerbations were defined as events that led a care provider to prescribe antibiotics or corticosteroids (or both) or that led to hospitalization (severe exacerbations). Exacerbation frequency was observed over a period of 3 years.
 RESULTS Exacerbations became more frequent (and more severe) as the severity of COPD increased; exacerbation rates in the first year of follow-up were 0.85 per person for patients with stage 2 COPD (with stage defined in accordance with Global Initiative for Chronic Obstructive Lung Disease [GOLD] stages), 1.34 for patients with stage 3, and 2.00 for patients with stage 4. Overall, 22% of patients with stage 2 disease, 33% with stage 3, and 47% with stage 4 had frequent exacerbations (two or more in the first year of follow-up). The single best predictor of exacerbations, across all GOLD stages, was a history of exacerbations. The frequent-exacerbation phenotype appeared to be relatively stable over a period of 3 years and could be predicted on the basis of the patient's recall of previous treated events. In addition to its association with more severe disease and prior exacerbations, the phenotype was independently associated with a history of gastroesophageal reflux or heartburn, poorer quality of life, and elevated white-cell count.
 CONCLUSIONS Although exacerbations become more frequent and more severe as COPD progresses, the rate at which they occur appears to reflect an independent susceptibility phenotype. This has implications for the targeting of exacerbation-prevention strategies across the spectrum of disease severity. (Funded by GlaxoSmithKline; ClinicalTrials.gov number, NCT00292552.)


Effects of risedronate treatment on vertebral and nonvertebral fractures in women with postmenopausal osteoporosis: a randomized controlled trial. Vertebral Efficacy With Risedronate Therapy (VERT) Study Group.

Abstract:
CONTEXT Risedronate, a potent bisphosphonate, has been shown to be effective in the treatment of Paget disease of bone and other metabolic bone diseases but, to our knowledge, it has not been evaluated in the treatment of established postmenopausal osteoporosis.
 OBJECTIVE To test the efficacy and safety of daily treatment with risedronate to reduce the risk of vertebral and other fractures in postmenopausal women with established osteoporosis.
 DESIGN, SETTING, AND PARTICIPANTS Randomized, double-blind, placebo-controlled trial of 2458 ambulatory postmenopausal women younger than 85 years with at least 1 vertebral fracture at baseline who were enrolled at 1 of 110 centers in North America conducted between December 1993 and January 1998.
 INTERVENTIONS Subjects were randomly assigned to receive oral treatment for 3 years with risedronate (2.5 or 5 mg/d) or placebo. All subjects received calcium, 1000 mg/d. Vitamin D (cholecalciferol, up to 500 IU/d) was provided if baseline levels of 25-hydroxyvitamin D were low.
 MAIN OUTCOME MEASURES Incidence of new vertebral fractures as detected by quantitative and semiquantitative assessments of radiographs; incidence of radiographically confirmed nonvertebral fractures and change from baseline in bone mineral density as determined by dual x-ray absorptiometry.
 RESULTS The 2.5 mg/d of risedronate arm was discontinued after 1 year; in the placebo and 5 mg/d of risedronate arms, 450 and 489 subjects, respectively, completed all 3 years of the trial. Treatment with 5 mg/d of risedronate, compared with placebo, decreased the cumulative incidence of new vertebral fractures by 41 % (95% confidence interval [CI], 18%-58%) over 3 years (11.3 % vs 16.3%; P= .003). A fracture reduction of 65% (95% CI, 38%-81 %) was observed after the first year (2.4% vs 6.4%; P<.001). The cumulative incidence of nonvertebral fractures over 3 years was reduced by 39% (95% CI, 6%-61 %) (5.2 % vs 8.4%; P = .02). Bone mineral density increased significantly compared with placebo at the lumbar spine (5.4% vs 1.1 %), femoral neck (1.6% vs -1.2%), femoral trochanter (3.3% vs -0.7%), and midshaft of the radius (0.2% vs -1.4%). Bone formed during risedronate treatment was histologically normal. The overall safety profile of risedronate, including gastrointestinal safety, was similar to that of placebo.
 CONCLUSIONS These data suggest that risedronate therapy is effective and well tolerated in the treatment of women with established postmenopausal osteoporosis.


In vitro splicing pathways of pre-mRNAs containing multiple intervening sequences?

Abstract: We analyzed the in vitro splicing pathways of three multi-intervening-sequence (IVS) pre-mRNAs: human beta-globin, which contains two IVSs (K. M. Lang, V. L. van Santen, and R. A. Spritz, EMBO J. 4:1991-1996, 1985); rat alpha-lactalbumin, which contains three IVSs; and murine interleukin-3, which contains four IVSs. We found that there are highly preferred pathways of IVS removal from these multi-IVS pre-mRNAs in vitro. The three IVSs of rat alpha-lactalbumin pre-mRNA were excised sequentially from 5' to 3'; in most molecules, IVS1 was removed first, followed by IVS2 and finally by IVS3. The splicing pathway of interleukin-3 pre-mRNA in vitro was more complex. The four IVSs were excised in a highly preferred temporal order, but the order was not strictly sequential or directional. In most molecules, IVS1 and IVS4 were removed first, either simultaneously or in rapid succession. Subsequently, IVS2 was excised, followed by IVS3. The observed splicing pathways apparently resulted from differences in lag times and maximum excision rates of the different IVSs. We detected no exon skipping during splicing of these transcripts in vitro. These observations have implication for proposed models of splice site selection.


Gut-expressed gustducin and taste receptors regulate secretion of glucagon-like peptide-1.

Abstract: Glucagon-like peptide-1 (GLP-1), released from gut endocrine L cells in response to glucose, regulates appetite, insulin secretion, and gut motility. How glucose given orally, but not systemically, induces GLP-1 secretion is unknown. We show that human duodenal L cells express sweet taste receptors, the taste G protein gustducin, and several other taste transduction elements. Mouse intestinal L cells also express alpha-gustducin. Ingestion of glucose by alpha-gustducin null mice revealed deficiencies in secretion of GLP-1 and the regulation of plasma insulin and glucose. Isolated small bowel and intestinal villi from alpha-gustducin null mice showed markedly defective GLP-1 secretion in response to glucose. The human L cell line NCI-H716 expresses alpha-gustducin, taste receptors, and several other taste signaling elements. GLP-1 release from NCI-H716 cells was promoted by sugars and the noncaloric sweetener sucralose, and blocked by the sweet receptor antagonist lactisole or siRNA for alpha-gustducin. We conclude that L cells of the gut "taste" glucose through the same mechanisms used by taste cells of the tongue. Modulating GLP-1 secretion in gut "taste cells" may provide an important treatment for obesity, diabetes and abnormal gut motility.


Vitamin D and obesity: current perspectives and future directions.

Abstract: In recent years, new functional roles of vitamin D beyond its traditional role in calcium homoeostasis and bone metabolism have emerged linking the fat-soluble vitamin to various non-communicable diseases. Vitamin D deficiency (25-hydroxyvitamin D (25(OH)D) < 25-30 nmol/l) and sub-optimal status (25(OH)D < 50-100 nmol/l) are increasingly associated with unfavourable metabolic phenotypes, including insulin resistance, type 2 diabetes and CVD; conditions also commonly linked with overweight and obesity. Early studies reported poor vitamin D status in the morbidly obese. More recently, it has been observed that a graded relationship between vitamin D status and BMI, or specifically adiposity, exists in the general population. A number of hypotheses have been proposed to explain the potential mechanisms whereby alterations in the vitamin D endocrine system occur in the obese state. Plausible explanations include sequestration in adipose tissue, volumetric dilution or negative feedback mechanisms from increased circulating 1,25-dihydroxyvitamin D3. Others hypothesise that heavier individuals may partake in less outdoor activity, may also cover-up and wear more clothing than leaner individuals, thus decreasing sun exposure and limiting endogenous production of cholecalciferol in the skin. Moreover, in some but not all studies, BMI and adiposity have been negatively associated with the change in vitamin D status following vitamin D supplementation. It therefore remains unclear if body size and/or adiposity should be taken into account when determining the dietary requirements for vitamin D. This review will evaluate the current evidence linking vitamin D status and supplementation to overweight and obesity, and discuss the implications for setting dietary requirements.


Hypoxia in cancer: significance and impact on clinical outcome

Abstract: Hypoxia, a characteristic feature of locally advanced solid tumors, has emerged as a pivotal factor of the tumor (patho-)physiome since it can promote tumor progression and resistance to therapy. Hypoxia represents a “Janus face” in tumor biology because (a) it is associated with restrained proliferation, differentiation, necrosis or apoptosis, and (b) it can also lead to the development of an aggressive phenotype. Independent of standard prognostic factors, such as tumor stage and nodal status, hypoxia has been suggested as an adverse prognostic factor for patient outcome. Studies of tumor hypoxia involving the direct assessment of the oxygenation status have suggested worse disease-free survival for patients with hypoxic cervical cancers or soft tissue sarcomas. In head & neck cancers the studies suggest that hypoxia is prognostic for survival and local control. Technical limitations of the direct O2 sensing technique have prompted the use of surrogate markers for tumor hypoxia, such as hypoxia-related endogenous proteins (e.g., HIF-1α, GLUT-1, CA IX) or exogenous bioreductive drugs. In many—albeit not in all—studies endogenous markers showed prognostic significance for patient outcome. The prognostic relevance of exogenous markers, however, appears to be limited. Noninvasive assessment of hypoxia using imaging techniques can be achieved with PET or SPECT detection of radiolabeled tracers or with MRI techniques (e.g., BOLD). Clinical experience with these methods regarding patient prognosis is so far only limited. In the clinical studies performed up until now, the lack of standardized treatment protocols, inconsistencies of the endpoints characterizing the oxygenation status and methodological differences (e.g., different immunohistochemical staining procedures) may compromise the power of the prognostic parameter used.


Late-life depression and microstructural abnormalities in dorsolateral prefrontal cortex white matter.

Abstract:
OBJECTIVE The purpose of this study was to determine whether microstructural abnormalities in the white matter of the dorsolateral prefrontal cortex are associated with late-life depression.
 METHOD Seventeen elderly depressed subjects were compared with 16 elderly subjects who were not depressed. Diffusion tensor imaging was used to measure the fractional anisotropy of the white matter in the dorsolateral prefrontal cortex's superior and middle frontal gyri bilaterally and in the left occipital lobe as a control region. The authors compared results between groups while controlling for age, sex, and comorbid medical disorders.
 RESULTS Even after controlling for age, sex, hypertension, and heart disease, the authors found significantly lower fractional anisotropy values in the right superior frontal gyrus white matter of depressed patients than comparison subjects.
 CONCLUSIONS Microstructural changes in the white matter of the right superior frontal gyrus are associated with late-life depression. Further work is needed to determine how these changes contribute to depression outcomes.


The role of the immune response during SIVagm infection of the African green monkey natural host.

Abstract: The African green monkey (AGM) is one of many African species endemically infected with simian immunodeficiency virus (SIV). Like the other natural hosts, AGMs do not succumb to AIDS and understanding the basis for this resistance to disease progression would be of enormous theoretical and practical importance. Early efforts by our group that concentrated on identifying immune mechanisms presumed to keep the virus under control failed to find any obvious candidates. The presumption of virus control was invalidated by the finding that SIVagm replicates in AGMs with the same vigor as HIV-1 does in humans. Focus therefore shifted to identifying possible immunopathologic features present in disease susceptible hosts but absent in the AGM natural host. The apparent immunologic tolerance of AGMs to the SIVagm core protein led to the development of a hypothesis implicating anti-Gag antibodies in the formation of immune complexes, virus trapping in the lymph nodes and immune dysfunction. The idea proved difficult to test in vivo and present work focuses on the possibility that Gag tolerance at the T-cell level plays an important role in preventing the catastrophic demise of the immune system characteristic of immunodeficiency virus infection of the heterologous primate host.


Epstein-Barr virus and virus human protein interaction maps.

Abstract: A comprehensive mapping of interactions among Epstein-Barr virus (EBV) proteins and interactions of EBV proteins with human proteins should provide specific hypotheses and a broad perspective on EBV strategies for replication and persistence. Interactions of EBV proteins with each other and with human proteins were assessed by using a stringent high-throughput yeast two-hybrid system. Overall, 43 interactions between EBV proteins and 173 interactions between EBV and human proteins were identified. EBV-EBV and EBV-human protein interaction, or "interactome" maps provided a framework for hypotheses of protein function. For example, LF2, an EBV protein of unknown function interacted with the EBV immediate early R transactivator (Rta) and was found to inhibit Rta transactivation. From a broader perspective, EBV genes can be divided into two evolutionary classes, "core" genes, which are conserved across all herpesviruses and subfamily specific, or "noncore" genes. Our EBV-EBV interactome map is enriched for interactions among proteins in the same evolutionary class. Furthermore, human proteins targeted by EBV proteins were enriched for highly connected or "hub" proteins and for proteins with relatively short paths to all other proteins in the human interactome network. Targeting of hubs might be an efficient mechanism for EBV reorganization of cellular processes.


The ketogenic diet for the treatment of childhood epilepsy: a randomised controlled trial.

Abstract:
BACKGROUND The ketogenic diet has been widely and successfully used to treat children with drug-resistant epilepsy since the 1920s. The aim of this study was to test the efficacy of the ketogenic diet in a randomised controlled trial.
 METHODS 145 children aged between 2 and 16 years who had at least daily seizures (or more than seven seizures per week), had failed to respond to at least two antiepileptic drugs, and had not been treated previously with the ketogenic diet participated in a randomised controlled trial of its efficacy to control seizures. Enrolment for the trial ran between December, 2001, and July, 2006. Children were seen at one of two hospital centres or a residential centre for young people with epilepsy. Children were randomly assigned to receive a ketogenic diet, either immediately or after a 3-month delay, with no other changes to treatment (control group). Neither the family nor investigators were blinded to the group assignment. Early withdrawals were recorded, and seizure frequency on the diet was assessed after 3 months and compared with that of the controls. The primary endpoint was a reduction in seizures; analysis was intention to treat. Tolerability of the diet was assessed by questionnaire at 3 months. The trial is registered with ClinicalTrials.gov, number NCT00564915.
 FINDINGS 73 children were assigned to the ketogenic diet and 72 children to the control group. Data from 103 children were available for analysis: 54 on the ketogenic diet and 49 controls. Of those who did not complete the trial, 16 children did not receive their intervention, 16 did not provide adequate data, and ten withdrew from the treatment before the 3-month review, six because of intolerance. After 3 months, the mean percentage of baseline seizures was significantly lower in the diet group than in the controls (62.0%vs 136.9%, 75% decrease, 95% CI 42.4-107.4%; p<0.0001). 28 children (38%) in the diet group had greater than 50% seizure reduction compared with four (6%) controls (p<0.0001), and five children (7%) in the diet group had greater than 90% seizure reduction compared with no controls (p=0.0582). There was no significant difference in the efficacy of the treatment between symptomatic generalised or symptomatic focal syndromes. The most frequent side-effects reported at 3-month review were constipation, vomiting, lack of energy, and hunger.
 INTERPRETATION The results from this trial of the ketogenic diet support its use in children with treatment-intractable epilepsy.
 FUNDING HSA Charitable Trust; Smiths Charity; Scientific Hospital Supplies; Milk Development Council.


Transcriptional profiling of the heart reveals chamber-specific gene expression patterns.

Abstract: Cardiac chamber-specific gene expression is critical for the normal development and function of the heart. To investigate the genetic basis of cardiac anatomical specialization, we have undertaken a nearly genome-wide transcriptional profiling of the four heart chambers and the interventricular septum. Rigorous statistical analysis has allowed the identification of known and novel members of gene families that are felt to be important in cardiac development and function, including LIM proteins, homeobox proteins, wnt and T-box pathway proteins, as well as structural proteins like actins and myosins. In addition, these studies have allowed the identification of thousands of additional differentially expressed genes, for which there is little structural or functional information. Clustering of genes with known and unknown functions provides insights into signaling pathways that are essential for development and maintenance of chamber-specific features. To facilitate future research in this area, a searchable internet database has been constructed that allows study of the chamber-specific expression of any gene represented on this comprehensive microarray. It is anticipated that further study of genes identified through this effort will provide insights into the specialization of heart chamber tissues, and their specific roles in cardiac development, aging, and disease.


Phosphorylation of the autophagy receptor optineurin restricts Salmonella growth.

Abstract: Selective autophagy can be mediated via receptor molecules that link specific cargoes to the autophagosomal membranes decorated by ubiquitin-like microtubule-associated protein light chain 3 (LC3) modifiers. Although several autophagy receptors have been identified, little is known about mechanisms controlling their functions in vivo. In this work, we found that phosphorylation of an autophagy receptor, optineurin, promoted selective autophagy of ubiquitin-coated cytosolic Salmonella enterica. The protein kinase TANK binding kinase 1 (TBK1) phosphorylated optineurin on serine-177, enhancing LC3 binding affinity and autophagic clearance of cytosolic Salmonella. Conversely, ubiquitin- or LC3-binding optineurin mutants and silencing of optineurin or TBK1 impaired Salmonella autophagy, resulting in increased intracellular bacterial proliferation. We propose that phosphorylation of autophagy receptors might be a general mechanism for regulation of cargo-selective autophagy.


A paediatric case of sideroblastic anaemia. Ultrastructural studies of erythroblasts cultured from marrow BFU-E in a methylcellulose micromethod.

Abstract: We examined the morphological and functional characteristics of erythroblasts derived from marrow erythroid progenitor cells grown in a methylcellulose microculture, which were taken from a female child with rare atypical sideroblastic anaemia (SA) partially responsive to pyridoxine. Colony formation was within the normal range in three successive cultures (median values: 82.25 CFU-E and 16.4 BFU-E derived colonies/6.6 X 10(4) cells) compared to growth by normal cells (65-315 CFU-E and 9-40 BFU-E). We evaluated in vitro differentiation by biochemical microassay of a cytosol enzyme involved in the haem pathway: uroporphyrinogen I synthase (UROS). The UROS values in the erythroid colonies from SA marrow were at the lowere end of the normal range (median values: 6.7 +/- 0.3 and 14.4 +/- 3.8 pmol uroporphyrinogen/h in CFU-E and BFU-E-derived colonies respectively versus 17.4 +/- 7.3 and 25 +/- 7.2 pmol/h in CFU-E and BFU-E colonies from normal subjects. Ultrastructural examination of the SA erythroblasts from non-cultured bone marrow or derived from cultured BFU-E revealed the characteristic deposition of iron in mitochondria around the nucleus of most cells (ringed sideroblasts). However, the majority of cultured cells had marked dyserythropoietic features, with a large number of bilobulated or trilobulated erythroblasts, multiple cytoplasmic vacuoles, numerous abnormalities of the nucleus, and excessive membrane material beneath the plasma membrane, all features difficult to observe in non-cultured marrows.


The epidemiology of major depressive disorder: results from the National Comorbidity Survey Replication (NCS-R).

Abstract:
CONTEXT Uncertainties exist about prevalence and correlates of major depressive disorder (MDD).
 OBJECTIVE To present nationally representative data on prevalence and correlates of MDD by Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) criteria, and on study patterns and correlates of treatment and treatment adequacy from the recently completed National Comorbidity Survey Replication (NCS-R).
 DESIGN Face-to-face household survey conducted from February 2001 to December 2002.
 SETTING The 48 contiguous United States.
 PARTICIPANTS Household residents ages 18 years or older (N = 9090) who responded to the NCS-R survey.
 MAIN OUTCOME MEASURES Prevalence and correlates of MDD using the World Health Organization's (WHO) Composite International Diagnostic Interview (CIDI), 12-month severity with the Quick Inventory of Depressive Symptomatology Self-Report (QIDS-SR), the Sheehan Disability Scale (SDS), and the WHO disability assessment scale (WHO-DAS). Clinical reinterviews used the Structured Clinical Interview for DSM-IV.
 RESULTS The prevalence of CIDI MDD for lifetime was 16.2% (95% confidence interval [CI], 15.1-17.3) (32.6-35.1 million US adults) and for 12-month was 6.6% (95% CI, 5.9-7.3) (13.1-14.2 million US adults). Virtually all CIDI 12-month cases were independently classified as clinically significant using the QIDS-SR, with 10.4% mild, 38.6% moderate, 38.0% severe, and 12.9% very severe. Mean episode duration was 16 weeks (95% CI, 15.1-17.3). Role impairment as measured by SDS was substantial as indicated by 59.3% of 12-month cases with severe or very severe role impairment. Most lifetime (72.1%) and 12-month (78.5%) cases had comorbid CIDI/DSM-IV disorders, with MDD only rarely primary. Although 51.6% (95% CI, 46.1-57.2) of 12-month cases received health care treatment for MDD, treatment was adequate in only 41.9% (95% CI, 35.9-47.9) of these cases, resulting in 21.7% (95% CI, 18.1-25.2) of 12-month MDD being adequately treated. Sociodemographic correlates of treatment were far less numerous than those of prevalence.
 CONCLUSIONS Major depressive disorder is a common disorder, widely distributed in the population, and usually associated with substantial symptom severity and role impairment. While the recent increase in treatment is encouraging, inadequate treatment is a serious concern. Emphasis on screening and expansion of treatment needs to be accompanied by a parallel emphasis on treatment quality improvement.


Selective enhancement of endothelial BMPR-II with BMP9 reverses pulmonary arterial hypertension

Abstract: Genetic evidence implicates the loss of bone morphogenetic protein type II receptor (BMPR-II) signaling in the endothelium as an initiating factor in pulmonary arterial hypertension (PAH). However, selective targeting of this signaling pathway using BMP ligands has not yet been explored as a therapeutic strategy. Here, we identify BMP9 as the preferred ligand for preventing apoptosis and enhancing monolayer integrity in both pulmonary arterial endothelial cells and blood outgrowth endothelial cells from subjects with PAH who bear mutations in the gene encoding BMPR-II, BMPR2. Mice bearing a heterozygous knock-in allele of a human BMPR2 mutation, R899X, which we generated as an animal model of PAH caused by BMPR-II deficiency, spontaneously developed PAH. Administration of BMP9 reversed established PAH in these mice, as well as in two other experimental PAH models, in which PAH develops in response to either monocrotaline or VEGF receptor inhibition combined with chronic hypoxia. These results demonstrate the promise of direct enhancement of endothelial BMP signaling as a new therapeutic strategy for PAH.


Two structurally independent domains of E. coli NusG create regulatory plasticity via distinct interactions with RNA polymerase and regulators.

Abstract: NusG is a conserved regulatory protein that interacts with elongation complexes (ECs) of RNA polymerase, DNA, and RNA to modulate transcription in multiple and sometimes opposite ways. In Escherichia coli, NusG suppresses pausing and increases elongation rate, enhances termination by E. coli rho and phage HK022 Nun protein, and promotes antitermination by lambdaN and in ribosomal RNA operons. We report NMR studies that suggest that E. coli NusG consists of two largely independent N- and C-terminal structural domains, NTD and CTD, respectively. Based on tests of the functions of the NTD and CTD and variants of NusG in vivo and in vitro, we find that NTD alone is sufficient to suppress pausing and enhance transcript elongation in vitro. However, neither domain alone can enhance rho-dependent termination or support antitermination, indicating that interactions of both domains with ECs are required for these processes. We propose that the two domains of NusG mediate distinct interactions with ECs: the NTD interacts with RNA polymerase and the CTD interacts with rho and other regulators, providing NusG with different combinations of interactions to effect different regulatory outcomes.


Growth control and ribosomopathies.

Abstract: Ribosome biogenesis and protein synthesis are two of the most energy consuming processes in a growing cell. Moreover, defects in their molecular components can alter the pattern of gene expression. Thus it is understandable that cells have developed a surveillance system to monitor the status of the translational machinery. Recent discoveries of causative mutations and deletions in genes linked to ribosome biogenesis have defined a group of similar pathologies termed ribosomopathies. Over the past decade, much has been learned regarding the relationship between growth control and ribosome biogenesis. The discovery of extra-ribosomal functions of several ribosome proteins and their regulation of p53 levels has provided a link from ribosome impairment to cell cycle regulation. Yet, evidence suggesting p53 and/or Hdm2 independent pathways also exists. In this review, we summarize recent advances in understanding the mechanisms underlying the pathologies of ribosomopathies and discuss the relationship between ribosome production and tumorigenesis.


Distinct memory CD4+ T cells with commitment to T follicular helper- and T helper 1-cell lineages are generated after acute viral infection.

Abstract: CD4(+) T follicular helper (Tfh) cells provide the required signals to B cells for germinal center reactions that are necessary for long-lived antibody responses. However, it remains unclear whether there are CD4(+) memory T cells committed to the Tfh cell lineage after antigen clearance. By using adoptive transfer of antigen-specific memory CD4(+) T cell subpopulations in the lymphocytic choriomeningitis virus infection model, we found that there are distinct memory CD4(+) T cell populations with commitment to either Tfh- or Th1-cell lineages. Our conclusions are based on gene expression profiles, epigenetic studies, and phenotypic and functional analyses. Our findings indicate that CD4(+) memory T cells "remember" their previous effector lineage after antigen clearance, being poised to reacquire their lineage-specific effector functions upon antigen reencounter. These findings have important implications for rational vaccine design, where improving the generation and engagement of memory Tfh cells could be used to enhance vaccine-induced protective immunity.