query: HNF4A mutations are associated with macrosomia in infancy.

title: Macrosomia and Hyperinsulinaemic Hypoglycaemia in Patients with Heterozygous Mutations in the HNF4A Gene

text: Background  Macrosomia is associated with considerable neonatal and maternal morbidity. Factors that predict macrosomia are poorly understood. The increased rate of macrosomia in the offspring of pregnant women with diabetes and in congenital hyperinsulinaemia is mediated by increased foetal insulin secretion. We assessed the in utero and neonatal role of two key regulators of pancreatic insulin secretion by studying birthweight and the incidence of neonatal hypoglycaemia in patients with heterozygous mutations in the maturity-onset diabetes of the young (MODY) genes HNF4A (encoding HNF-4α) and HNF1A/TCF1 (encoding HNF-1α), and the effect of pancreatic deletion of Hnf4a on foetal and neonatal insulin secretion in mice.

title: Atopy risk in infants and children in relation to early exposure to fish, oily fish, or long-chain omega-3 fatty acids: a systematic review.

text: There are two main families of polyunsaturated fatty acids (PUFAs), the n-6 and the n-3 families. It has been suggested that there is a causal relationship between n-6 PUFA intake and allergic disease, and there are biologically plausible mechanisms, involving eicosanoid mediators of the n-6 PUFA arachidonic acid, that could explain this. Fish and fish oils are sources of long-chain n-3 PUFAs and these fatty acids act to oppose the actions of n-6 PUFAs. Thus, it is considered that n-3 PUFAs will protect against atopic sensitization and against the clinical manifestations of atopy. Evidence to examine this has been acquired from epidemiologic studies investigating associations between fish intake in pregnancy, lactation, infancy, and childhood, and atopic outcomes in infants and children and from intervention studies with fish oil supplements in pregnancy, lactation, infancy, and childhood, and atopic outcomes in infants and children. All five epidemiological studies investigating the effect of maternal fish intake during pregnancy on atopic or allergic outcomes in infants/children of those pregnancies concluded protective associations. One study investigating the effects of maternal fish intake during lactation did not observe any significant associations. The evidence from epidemiological studies investigating the effects of fish intake during infancy and childhood on atopic outcomes in those infants or children is inconsistent, although the majority of the studies (nine of 14) showed a protective effect of fish intake during infancy or childhood on atopic outcomes in those infants/children. Fish oil supplementation during pregnancy and lactation or during infancy or childhood results in a higher n-3 PUFA status in the infants or children. Fish oil provision to pregnant women is associated with immunologic changes in cord blood and such changes may persist. Studies performed to date indicate that provision of fish oil during pregnancy may reduce sensitization to common food allergens and reduce prevalence and severity of atopic dermatitis in the first year of life, with a possible persistence until adolescence with a reduction in eczema, hay fever, and asthma. Fish oil provision to infants or children may be associated with immunologic changes in the blood but it is not clear if these are of clinical significance and whether they persist. Fish oil supplementation in infancy may decrease the risk of developing some manifestations of allergic disease, but this benefit may not persist as other factors come into play. It is not clear whether fish oil can be used to treat children with asthma as the two studies conducted to date give divergent results. Further studies of increased long-chain n-3 PUFA provision in during pregnancy, lactation, and infancy are needed to more clearly identify the immunologic and clinical effects in infants and children and to identify protective and therapeutic effects and their persistence.

title: Effect of low doses of ionising radiation in infancy on cognitive function in adulthood: Swedish population based cohort study.

text:
OBJECTIVE To determine whether exposure to low doses of ionising radiation in infancy affects cognitive function in adulthood.
 DESIGN Population based cohort study.
 SETTING Sweden.
 PARTICIPANTS 3094 men who had received radiation for cutaneous haemangioma before age 18 months during 1930-59.
 MAIN OUTCOME MEASURES Radiation dose to frontal and posterior parts of the brain, and association between dose and intellectual capacity at age 18 or 19 years based on cognitive tests (learning ability, logical reasoning, spatial recognition) and high school attendance.
 RESULTS The proportion of boys who attended high school decreased with increasing doses of radiation to both the frontal and the posterior parts of the brain from about 32% among those not exposed to around 17% in those who received > 250 mGy. For the frontal dose, the multivariate odds ratio was 0.47 (95% confidence interval 0.26 to 0.85, P for trend 0.0003) and for the posterior dose it was 0.59 (0.23 to 1.47, 0.0005). A negative dose-response relation was also evident for the three cognitive tests for learning ability and logical reasoning but not for the test of spatial recognition.
 CONCLUSIONS Low doses of ionising radiation to the brain in infancy influence cognitive abilities in adulthood.

title: EIF2AK3, encoding translation initiation factor 2-α kinase 3, is mutated in patients with Wolcott-Rallison syndrome

text: Wolcott-Rallison syndrome (WRS) is a rare, autosomal recessive disorder characterized by permanent neonatal or early infancy insulin-dependent diabetes. Epiphyseal dysplasia, osteoporosis and growth retardation occur at a later age. Other frequent multisystemic manifestations include hepatic and renal dysfunction, mental retardation and cardiovascular abnormalities. On the basis of two consanguineous families, we mapped WRS to a region of less than 3 cM on chromosome 2p12, with maximal evidence of linkage and homozygosity at 4 microsatellite markers within an interval of approximately 1 cM. The gene encoding the eukaryotic translation initiation factor 2-α kinase 3 (EIF2AK3) resides in this interval; thus we explored it as a candidate. We identified distinct mutations of EIF2AK3 that segregated with the disorder in each of the families. The first mutation produces a truncated protein in which the entire catalytic domain is missing. The other changes an amino acid, located in the catalytic domain of the protein, that is highly conserved among kinases from the same subfamily. Our results provide evidence for the role of EIF2AK3 in WRS. The identification of this gene may provide insight into the understanding of the more common forms of diabetes and other pathologic manifestations of WRS.

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

text: 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.

title: Direct Reprogramming of Hepatic Myofibroblasts into Hepatocytes In Vivo Attenuates Liver Fibrosis.

text: Direct induction of induced hepatocytes (iHeps) from fibroblasts holds potential as a strategy for regenerative medicine but until now has only been shown in culture settings. Here, we describe in vivo iHep formation using transcription factor induction and genetic fate tracing in mouse models of chronic liver disease. We show that ectopic expression of the transcription factors FOXA3, GATA4, HNF1A, and HNF4A from a polycistronic lentiviral vector converts mouse myofibroblasts into cells with a hepatocyte phenotype. In vivo expression of the same set of transcription factors from a p75 neurotrophin receptor peptide (p75NTRp)-tagged adenovirus enabled the generation of hepatocyte-like cells from myofibroblasts in fibrotic mouse livers and reduced liver fibrosis. We have therefore been able to convert pro-fibrogenic myofibroblasts in the liver into hepatocyte-like cells with positive functional benefits. This direct in vivo reprogramming approach may open new avenues for the treatment of chronic liver disease.

title: Cardiorespiratory adaptation during sleep in infants and children.

text: The cardiorespiratory control system undergoes functional maturation after birth. Until this process is completed, the cardiorespiratory system is unstable, placing infants at risk for cardiorespiratory disturbances, especially during sleep. The profound influence of states of alertness on respiratory and cardiac control has been the focus of intense scrutiny during the last decade. The effects of rapid-eye movement (REM) sleep on various mechanisms involved in cardiorespiratory control are of particular significance during the postnatal period since newborns spend much of their time in this sleep state. In fullterm newborns, REM sleep occupies more than 50% of total sleep time, and this percentage is even greater in preterm newborns. From term to six months of age, the proportion of REM sleep decreases. Since respiratory and cardiac disturbances are known to occur selectively during REM sleep, the predominance of REM sleep may be a risk factor for abnormal sleep-related events during early infancy. Awareness of these developmental changes in sleep patterns is important for clinicians dealing with problems such as apparent life-threatening events (ALTE), sudden infant death syndrome (SIDS), and/or cardiorespiratory responses to respiratory disorders. Our current understanding of respiratory and cardiac control rests mainly on studies conducted during the first months of life. There is a paucity of data on late infancy and early childhood. The present paper will review available data on how sleep affects 1) ventilatory mechanics, in particular of the upper airways and the chest wall; ventilation and apnea; gas exchange; chemoreceptor function; and arousal responses; 2) changes in heart rate and heart rate variability, and the occurrence and mechanisms of bradycardia.

title: Can genome engineering be used to target cancer-associated enhancers?

text: Transcriptional misregulation is involved in the development of many diseases, especially neoplastic transformation. Distal regulatory elements, such as enhancers, play a major role in specifying cell-specific transcription patterns in both normal and diseased tissues, suggesting that enhancers may be prime targets for therapeutic intervention. By focusing on modulating gene regulation mediated by cell type-specific enhancers, there is hope that normal epigenetic patterning in an affected tissue could be restored with fewer side effects than observed with treatments employing relatively nonspecific inhibitors such as epigenetic drugs. New methods employing genomic nucleases and site-specific epigenetic regulators targeted to specific genomic regions, using either artificial DNA-binding proteins or RNA-DNA interactions, may allow precise genome engineering at enhancers. However, this field is still in its infancy and further refinements that increase specificity and efficiency are clearly required.

title: Percentile curves for hemoglobin and red cell volume in infancy and childhood.

text: Percentile curves were calculated for hemoglobin and mean corpuscular volume in children between 0.5 and 16 years of age. The curves were derived from several populations of non-indigent white children who lived near sea level. Subjects were excluded from the reference population if they had laboratory evidence of iron deficiency, thalassemia minor, and/or hemoglobinopathy. The final reference populations included 9,946 children for the derivation of the hemoglobin curves and 2,314 for the MCV curves. The percentile curves should be particularly applicable to the diagnosis and screening of iron deficiency and thalassemia minor.

title: Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype

text: Point mutations of the transcription factor AML1 are associated with leukemogenesis in acute myeloblastic leukemia (AML). Internal tandem duplications (ITDs) in the juxtamembrane domain and mutations in the second tyrosine kinase domain of the Fms-like tyrosine kinase 3 (FLT3) gene represent the most frequent genetic alterations in AML. However, such mutations per se appear to be insufficient for leukemic transformation. To evaluate whether both AML1 and FLT3 mutations contribute to leukemogenesis, we analyzed mutations of these genes in AML M0 subtype in whom AML1 mutations were predominantly observed. Of 51 patients, eight showed a mutation in the Runt domain of the AML1 gene: one heterozygous missense mutation with normal function, five heterozygous frameshift mutations and two biallelic nonsense or frameshift mutations, resulting in haploinsufficiency or complete loss of the AML1 activities. On the other hand, a total of 10 of 49 patients examined had the FLT3 mutation. We detected the FLT3 mutation in five of eight (63%) patients with AML1 mutation, whereas five of 41 (12%) without AML1 mutation showed the FLT3 mutation (P=0.0055). These observations suggest that reduced AML1 activities predispose cells to the acquisition of the activating FLT3 mutation as a secondary event leading to full transformation in AML M0.

title: Psychobiological mechanisms underlying the social buffering of the hypothalamic-pituitary-adrenocortical axis: a review of animal models and human studies across development.

text: Discovering the stress-buffering effects of social relationships has been one of the major findings in psychobiology in the last century. However, an understanding of the underlying neurobiological and psychological mechanisms of this buffering is only beginning to emerge. An important avenue of this research concerns the neurocircuitry that can regulate the activity of the hypothalamic-pituitary-adrenocortical (HPA) axis. The present review is a translational effort aimed at integrating animal models and human studies of the social regulation of the HPA axis from infancy to adulthood, specifically focusing on the process that has been named social buffering. This process has been noted across species and consists of a dampened HPA axis stress response to threat or challenge that occurs with the presence or assistance of a conspecific. We describe aspects of the relevant underlying neurobiology when enough information exists and expose major gaps in our understanding across all domains of the literatures we aimed to integrate. We provide a working conceptual model focused on the role of oxytocinergic systems and prefrontal neural networks as 2 of the putative biological mediators of this process, and propose that the role of early experiences is critical in shaping later social buffering effects. This synthesis points to both general future directions and specific experiments that need to be conducted to build a more comprehensive model of the HPA social buffering effect across the life span that incorporates multiple levels of analysis: neuroendocrine, behavioral, and social.

title: Neurological development of 5-year-old children receiving a low-saturated fat, low-cholesterol diet since infancy: A randomized controlled trial.

text:
CONTEXT Early childhood introduction of nutritional habits aimed at atherosclerosis prevention is compatible with normal growth, but its effect on neurological development is unknown.
 OBJECTIVE To analyze how parental counseling aimed at keeping children's diets low in saturated fat and cholesterol influences neurodevelopment during the first 5 years of life.
 DESIGN Randomized controlled trial conducted between February 1990 and November 1996.
 SETTING Outpatient clinic of a university department in Turku, Finland.
 PARTICIPANTS A total of 1062 seven-month-old infants and their parents, recruited at well-baby clinics between 1990 and 1992. At age 5 years, 496 children still living in the city of Turku were available to participate in neurodevelopmental testing.
 INTERVENTION Participants were randomly assigned to receive individualized counseling aimed at limiting the child's fat intake to 30% to 35% of daily energy, with a saturated:monounsaturated:polyunsaturated fatty acid ratio of 1:1:1 and a cholesterol intake of less than 200 mg/d (n = 540) or usual health education (control group, n = 522).
 MAIN OUTCOME MEASURES Nutrient intake, serum lipid concentrations, and neurological development at 5 years, among children in the intervention vs control groups.
 RESULTS Absolute and relative intakes of fat, saturated fatty acids, and cholesterol among children in the intervention group were markedly less than the respective values of control children. Mean (SD) percentages of daily energy at age 5 years for the intervention vs control groups were as follows: for total fat, 30.6% (4.5%) vs 33.4% (4.4%) (P<. 001); and for saturated fat, 11.7% (2.3%) vs 14.5% (2.4%) (P<.001). Mean intakes of cholesterol were 164.2 mg (60.1 mg) and 192.5 mg (71. 9 mg) (P<.001) for the intervention and control groups, respectively. Serum cholesterol concentrations were continuously 3% to 5% lower in children in the intervention group than in children in the control group. At age 5 years, mean (SD) serum cholesterol concentration of the intervention group was 4.27 (0.63) mmol/L (165 [24] mg/dL) and of the control group, 4.41 (0.74) mmol/L (170 [29] mg/dL) (P =.04). Neurological development of children in the intervention group was at least as good as that of children in the control group. Relative risks for children in the intervention group to fail tests of speech and language skills, gross motor functioning plus perception, and visual motor skills were 0.95 (90% confidence interval [CI], 0.60-1.49), 0.95 (90% CI, 0.58-1.55), and 0.65 (90% CI, 0.39-1.08), respectively (P =.85,.86, and.16, respectively, vs control children).
 CONCLUSION Our data indicate that repeated child-targeted dietary counseling of parents during the first 5 years of a child's life lessens age-associated increases in children's serum cholesterol and is compatible with normal neurological development. JAMA. 2000;284:993-1000

title: N348I in the Connection Domain of HIV-1 Reverse Transcriptase Confers Zidovudine and Nevirapine Resistance

text: Background The catalytically active 66-kDa subunit of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) consists of DNA polymerase, connection, and ribonuclease H (RNase H) domains. Almost all known RT inhibitor resistance mutations identified to date map to the polymerase domain of the enzyme. However, the connection and RNase H domains are not routinely analysed in clinical samples and none of the genotyping assays available for patient management sequence the entire RT coding region. The British Columbia Centre for Excellence in HIV/AIDS (the Centre) genotypes clinical isolates up to codon 400 in RT, and our retrospective statistical analyses of the Centre’s database have identified an N348I mutation in the RT connection domain in treatment-experienced individuals. The objective of this multidisciplinary study was to establish the in vivo relevance of this mutation and its role in drug resistance. Methods and Findings The prevalence of N348I in clinical isolates, the time taken for it to emerge under selective drug pressure, and its association with changes in viral load, specific drug treatment, and known drug resistance mutations was analysed from genotypes, viral loads, and treatment histories from the Centre’s database. N348I increased in prevalence from below 1% in 368 treatmentnao ¨ve individuals to 12.1% in 1,009 treatment-experienced patients (p ¼ 7.7 3 10 � 12 ). N348I appeared early in therapy and was highly associated with thymidine analogue mutations (TAMs) M41L and T215Y/F (p , 0.001), the lamivudine resistance mutations M184V/I (p , 0.001), and non-nucleoside RTI (NNRTI) resistance mutations K103N and Y181C/I (p , 0.001). The association with TAMs and NNRTI resistance mutations was consistent with the selection of N348I in patients treated with regimens that included both zidovudine and nevirapine (odds ratio 2.62, 95% confidence interval 1.43–4.81). The appearance of N348I was associated with a significant increase in viral load (p , 0.001), which was as large as the viral load increases observed for any of the TAMs. However, this analysis did not account for the simultaneous selection of other RT or protease inhibitor resistance mutations on viral load. To delineate the role of this mutation in RT inhibitor resistance, N348I was introduced into HIV-1 molecular clones containing different genetic backbones. N348I decreased zidovudine susceptibility 2- to 4-fold in the context of wildtype HIV-1 or when combined with TAMs. N348I also decreased susceptibility to nevirapine (7.4fold) and efavirenz (2.5-fold) and significantly potentiated resistance to these drugs when combined with K103N. Biochemical analyses of recombinant RT containing N348I provide supporting evidence for the role of this mutation in zidovudine and NNRTI resistance and give some insight into the molecular mechanism of resistance. Conclusions

title: Association of type and location of BRCA1 and BRCA2 mutations with risk of breast and ovarian cancer.

text:
IMPORTANCE Limited information about the relationship between specific mutations in BRCA1 or BRCA2 (BRCA1/2) and cancer risk exists.
 OBJECTIVE To identify mutation-specific cancer risks for carriers of BRCA1/2.
 DESIGN, SETTING, AND PARTICIPANTS Observational study of women who were ascertained between 1937 and 2011 (median, 1999) and found to carry disease-associated BRCA1 or BRCA2 mutations. The international sample comprised 19,581 carriers of BRCA1 mutations and 11,900 carriers of BRCA2 mutations from 55 centers in 33 countries on 6 continents. We estimated hazard ratios for breast and ovarian cancer based on mutation type, function, and nucleotide position. We also estimated RHR, the ratio of breast vs ovarian cancer hazard ratios. A value of RHR greater than 1 indicated elevated breast cancer risk; a value of RHR less than 1 indicated elevated ovarian cancer risk.    EXPOSURES Mutations of BRCA1 or BRCA2.
 MAIN OUTCOMES AND MEASURES Breast and ovarian cancer risks.
 RESULTS Among BRCA1 mutation carriers, 9052 women (46%) were diagnosed with breast cancer, 2317 (12%) with ovarian cancer, 1041 (5%) with breast and ovarian cancer, and 7171 (37%) without cancer. Among BRCA2 mutation carriers, 6180 women (52%) were diagnosed with breast cancer, 682 (6%) with ovarian cancer, 272 (2%) with breast and ovarian cancer, and 4766 (40%) without cancer. In BRCA1, we identified 3 breast cancer cluster regions (BCCRs) located at c.179 to c.505 (BCCR1; RHR = 1.46; 95% CI, 1.22-1.74; P = 2 × 10(-6)), c.4328 to c.4945 (BCCR2; RHR = 1.34; 95% CI, 1.01-1.78; P = .04), and c. 5261 to c.5563 (BCCR2', RHR = 1.38; 95% CI, 1.22-1.55; P = 6 × 10(-9)). We also identified an ovarian cancer cluster region (OCCR) from c.1380 to c.4062 (approximately exon 11) with RHR = 0.62 (95% CI, 0.56-0.70; P = 9 × 10(-17)). In BRCA2, we observed multiple BCCRs spanning c.1 to c.596 (BCCR1; RHR = 1.71; 95% CI, 1.06-2.78; P = .03), c.772 to c.1806 (BCCR1'; RHR = 1.63; 95% CI, 1.10-2.40; P = .01), and c.7394 to c.8904 (BCCR2; RHR = 2.31; 95% CI, 1.69-3.16; P = .00002). We also identified 3 OCCRs: the first (OCCR1) spanned c.3249 to c.5681 that was adjacent to c.5946delT (6174delT; RHR = 0.51; 95% CI, 0.44-0.60; P = 6 × 10(-17)). The second OCCR spanned c.6645 to c.7471 (OCCR2; RHR = 0.57; 95% CI, 0.41-0.80; P = .001). Mutations conferring nonsense-mediated decay were associated with differential breast or ovarian cancer risks and an earlier age of breast cancer diagnosis for both BRCA1 and BRCA2 mutation carriers.
 CONCLUSIONS AND RELEVANCE Breast and ovarian cancer risks varied by type and location of BRCA1/2 mutations. With appropriate validation, these data may have implications for risk assessment and cancer prevention decision making for carriers of BRCA1 and BRCA2 mutations.

title: Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma

text: Glioblastoma multiforme (GBM) is a lethal brain tumour in adults and children. However, DNA copy number and gene expression signatures indicate differences between adult and paediatric cases. To explore the genetic events underlying this distinction, we sequenced the exomes of 48 paediatric GBM samples. Somatic mutations in the H3.3-ATRX-DAXX chromatin remodelling pathway were identified in 44% of tumours (21/48). Recurrent mutations in H3F3A, which encodes the replication-independent histone 3 variant H3.3, were observed in 31% of tumours, and led to amino acid substitutions at two critical positions within the histone tail (K27M, G34R/G34V) involved in key regulatory post-translational modifications. Mutations in ATRX (α-thalassaemia/mental retardation syndrome X-linked) and DAXX (death-domain associated protein), encoding two subunits of a chromatin remodelling complex required for H3.3 incorporation at pericentric heterochromatin and telomeres, were identified in 31% of samples overall, and in 100% of tumours harbouring a G34R or G34V H3.3 mutation. Somatic TP53 mutations were identified in 54% of all cases, and in 86% of samples with H3F3A and/or ATRX mutations. Screening of a large cohort of gliomas of various grades and histologies (n = 784) showed H3F3A mutations to be specific to GBM and highly prevalent in children and young adults. Furthermore, the presence of H3F3A/ATRX-DAXX/TP53 mutations was strongly associated with alternative lengthening of telomeres and specific gene expression profiles. This is, to our knowledge, the first report to highlight recurrent mutations in a regulatory histone in humans, and our data suggest that defects of the chromatin architecture underlie paediatric and young adult GBM pathogenesis.

title: In vivo effect of chronic hypoxia on the neurochemical profile of the developing rat hippocampus.

text: The cognitive deficits observed in children with cyanotic congenital heart disease suggest involvement of the developing hippocampus. Chronic postnatal hypoxia present during infancy in these children may play a role in these impairments. To understand the biochemical mechanisms of hippocampal injury in chronic hypoxia, a neurochemical profile consisting of 15 metabolite concentrations and 2 metabolite ratios in the hippocampus was evaluated in a rat model of chronic postnatal hypoxia using in vivo 1H NMR spectroscopy at 9.4 T. Chronic hypoxia was induced by continuously exposing rats (n = 23) to 10% O2 from postnatal day (P) 3 to P28. Fifteen metabolites were quantified from a volume of 9-11 microl centered on the left hippocampus on P14, P21, and P28 and were compared with normoxic controls (n = 14). The developmental trajectory of neurochemicals in chronic hypoxia was similar to that seen in normoxia. However, chronic hypoxia had an effect on the concentrations of the following neurochemicals: aspartate, creatine, phosphocreatine, GABA, glutamate, glutamine, glutathione, myoinositol, N-acetylaspartate (NAA), phosphorylethanolamine, and phosphocreatine/creatine (PCr/Cr) and glutamate/glutamine (Glu/Gln) ratios (P < 0.001 each, except glutamate, P = 0.04). The increased PCr/Cr ratio is consistent with decreased brain energy consumption. Given the well-established link between excitatory neurotransmission and brain energy metabolism, we postulate that elevated glutamate, Glu/Gln ratio, and GABA indicate suppressed excitatory neurotransmission in an energy-limited environment. Decreased NAA and phosphorylethanolamine suggest reduced neuronal integrity and phospholipid metabolism. The altered hippocampal neurochemistry during its development may underlie some of the cognitive deficits present in human infants at risk of chronic hypoxia.

title: ALS-FUS pathology revisited: singleton FUS mutations and an unusual case with both a FUS and TARDBP mutation

text:
INTRODUCTION Mutations in the FUS gene have been shown to be a rare cause of amyotrophic lateral sclerosis (ALS-FUS) and whilst well documented clinically and genetically there have been relatively few neuropathological studies. Recent work suggested a possible correlation between pathological features such as frequency of basophilic inclusions in neurons and rate of clinical decline, other studies have revealed a discrepancy between the upper motor neuron features detected clinically and the associated pathology. The purpose of this study was to describe the pathological features associated with more recently discovered FUS mutations and reinvestigate those with well recognised mutations in an attempt to correlate the pathology with mutation and/or clinical phenotype. The brains and spinal cords of seven cases of ALS-FUS were examined neuropathologically, including cases with the newly described p. K510E mutation and a case with both a known p. P525L mutation in the FUS gene and a truncating p. Y374X mutation in the TARDBP gene.
 RESULTS The neuropathology in all cases revealed basophilic and FUS inclusions in the cord. The density and type of inclusions varied markedly between cases, but did not allow a clear correlation with clinical progression. Only one case showed significant motor cortical pathology despite the upper motor neuron clinical features being evident in 4 patients. The case with both a FUS and TARDBP mutation revealed FUS positive inclusions but no TDP-43 pathology. Instead there were unusual p62 positive, FUS negative neuronal and glial inclusions as well as dot-like neurites.
 CONCLUSIONS The study confirms cases of ALS-FUS to be mainly a lower motor neuron disease and to have pathology that does not appear to neatly correlate with clinical features or genetics. Furthermore, the case with both a FUS and TARDBP mutation reveals an intriguing pathological profile which at least in part involves a very unusual staining pattern for the ubiquitin-binding protein p62.

title: Mosaic PPM1D mutations are associated with predisposition to breast and ovarian cancer

text: Improved sequencing technologies offer unprecedented opportunities for investigating the role of rare genetic variation in common disease. However, there are considerable challenges with respect to study design, data analysis and replication. Using pooled next-generation sequencing of 507 genes implicated in the repair of DNA in 1,150 samples, an analytical strategy focused on protein-truncating variants (PTVs) and a large-scale sequencing case–control replication experiment in 13,642 individuals, here we show that rare PTVs in the p53-inducible protein phosphatase PPM1D are associated with predisposition to breast cancer and ovarian cancer. PPM1D PTV mutations were present in 25 out of 7,781 cases versus 1 out of 5,861 controls (P = 1.12 × 10−5), including 18 mutations in 6,912 individuals with breast cancer (P = 2.42 × 10−4) and 12 mutations in 1,121 individuals with ovarian cancer (P = 3.10 × 10−9). Notably, all of the identified PPM1D PTVs were mosaic in lymphocyte DNA and clustered within a 370-base-pair region in the final exon of the gene, carboxy-terminal to the phosphatase catalytic domain. Functional studies demonstrate that the mutations result in enhanced suppression of p53 in response to ionizing radiation exposure, suggesting that the mutant alleles encode hyperactive PPM1D isoforms. Thus, although the mutations cause premature protein truncation, they do not result in the simple loss-of-function effect typically associated with this class of variant, but instead probably have a gain-of-function effect. Our results have implications for the detection and management of breast and ovarian cancer risk. More generally, these data provide new insights into the role of rare and of mosaic genetic variants in common conditions, and the use of sequencing in their identification.

title: The ATM missense mutation p.Ser49Cys (c.146C>G) and the risk of breast cancer.

text: Homozygous mutation in the ATM gene causes ataxia telangiectasia and heterozygous mutation carriers may be at increased risk of breast cancer. We studied a total of 22 ATM variants; 18 variants were analyzed in one of two large population-based studies from the U.S. and Poland, and four variants were analyzed in all 2,856 breast cancer cases and 3,344 controls from the two studies. The missense mutation Ser49Cys (c.146C>G, p. S49C), carried by approximately 2% of subjects, was more common in cases than controls in both study populations, combined odds ratio (OR) 1.69 (95% CI, 1.19-2.40; P=0.004). Another missense mutation at approximately 2% frequency, Phe858Leu (c.2572T>C, p. F858L), was associated with a significant increased risk in the U.S. study but not in Poland, and had a combined OR of 1.44 (95% CI, 0.98-2.11; P=0.06). These analyses provide the most convincing evidence thus far that missense mutations in ATM, particularly p. S49C, may be breast cancer susceptibility alleles. Because of their low frequency, even larger sample sizes are required to more firmly establish these associations.

title: Aberrant luminal progenitors as the candidate target population for basal tumor development in BRCA1 mutation carriers

text: Basal-like breast cancers arising in women carrying mutations in the BRCA1 gene, encoding the tumor suppressor protein BRCA1, are thought to develop from the mammary stem cell. To explore early cellular changes that occur in BRCA1 mutation carriers, we have prospectively isolated distinct epithelial subpopulations from normal mammary tissue and preneoplastic specimens from individuals heterozygous for a BRCA1 mutation. We describe three epithelial subsets including basal stem/progenitor, luminal progenitor and mature luminal cells. Unexpectedly, we found that breast tissue from BRCA1 mutation carriers harbors an expanded luminal progenitor population that shows factor-independent growth in vitro. Moreover, gene expression profiling revealed that breast tissue heterozygous for a BRCA1 mutation and basal breast tumors were more similar to normal luminal progenitor cells than any other subset, including the stem cell–enriched population. The c-KIT tyrosine kinase receptor (encoded by KIT) emerged as a key marker of luminal progenitor cells and was more highly expressed in BRCA1-associated preneoplastic tissue and tumors. Our findings suggest that an aberrant luminal progenitor population is a target for transformation in BRCA1-associated basal tumors .


query: HOXB4 facilitates HSC expansion when co-cultured with HSCs.

title: Angiopoietin-like proteins stimulate ex vivo expansion of hematopoietic stem cells

text: Successful ex vivo expansion of hematopoietic stem cells (HSCs) would greatly benefit the treatment of disease and the understanding of crucial questions of stem cell biology. Here we show, using microarray studies, that the HSC-supportive mouse fetal liver CD3+ cells specifically express the proteins angiopoietin-like 2 (Angptl2) and angiopoietin-like 3 (Angptl3). We observed a 24- or 30-fold net expansion of long-term HSCs by reconstitution analysis when we cultured highly enriched HSCs for 10 days in the presence of Angptl2 or Angptl3 together with saturating levels of other growth factors. The coiled-coil domain of Angptl2 was capable of stimulating expansion of HSCs. Furthermore, angiopoietin-like 5, angiopoietin-like 7 and microfibril-associated glycoprotein 4 also supported expansion of HSCs in culture.

title: The journey of developing hematopoietic stem cells.

text: Hematopoietic stem cells (HSCs) develop during embryogenesis in a complex process that involves multiple anatomical sites. Once HSC precursors have been specified from mesoderm, they have to mature into functional HSCs and undergo self-renewing divisions to generate a pool of HSCs. During this process, developing HSCs migrate through various embryonic niches, which provide signals for their establishment and the conservation of their self-renewal ability. These processes have to be recapitulated to generate HSCs from embryonic stem cells. Elucidating the interactions between developing HSCs and their niches should facilitate the generation and expansion of HSCs in vitro to exploit their clinical potential.

title: In vivo proliferation and cell cycle kinetics of long-term self-renewing hematopoietic stem cells.

text: A rare set of hematopoietic stem cells (HSC) must undergo a massive expansion to produce mature blood cells. The phenotypic isolation of HSC from mice offers the opportunity to determine directly their proliferation kinetics. We analyzed the proliferation and cell cycle kinetics of long-term self-renewing HSC (LT-HSC) in normal adult mice. At any one time, approximately 5% of LT-HSC were in S/G2/M phases of the cell cycle and another 20% were in G1 phase. BrdUrd incorporation was used to determine the rate at which different cohorts of HSC entered the cell cycle over time. About 50% of LT-HSC incorporated BrdUrd by 6 days and >90% incorporated BrdUrd by 30 days. By 6 months, 99% of LT-HSC had incorporated BrdUrd. We calculated that approximately 8% of LT-HSC asynchronously entered the cell cycle per day. Nested reverse transcription-PCR analysis revealed cyclin D2 expression in a high proportion of LT-HSC. Although approximately 75% of LT-HSC are quiescent in G0 at any one time, all HSC are recruited into cycle regularly such that 99% of LT-HSC divide on average every 57 days.

title: Angiopoietin-like protein 3 supports the activity of hematopoietic stem cells in the bone marrow niche.

text: The physiologic roles of angiopoietin-like proteins (Angptls) in the hematopoietic system remain unknown. Here we show that hematopoietic stem cells (HSCs) in Angptl3-null mice are decreased in number and quiescence. HSCs transplanted into Angptl3-null recipient mice exhibited impaired repopulation. Bone marrow sinusoidal endothelial cells express high levels of Angptl3 and are adjacent to HSCs. Importantly, bone marrow stromal cells or endothelium deficient in Angptl3 have a significantly decreased ability to support the expansion of repopulating HSCs. Angptl3 represses the expression of the transcription factor Ikaros, whose unregulated overexpression diminishes the repopulation activity of HSCs. Angptl3, as an extrinsic factor, thus supports the stemness of HSCs in the bone marrow niche.

title: Haematopoietic stem cells do not asymmetrically segregate chromosomes or retain BrdU

text: Stem cells are proposed to segregate chromosomes asymmetrically during self-renewing divisions so that older (‘immortal’) DNA strands are retained in daughter stem cells whereas newly synthesized strands segregate to differentiating cells. Stem cells are also proposed to retain DNA labels, such as 5-bromo-2-deoxyuridine (BrdU), either because they segregate chromosomes asymmetrically or because they divide slowly. However, the purity of stem cells among BrdU-label-retaining cells has not been documented in any tissue, and the ‘immortal strand hypothesis’ has not been tested in a system with definitive stem cell markers. Here we tested these hypotheses in haematopoietic stem cells (HSCs), which can be highly purified using well characterized markers. We administered BrdU to newborn mice, mice treated with cyclophosphamide and granulocyte colony-stimulating factor, and normal adult mice for 4 to 10 days, followed by 70 days without BrdU. In each case, less than 6% of HSCs retained BrdU and less than 0.5% of all BrdU-retaining haematopoietic cells were HSCs, revealing that BrdU has poor specificity and poor sensitivity as an HSC marker. Sequential administration of 5-chloro-2-deoxyuridine and 5-iodo-2-deoxyuridine indicated that all HSCs segregate their chromosomes randomly. Division of individual HSCs in culture revealed no asymmetric segregation of the label. Thus, HSCs cannot be identified on the basis of BrdU-label retention and do not retain older DNA strands during division, indicating that these are not general properties of stem cells.

title: Asymmetric Cell Divisions Sustain Long-Term  Hematopoiesis from Single-sorted Human Fetal Liver Cells

text: Hematopoietic stem cells (HSCs) in adult marrow are believed to be derived from fetal liver precursors. To study cell kinetics involved in long-term hematopoiesis, we studied single-sorted candidate HSCs from fetal liver that were cultured in the presence of a mixture of stimulatory cytokines. After 8–10 d, the number of cells in primary cultures varied from 10,000 cells. Single cells in slow growing colonies were recloned upon reaching a 100–200 cell stage. Strikingly, the number of cells in subclones varied widely again. These results are indicative of asymmetric divisions in primitive hematopoietic cells in which proliferative potential and cell cycle properties are unevenly distributed among daughter cells. The continuous generation of functional heterogeneity among the clonal progeny of HSCs is in support of intrinsic control of stem cell fate and provides a model for the long-term maintenance of hematopoiesis in vitro and in vivo.

title: Hematopoietic stem cells and other hematopoietic cells show broad resistance to chemotherapeutic agents in vivo when overexpressing bcl-2.

text: Objective. Chemotherapeutic agents function by inducing apoptosis and their effectiveness depends on the balance of pro- and anti-apoptotic proteins in cells. Due to the complicated interactions of the many proteins involved, it has been difficult to determine in tumors whether overexpression of single genes is prognostic for increased resistance. Therefore, we studied the influence of bcl-2 overexpression on resistance to chemotherapeutics in a transgenic mouse system. This allowed us to study a wide variety of cells, including important but rare populations such as hematopoietic stem cells (HSC).Methods. H2K-bcl-2 transgenic and wild-type (WT) mice were treated with several agents(5-fluoruracil, cyclophosphamide, and busulfan) to determine the contribution of increased amounts of bcl-2 to the response to these chemotherapeutics in vivo. Populations were enumerated using flow cytometry. HSC were studied by FACS purification and long-term reconstitution assays in vivo and resistance was confirmed by short-term proliferation assays with different amounts of chemotherapeutics in vitro. Results. bcl-2 overexpression alone protects many cell types, though protection levels differ between populations and agents. However, even sensitive populations return to pretreatment levels faster in transgenic mice. bcl-2 overexpression also prevents the dramatic changes in HSC following 5-FU treatment (downregulation of c-kit, upregulation of Lin, less efficient long-term reconstitution). In vitro studies directly demonstrate increased resistance of bcl-2 overexpressing HSC to chemotherapeutic agents. Conclusions. Increased expression of bcl-2 in HSC and their progeny endows these cells with broad resistance to chemotherapeutic agents. The ability to (differentially) regulate sensitivity to apoptosis of bystander and tumor cells is clinically important.

title: Tie2/Angiopoietin-1 Signaling Regulates Hematopoietic Stem Cell Quiescence in the Bone Marrow Niche

text: The quiescent state is thought to be an indispensable property for the maintenance of hematopoietic stem cells (HSCs). Interaction of HSCs with their particular microenvironments, known as the stem cell niches, is critical for adult hematopoiesis in the bone marrow (BM). Here, we demonstrate that HSCs expressing the receptor tyrosine kinase Tie2 are quiescent and antiapoptotic, and comprise a side-population (SP) of HSCs, which adhere to osteoblasts (OBs) in the BM niche. The interaction of Tie2 with its ligand Angiopoietin-1 (Ang-1) induced cobblestone formation of HSCs in vitro and maintained in vivo long-term repopulating activity of HSCs. Furthermore, Ang-1 enhanced the ability of HSCs to become quiescent and induced adhesion to bone, resulting in protection of the HSC compartment from myelosuppressive stress. These data suggest that the Tie2/Ang-1 signaling pathway plays a critical role in the maintenance of HSCs in a quiescent state in the BM niche.

title: Thrombopoietin/MPL signaling regulates hematopoietic stem cell quiescence and interaction with the osteoblastic niche.

text: Maintenance of hematopoietic stem cells (HSCs) depends on interaction with their niche. Here we show that the long-term (LT)-HSCs expressing the thrombopoietin (THPO) receptor, MPL, are a quiescent population in adult bone marrow (BM) and are closely associated with THPO-producing osteoblastic cells. THPO/MPL signaling upregulated beta1-integrin and cyclin-dependent kinase inhibitors in HSCs. Furthermore, inhibition and stimulation of THPO/MPL pathway by treatments with anti-MPL neutralizing antibody, AMM2, and with THPO showed reciprocal regulation of quiescence of LT-HSC. AMM2 treatment reduced the number of quiescent LT-HSCs and allowed exogenous HSC engraftment without irradiation. By contrast, exogenous THPO transiently increased quiescent HSC population and subsequently induced HSC proliferation in vivo. Altogether, these observations suggest that THPO/MPL signaling plays a critical role of LT-HSC regulation in the osteoblastic niche.

title: p62/SQSTM1 by Binding to Vitamin D Receptor Inhibits Hepatic Stellate Cell Activity, Fibrosis, and Liver Cancer.

text: Hepatic stellate cells (HSCs) play critical roles in liver fibrosis and hepatocellular carcinoma (HCC). Vitamin D receptor (VDR) activation in HSCs inhibits liver inflammation and fibrosis. We found that p62/SQSTM1, a protein upregulated in liver parenchymal cells but downregulated in HCC-associated HSCs, negatively controls HSC activation. Total body or HSC-specific p62 ablation potentiates HSCs and enhances inflammation, fibrosis, and HCC progression. p62 directly interacts with VDR and RXR promoting their heterodimerization, which is critical for VDR:RXR target gene recruitment. Loss of p62 in HSCs impairs the repression of fibrosis and inflammation by VDR agonists. This demonstrates that p62 is a negative regulator of liver inflammation and fibrosis through its ability to promote VDR signaling in HSCs, whose activation supports HCC.

title: Haematopoietic stem cells and early lymphoid progenitors occupy distinct bone marrow niches

text: Although haematopoietic stem cells (HSCs) are commonly assumed to reside within a specialized microenvironment, or niche, most published experimental manipulations of the HSC niche have affected the function of diverse restricted progenitors. This raises the fundamental question of whether HSCs and restricted progenitors reside within distinct, specialized niches or whether they share a common niche. Here we assess the physiological sources of the chemokine CXCL12 for HSC and restricted progenitor maintenance. Cxcl12(DsRed) knock-in mice (DsRed-Express2 recombined into the Cxcl12 locus) showed that Cxcl12 was primarily expressed by perivascular stromal cells and, at lower levels, by endothelial cells, osteoblasts and some haematopoietic cells. Conditional deletion of Cxcl12 from haematopoietic cells or nestin-cre-expressing cells had little or no effect on HSCs or restricted progenitors. Deletion of Cxcl12 from endothelial cells depleted HSCs but not myeloerythroid or lymphoid progenitors. Deletion of Cxcl12 from perivascular stromal cells depleted HSCs and certain restricted progenitors and mobilized these cells into circulation. Deletion of Cxcl12 from osteoblasts depleted certain early lymphoid progenitors but not HSCs or myeloerythroid progenitors, and did not mobilize these cells into circulation. Different stem and progenitor cells thus reside in distinct cellular niches in bone marrow: HSCs occupy a perivascular niche and early lymphoid progenitors occupy an endosteal niche.

title: Hematopoietic stem cell: self-renewal versus differentiation.

text: The mammalian blood system, containing more than 10 distinct mature cell types, stands on one specific cell type, hematopoietic stem cell (HSC). Within the system, only HSCs possess the ability of both multipotency and self-renewal. Multipotency is the ability to differentiate into all functional blood cells. Self-renewal is the ability to give rise to HSC itself without differentiation. Since mature blood cells (MBCs) are predominantly short-lived, HSCs continuously provide more differentiated progenitors while properly maintaining the HSC pool size throughout life by precisely balancing self-renewal and differentiation. Thus, understanding the mechanisms of self-renewal and differentiation of HSC has been a central issue. In this review, we focus on the hierarchical structure of the hematopoietic system, the current understanding of microenvironment and molecular cues regulating self-renewal and differentiation of adult HSCs, and the currently emerging systems approaches to understand HSC biology.

title: GATA-3 regulates hematopoietic stem cell maintenance and cell-cycle entry.

text: Maintaining hematopoietic stem cell (HSC) quiescence is a critical property for the life-long generation of blood cells. Approximately 75% of cells in a highly enriched long-term repopulating HSC (LT-HSC) pool (Lin(-)Sca1(+)c-Kit(hi)CD150(+)CD48(-)) are quiescent, with only a small percentage of the LT-HSCs in cycle. Transcription factor GATA-3 is known to be vital for the development of T cells at multiple stages in the thymus and for Th2 differentiation in the peripheral organs. Although it is well documented that GATA-3 is expressed in HSCs, a role for GATA-3 in any prethymic progenitor cell has not been established. In the present study, we show that Gata3-null mutant mice generate fewer LT-HSCs and that fewer Gata3-null LT-HSCs are in cycle. Furthermore, Gata3 mutant hematopoietic progenitor cells fail to be recruited into an increased cycling state after 5-fluorouracil-induced myelosuppression. Therefore, GATA-3 is required for the maintenance of a normal number of LT-HSCs and for their entry into the cell cycle.

title: Combined Single-Cell Functional and Gene Expression Analysis Resolves Heterogeneity within Stem Cell Populations

text: Heterogeneity within the self-renewal durability of adult hematopoietic stem cells (HSCs) challenges our understanding of the molecular framework underlying HSC function. Gene expression studies have been hampered by the presence of multiple HSC subtypes and contaminating non-HSCs in bulk HSC populations. To gain deeper insight into the gene expression program of murine HSCs, we combined single-cell functional assays with flow cytometric index sorting and single-cell gene expression assays. Through bioinformatic integration of these datasets, we designed an unbiased sorting strategy that separates non-HSCs away from HSCs, and single-cell transplantation experiments using the enriched population were combined with RNA-seq data to identify key molecules that associate with long-term durable self-renewal, producing a single-cell molecular dataset that is linked to functional stem cell activity. Finally, we demonstrated the broader applicability of this approach for linking key molecules with defined cellular functions in another stem cell system.

title: A distinctive DNA damage response in human hematopoietic stem cells reveals an apoptosis-independent role for p53 in self-renewal.

text: Highly regenerative tissues such as blood must possess effective DNA damage responses (DDR) that balance long-term regeneration with protection from leukemogenesis. Hematopoietic stem cells (HSCs) sustain life-long blood production, yet their response to DNA damage remains largely unexplored. We report that human HSCs exhibit delayed DNA double-strand break rejoining, persistent gammaH2AX foci, and enhanced p53- and ASPP1-dependent apoptosis after gamma-radiation compared to progenitors. p53 inactivation or Bcl-2 overexpression reduced radiation-induced apoptosis and preserved in vivo repopulating HSC function. Despite similar protection from irradiation-induced apoptosis, only Bcl-2-overexpressing HSCs showed higher self-renewal capacity, establishing that intact p53 positively regulates self-renewal independently from apoptosis. The reduced self-renewal of HSCs with inactivated p53 was associated with increased spontaneous gammaH2AX foci in secondary transplants of HSCs. Our data reveal distinct physiological roles of p53 that together ensure optimal HSC function: apoptosis regulation and prevention of gammaH2AX foci accumulation upon HSC self-renewal.

title: Osteoclasts promote the formation of hematopoietic stem cell niches in the bone marrow

text: Formation of the hematopoietic stem cell (HSC) niche in bone marrow (BM) is tightly associated with endochondral ossification, but little is known about the mechanisms involved. We used the oc/oc mouse, a mouse model with impaired endochondral ossification caused by a loss of osteoclast (OCL) activity, to investigate the role of osteoblasts (OBLs) and OCLs in the HSC niche formation. The absence of OCL activity resulted in a defective HSC niche associated with an increased proportion of mesenchymal progenitors but reduced osteoblastic differentiation, leading to impaired HSC homing to the BM. Restoration of OCL activity reversed the defect in HSC niche formation. Our data demonstrate that OBLs are required for establishing HSC niches and that osteoblastic development is induced by OCLs. These findings broaden our knowledge of the HSC niche formation, which is critical for understanding normal and pathological hematopoiesis.

title: Mesenchymal and haematopoietic stem cells form a unique bone marrow niche

text: The cellular constituents forming the haematopoietic stem cell (HSC) niche in the bone marrow are unclear, with studies implicating osteoblasts, endothelial and perivascular cells. Here we demonstrate that mesenchymal stem cells (MSCs), identified using nestin expression, constitute an essential HSC niche component. Nestin+ MSCs contain all the bone-marrow colony-forming-unit fibroblastic activity and can be propagated as non-adherent ‘mesenspheres’ that can self-renew and expand in serial transplantations. Nestin+ MSCs are spatially associated with HSCs and adrenergic nerve fibres, and highly express HSC maintenance genes. These genes, and others triggering osteoblastic differentiation, are selectively downregulated during enforced HSC mobilization or β3 adrenoreceptor activation. Whereas parathormone administration doubles the number of bone marrow nestin+ cells and favours their osteoblastic differentiation, in vivo nestin+ cell depletion rapidly reduces HSC content in the bone marrow. Purified HSCs home near nestin+ MSCs in the bone marrow of lethally irradiated mice, whereas in vivo nestin+ cell depletion significantly reduces bone marrow homing of haematopoietic progenitors. These results uncover an unprecedented partnership between two distinct somatic stem-cell types and are indicative of a unique niche in the bone marrow made of heterotypic stem-cell pairs.

title: Hematopoietic stem cell quiescence promotes error-prone DNA repair and mutagenesis.

text: Most adult stem cells, including hematopoietic stem cells (HSCs), are maintained in a quiescent or resting state in vivo. Quiescence is widely considered to be an essential protective mechanism for stem cells that minimizes endogenous stress caused by cellular respiration and DNA replication. We demonstrate that HSC quiescence can also have detrimental effects. We found that HSCs have unique cell-intrinsic mechanisms ensuring their survival in response to ionizing irradiation (IR), which include enhanced prosurvival gene expression and strong activation of p53-mediated DNA damage response. We show that quiescent and proliferating HSCs are equally radioprotected but use different types of DNA repair mechanisms. We describe how nonhomologous end joining (NHEJ)-mediated DNA repair in quiescent HSCs is associated with acquisition of genomic rearrangements, which can persist in vivo and contribute to hematopoietic abnormalities. Our results demonstrate that quiescence is a double-edged sword that renders HSCs intrinsically vulnerable to mutagenesis following DNA damage.

title: A lineage of myeloid cells independent of Myb and hematopoietic stem cells.

text: Macrophages and dendritic cells (DCs) are key components of cellular immunity and are thought to originate and renew from hematopoietic stem cells (HSCs). However, some macrophages develop in the embryo before the appearance of definitive HSCs. We thus reinvestigated macrophage development. We found that the transcription factor Myb was required for development of HSCs and all CD11b(high) monocytes and macrophages, but was dispensable for yolk sac (YS) macrophages and for the development of YS-derived F4/80(bright) macrophages in several tissues, such as liver Kupffer cells, epidermal Langerhans cells, and microglia--cell populations that all can persist in adult mice independently of HSCs. These results define a lineage of tissue macrophages that derive from the YS and are genetically distinct from HSC progeny.

title: Blood stem cells emerge from aortic endothelium by a novel type of cell transition

text: The ontogeny of haematopoietic stem cells (HSCs) during embryonic development is still highly debated, especially their possible lineage relationship to vascular endothelial cells. The first anatomical site from which cells with long-term HSC potential have been isolated is the aorta-gonad-mesonephros (AGM), more specifically the vicinity of the dorsal aortic floor. But although some authors have presented evidence that HSCs may arise directly from the aortic floor into the dorsal aortic lumen, others support the notion that HSCs first emerge within the underlying mesenchyme. Here we show by non-invasive, high-resolution imaging of live zebrafish embryos, that HSCs emerge directly from the aortic floor, through a stereotyped process that does not involve cell division but a strong bending then egress of single endothelial cells from the aortic ventral wall into the sub-aortic space, and their concomitant transformation into haematopoietic cells. The process is polarized not only in the dorso-ventral but also in the rostro-caudal versus medio-lateral direction, and depends on Runx1 expression: in Runx1-deficient embryos, the exit events are initially similar, but much rarer, and abort into violent death of the exiting cell. These results demonstrate that the aortic floor is haemogenic and that HSCs emerge from it into the sub-aortic space, not by asymmetric cell division but through a new type of cell behaviour, which we call an endothelial haematopoietic transition.


query: HOXB4 is a highly expressed component of cellular secretome from fetal liver cell populations.

title: Angiopoietin-like proteins stimulate ex vivo expansion of hematopoietic stem cells

text: Successful ex vivo expansion of hematopoietic stem cells (HSCs) would greatly benefit the treatment of disease and the understanding of crucial questions of stem cell biology. Here we show, using microarray studies, that the HSC-supportive mouse fetal liver CD3+ cells specifically express the proteins angiopoietin-like 2 (Angptl2) and angiopoietin-like 3 (Angptl3). We observed a 24- or 30-fold net expansion of long-term HSCs by reconstitution analysis when we cultured highly enriched HSCs for 10 days in the presence of Angptl2 or Angptl3 together with saturating levels of other growth factors. The coiled-coil domain of Angptl2 was capable of stimulating expansion of HSCs. Furthermore, angiopoietin-like 5, angiopoietin-like 7 and microfibril-associated glycoprotein 4 also supported expansion of HSCs in culture.

title: Oncofetal long noncoding RNA PVT1 promotes proliferation and stem cell-like property of hepatocellular carcinoma cells by stabilizing NOP2.

text:
UNLABELLED Many protein-coding oncofetal genes are highly expressed in murine and human fetal liver and silenced in adult liver. The protein products of these hepatic oncofetal genes have been used as clinical markers for the recurrence of hepatocellular carcinoma (HCC) and as therapeutic targets for HCC. Herein we examined the expression profiles of long noncoding RNAs (lncRNAs) found in fetal and adult liver in mice. Many fetal hepatic lncRNAs were identified; one of these, lncRNA-mPvt1, is an oncofetal RNA that was found to promote cell proliferation, cell cycling, and the expression of stem cell-like properties of murine cells. Interestingly, we found that human lncRNA-hPVT1 was up-regulated in HCC tissues and that patients with higher lncRNA-hPVT1 expression had a poor clinical prognosis. The protumorigenic effects of lncRNA-hPVT1 on cell proliferation, cell cycling, and stem cell-like properties of HCC cells were confirmed both in vitro and in vivo by gain-of-function and loss-of-function experiments. Moreover, mRNA expression profile data showed that lncRNA-hPVT1 up-regulated a series of cell cycle genes in SMMC-7721 cells. By RNA pulldown and mass spectrum experiments, we identified NOP2 as an RNA-binding protein that binds to lncRNA-hPVT1. We confirmed that lncRNA-hPVT1 up-regulated NOP2 by enhancing the stability of NOP2 proteins and that lncRNA-hPVT1 function depends on the presence of NOP2.
 CONCLUSION Our study demonstrates that the expression of many lncRNAs is up-regulated in early liver development and that the fetal liver can be used to search for new diagnostic markers for HCC. LncRNA-hPVT1 promotes cell proliferation, cell cycling, and the acquisition of stem cell-like properties in HCC cells by stabilizing NOP2 protein. Regulation of the lncRNA-hPVT1/NOP2 pathway may have beneficial effects on the treatment of HCC.

title: Identification of Senescent Cells in the Bone Microenvironment.

text: Cellular senescence is a fundamental mechanism by which cells remain metabolically active yet cease dividing and undergo distinct phenotypic alterations, including upregulation of p16Ink4a , profound secretome changes, telomere shortening, and decondensation of pericentromeric satellite DNA. Because senescent cells accumulate in multiple tissues with aging, these cells and the dysfunctional factors they secrete, termed the senescence-associated secretory phenotype (SASP), are increasingly recognized as promising therapeutic targets to prevent age-related degenerative pathologies, including osteoporosis. However, the cell type(s) within the bone microenvironment that undergoes senescence with aging in vivo has remained poorly understood, largely because previous studies have focused on senescence in cultured cells. Thus in young (age 6 months) and old (age 24 months) mice, we measured senescence and SASP markers in vivo in highly enriched cell populations, all rapidly isolated from bone/marrow without in vitro culture. In both females and males, p16Ink4a expression by real-time quantitative polymerase chain reaction (rt-qPCR) was significantly higher with aging in B cells, T cells, myeloid cells, osteoblast progenitors, osteoblasts, and osteocytes. Further, in vivo quantification of senescence-associated distension of satellites (SADS), ie, large-scale unraveling of pericentromeric satellite DNA, revealed significantly more senescent osteocytes in old compared with young bone cortices (11% versus 2%, p < 0.001). In addition, primary osteocytes from old mice had sixfold more (p < 0.001) telomere dysfunction-induced foci (TIFs) than osteocytes from young mice. Corresponding with the age-associated accumulation of senescent osteocytes was significantly higher expression of multiple SASP markers in osteocytes from old versus young mice, several of which also showed dramatic age-associated upregulation in myeloid cells. These data show that with aging, a subset of cells of various lineages within the bone microenvironment become senescent, although senescent myeloid cells and senescent osteocytes predominantly develop the SASP. Given the critical roles of osteocytes in orchestrating bone remodeling, our findings suggest that senescent osteocytes and their SASP may contribute to age-related bone loss. © 2016 American Society for Bone and Mineral Research.

title: A developmental transition in definitive erythropoiesis: erythropoietin expression is sequentially regulated by retinoic acid receptors and HNF4.

text: The cytokine erythropoietin (Epo) promotes erythropoietic progenitor cell proliferation and is required for erythropoietic differentiation. We have found that the Epo gene is a direct transcriptional target gene of retinoic acid signaling during early erythropoiesis (prior to embryonic day E12.5) in the fetal liver. Mouse embryos lacking the retinoic acid receptor gene RXR alpha have a morphological and histological phenotype that is comparable with embryos in which the Epo gene itself has been mutated, and flow cytometric analysis indicates that RXR alpha-deficient embryos are deficient in erythroid differentiation. Epo mRNA levels are reduced substantially in the fetal livers of RXR alpha(-/-) embryos at E10.25 and E11.25, and genetic analysis shows that the RXR alpha and Epo genes are coupled in the same pathway. We furthermore show that the Epo gene is retinoic acid inducible in embryos, and that the Epo gene enhancer contains a DR2 sequence that represents a retinoic acid receptor-binding site and a retinoic acid receptor transcriptional response element. However, unlike Epo-deficient embryos that die from anemia, the erythropoietic deficiency in RXR alpha(-/-) embryos is transient; Epo mRNA is expressed at normal levels by E12.5, and erythropoiesis and liver morphology are normal by E14.5. We show that HNF4, like RXR alpha a member of the nuclear receptor family, is abundantly expressed in fetal liver hepatocytes, and is competitive with retinoic acid receptors for occupancy of the Epo gene enhancer DR2 element. We propose that Epo expression is regulated during the E9.5--E11.5 phase of fetal liver erythropoiesis by RXR alpha and retinoic acid, and that expression then becomes dominated by HNF4 activity from E11.5 onward. This transition may be responsible for switching regulation of Epo expression from retinoic acid control to hypoxic control, as is found throughout the remainder of life.

title: Protein restriction during pregnancy affects maternal liver lipid metabolism and fetal brain lipid composition in the rat.

text: Suboptimal developmental environments program offspring to lifelong metabolic problems. The aim of this study was to determine the impact of protein restriction in pregnancy on maternal liver lipid metabolism at 19 days of gestation (dG) and its effect on fetal brain development. Control (C) and restricted (R) mothers were fed with isocaloric diets containing 20 and 10% of casein. At 19 dG, maternal blood and livers and fetal livers and brains were collected. Serum insulin and leptin levels were determinate in mothers. Maternal and fetal liver lipid and fetal brain lipid quantification were performed. Maternal liver and fetal brain fatty acids were quantified by gas chromatography. In mothers, liver desaturase and elongase mRNAs were measured by RT-PCR. Maternal body and liver weights were similar in both groups. However, fat body composition, including liver lipids, was lower in R mothers. A higher fasting insulin at 19 dG in the R group was observed (C = 0.2 +/- 0.04 vs. R = 0.9 +/- 0.16 ng/ml, P < 0.01) and was inversely related to early growth retardation. Serum leptin in R mothers was significantly higher than that observed in C rats (C = 5 +/- 0.1 vs. R = 7 +/- 0.7 ng/ml, P < 0.05). In addition, protein restriction significantly reduced gene expression in maternal liver of desaturases and elongases and the concentration of arachidonic (AA) and docosahexanoic (DHA) acids. In fetus from R mothers, a low body weight (C = 3 +/- 0.3 vs. R = 2 +/- 0.1 g, P < 0.05), as well as liver and brain lipids, including the content of DHA in the brain, was reduced. This study showed that protein restriction during pregnancy may negatively impact normal fetal brain development by changes in maternal lipid metabolism.

title: Isolation and characterization of the cDNA encoding BKLF/TEF-2, a major CACCC-box-binding protein in erythroid cells and selected other cells.

text: CACCC boxes are among the critical sequences present in regulatory elements of genes expressed in erythroid cells, as well as in selected other cell types. While an erythroid cell-specific CACCC-box-binding protein, EKLF, has been shown to be required in vivo for proper expression of the adult beta-globin gene, it is dispensable for the regulation of several other globin and nonglobin erythroid cell-expressed genes. In the work described here, we searched for additional CACCC-box transcription factors that might be active in murine erythroid cells. We identified a major gel shift activity (termed BKLF), present in yolk sac and fetal liver erythroid cells, that could be distinguished from EKLF by specific antisera. Through relaxed-stringency hybridization, we obtained the cDNA encoding BKLF, a highly basic, novel zinc finger protein that is related to EKLF and other Krüppel-like members in its DNA-binding domain but unrelated elsewhere. BKLF, which is widely but not ubiquitously expressed in cell lines, is highly expressed in the midbrain region of embryonic mice and appears to correspond to the gel shift activity TEF-2, a transcriptional activator implicated in regulation of the simian virus 40 enhancer and other CACCC-box-containing regulatory elements. Because BKLF binds with high affinity and preferentially over Sp1 to many CACCC sequences of erythroid cell expressed genes, it is likely to participate in the control of many genes whose expression appears independent of the action of EKLF.

title: Reversal of hyperglycemia in mice by using human expandable insulin-producing cells differentiated from fetal liver progenitor cells.

text: Beta-cell replacement is considered to be the most promising approach for treatment of type 1 diabetes. Its application on a large scale is hindered by a shortage of cells for transplantation. Activation of insulin expression, storage, and regulated secretion in stem/progenitor cells offers novel ways to overcome this shortage. We explored whether fetal human progenitor liver cells (FH) could be induced to differentiate into insulin-producing cells after expression of the pancreatic duodenal homeobox 1 (Pdx1) gene, which is a key regulator of pancreatic development and insulin expression in beta cells. FH cells possess a considerable replication capacity, and this was further extended by introduction of the gene for the catalytic subunit of human telomerase. Immortalized FH cells expressing Pdx1 activated multiple beta-cell genes, produced and stored considerable amounts of insulin, and released insulin in a regulated manner in response to glucose. When transplanted into hyperglycemic immunodeficient mice, the cells restored and maintained euglycemia for prolonged periods. Quantitation of human C-peptide in the mouse serum confirmed that the glycemia was normalized by the transplanted human cells. This approach offers the potential of a novel source of cells for transplantation into patients with type 1 diabetes.

title: Liver receptor homolog-1 is essential for pregnancy

text: Successful pregnancy requires coordination of an array of signals and factors from multiple tissues. One such element, liver receptor homolog-1 (Lrh-1), is an orphan nuclear receptor that regulates metabolism and hormone synthesis. It is strongly expressed in granulosa cells of ovarian follicles and in the corpus luteum of rodents and humans. Germline ablation of Nr5a2 (also called Lrh-1), the gene coding for Lrh-1, in mice is embryonically lethal at gastrulation. Depletion of Lrh-1 in the ovarian follicle shows that it regulates genes required for both steroid synthesis and ovulation. To study the effects of Lrh-1 on mouse gestation, we genetically disrupted its expression in the corpus luteum, resulting in luteal insufficiency. Hormone replacement permitted embryo implantation but was followed by gestational failure with impaired endometrial decidualization, compromised placental formation, fetal growth retardation and fetal death. Lrh-1 is also expressed in the mouse and human endometrium, and in a primary culture of human endometrial stromal cells, reduction of NR5A2 transcript abundance by RNA interference abrogated decidualization. These findings show that Lrh-1 is necessary for maintenance of the corpus luteum, for promotion of decidualization and for formation of the placenta. It therefore has multiple, indispensible roles in establishing and sustaining pregnancy.

title: Kruppel-like factor 4 is essential for inflammatory monocyte differentiation in vivo.

text: Several members of the Kruppel-like factor (KLF) family of transcription factors play important roles in differentiation, survival, and trafficking of blood and immune cell types. We demonstrate in this study that hematopoietic cells from KLF4(-/-) fetal livers (FL) contained normal numbers of functional hematopoietic progenitor cells, were radioprotective, and performed as well as KLF4(+/+) cells in competitive repopulation assays. However, hematopoietic "KLF4(-/-) chimeras" generated by transplantation of KLF4(-/-) fetal livers cells into lethally irradiated wild-type mice completely lacked circulating inflammatory (CD115(+)Gr1(+)) monocytes, and had reduced numbers of resident (CD115(+)Gr1(-)) monocytes. Although the numbers and function of peritoneal macrophages were normal in KLF4(-/-) chimeras, bone marrow monocytic cells from KLF4(-/-) chimeras expressed lower levels of key trafficking molecules and were more apoptotic. Thus, our in vivo loss-of-function studies demonstrate that KLF4, previously shown to mediate proinflammatory signaling in human macrophages in vitro, is essential for differentiation of mouse inflammatory monocytes, and is involved in the differentiation of resident monocytes. In addition, inducible expression of KLF4 in the HL60 human acute myeloid leukemia cell line stimulated monocytic differentiation and enhanced 12-O-tetradecanoylphorbol 13-acetate induced macrophage differentiation, but blocked all-trans-retinoic acid induced granulocytic differentiation of HL60 cells. The inflammation-selective effects of loss-of-KLF4 and the gain-of-KLF4-induced monocytic differentiation in HL60 cells identify KLF4 as a key regulator of monocytic differentiation and a potential target for translational immune modulation.

title: RANK signals from CD4+3− inducer cells regulate development of Aire-expressing epithelial cells in the thymic medulla

text: Aire-expressing medullary thymic epithelial cells (mTECs) play a key role in preventing autoimmunity by expressing tissue-restricted antigens to help purge the emerging T cell receptor repertoire of self-reactive specificities. Here we demonstrate a novel role for a CD4+3− inducer cell population, previously linked to development of organized secondary lymphoid structures and maintenance of T cell memory in the functional regulation of Aire-mediated promiscuous gene expression in the thymus. CD4+3− cells are closely associated with mTECs in adult thymus, and in fetal thymus their appearance is temporally linked with the appearance of Aire+ mTECs. We show that RANKL signals from this cell promote the maturation of RANK-expressing CD80−Aire− mTEC progenitors into CD80+Aire+ mTECs, and that transplantation of RANK-deficient thymic stroma into immunodeficient hosts induces autoimmunity. Collectively, our data reveal cellular and molecular mechanisms leading to the generation of Aire+ mTECs and highlight a previously unrecognized role for CD4+3−RANKL+ inducer cells in intrathymic self-tolerance.

title: Generation of mice deficient in both KLF3/BKLF and KLF8 reveals a genetic interaction and a role for these factors in embryonic globin gene silencing.

text: Krüppel-like factors 3 and 8 (KLF3 and KLF8) are highly related transcriptional regulators that bind to similar sequences of DNA. We have previously shown that in erythroid cells there is a regulatory hierarchy within the KLF family, whereby KLF1 drives the expression of both the Klf3 and Klf8 genes and KLF3 in turn represses Klf8 expression. While the erythroid roles of KLF1 and KLF3 have been explored, the contribution of KLF8 to this regulatory network has been unknown. To investigate this, we have generated a mouse model with disrupted KLF8 expression. While these mice are viable, albeit with a reduced life span, mice lacking both KLF3 and KLF8 die at around embryonic day 14.5 (E14.5), indicative of a genetic interaction between these two factors. In the fetal liver, Klf3 Klf8 double mutant embryos exhibit greater dysregulation of gene expression than either of the two single mutants. In particular, we observe derepression of embryonic, but not adult, globin expression. Taken together, these results suggest that KLF3 and KLF8 have overlapping roles in vivo and participate in the silencing of embryonic globin expression during development.

title: Hmga2 Promotes Neural Stem Cell Self-Renewal in Young but Not Old Mice by Reducing p16Ink4a and p19Arf Expression

text: Stem cells persist throughout life in diverse tissues by undergoing self-renewing divisions. Self-renewal capacity declines with age, partly because of increasing expression of the tumor suppressor p16(Ink4a). We discovered that the Hmga2 transcriptional regulator is highly expressed in fetal neural stem cells but that expression declines with age. This decrease is partly caused by the increasing expression of let-7b microRNA, which is known to target HMGA2. Hmga2-deficient mice show reduced stem cell numbers and self-renewal throughout the central and peripheral nervous systems of fetal and young-adult mice but not old-adult mice. Furthermore, p16(Ink4a) and p19(Arf) expression were increased in Hmga2-deficient fetal and young-adult stem cells, and deletion of p16(Ink4a) and/or p19(Arf) partially restored self-renewal capacity. let-7b overexpression reduced Hmga2 and increased p16(Ink4a)/p19(Arf) expression. Hmga2 thus promotes fetal and young-adult stem cell self-renewal by decreasing p16(Ink4a)/p19(Arf) expression. Changes in let-7 and Hmga2 expression during aging contribute to the decline in neural stem cell function.

title: EpCAM-regulated intramembrane proteolysis induces a cancer stem cell-like gene signature in hepatitis B virus-infected hepatocytes.

text:
BACKGROUND & AIMS Hepatocytes in which the hepatitis B virus (HBV) is replicating exhibit loss of the chromatin modifying polycomb repressive complex 2 (PRC2), resulting in re-expression of specific, cellular PRC2-repressed genes. Epithelial cell adhesion molecule (EpCAM) is a PRC2-repressed gene, normally expressed in hepatic progenitors, but re-expressed in hepatic cancer stem cells (hCSCs). Herein, we investigated the functional significance of EpCAM re-expression in HBV-mediated hepatocarcinogenesis.
 METHODS Employing molecular approaches (transfections, fluorescence-activated cell sorting, immunoblotting, qRT-PCR), we investigated the role of EpCAM-regulated intramembrane proteolysis (RIP) in HBV replicating cells in vitro, and in liver tumors from HBV X/c-myc mice and chronically HBV infected patients.
 RESULTS EpCAM undergoes RIP in HBV replicating cells, activating canonical Wnt signaling. Transfection of Wnt-responsive plasmid expressing green fluorescent protein (GFP) identified a GFP + population of HBV replicating cells. These GFP+/Wnt+ cells exhibited cisplatin- and sorafenib-resistant growth resembling hCSCs, and increased expression of pluripotency genes NANOG, OCT4, SOX2, and hCSC markers BAMBI, CD44 and CD133. These genes are referred as EpCAM RIP and Wnt-induced hCSC-like gene signature. Interestingly, this gene signature is also overexpressed in liver tumors of X/c-myc bitransgenic mice. Clinically, a group of HBV-associated hepatocellular carcinomas was identified, exhibiting elevated expression of the hCSC-like gene signature and associated with reduced overall survival post-surgical resection.
 CONCLUSIONS The hCSC-like gene signature offers promise as prognostic tool for classifying subtypes of HBV-induced HCCs. Since EpCAM RIP and Wnt signaling drive expression of this hCSC-like signature, inhibition of these pathways can be explored as therapeutic strategy for this subtype of HBV-associated HCCs.    LAY SUMMARY In this study, we provide evidence for a molecular mechanism by which chronic infection by the hepatitis B virus results in the development of poor prognosis liver cancer. Based on this mechanism our results suggest possible therapeutic interventions.

title: A bipotential precursor population for pancreas and liver within the embryonic endoderm.

text: The pancreas emerges independently from dorsal and ventral domains of embryonic gut endoderm. Gene inactivation experiments in mice have identified factors required for dorsal pancreas development, but factors that initiate the ventral pancreas have remained elusive. In this study, we investigated the hypothesis that the emergence of the ventral pancreas is related to the emergence of the liver. We find that the liver and ventral pancreas are specified at the same time and in the same general domain of cells. Using embryo tissue explantation experiments, we find that the default fate of the ventral foregut endoderm is to activate the pancreas gene program. FGF signalling from the cardiac mesoderm diverts this endoderm to express genes for liver instead of those for pancreas. No evidence was found to indicate that the cell type choice for pancreas or liver involves a selection for growth or viability. Cardiac mesoderm or FGF induces the local expression of sonic hedgehog, which in turn is inhibitory to pancreas but not to liver. The bipotential precursor cell population for pancreas and liver in embryonic development and its fate selection by FGF has features that appear to be recapitulated in the adult pancreas and are reflected in the evolution of these organs.

title: Transcriptional repressor Blimp-1 is essential for T cell homeostasis and self-tolerance

text: T cell homeostasis is crucial for a functional immune system, as the accumulation of T cells resulting from lack of regulatory T cells or an inability to shut down immune responses can lead to inflammation and autoimmune pathology. Here we show that Blimp-1, a transcriptional repressor that is a 'master regulator' of terminal B cell differentiation, was expressed in a subset of antigen-experienced CD4+ and CD8+ T cells. Mice reconstituted with fetal liver stem cells expressing a mutant Blimp-1 lacking the DNA-binding domain developed a lethal multiorgan inflammatory disease caused by an accumulation of effector and memory T cells. These data identify Blimp-1 as an essential regulator of T cell homeostasis and suggest that Blimp-1 regulates both B cell and T cell differentiation.

title: Engineered FGF19 eliminates bile acid toxicity and lipotoxicity leading to resolution of steatohepatitis and fibrosis in mice

text: Nonalcoholic fatty liver disease (NAFLD) is an increasingly prevalent chronic liver disease for which no approved therapies are available. Despite intensive research, the cellular mechanisms that mediate NAFLD pathogenesis and progression are poorly understood. Although obesity, diabetes, insulin resistance, and related metabolic syndrome, all consequences of a Western diet lifestyle, are well-recognized risk factors for NAFLD development, dysregulated bile acid metabolism is emerging as a novel mechanism contributing to NAFLD pathogenesis. Notably, NAFLD patients exhibit a deficiency in fibroblast growth factor 19 (FGF19), an endocrine hormone in the gut-liver axis that controls de novo bile acid synthesis, lipogenesis, and energy homeostasis. Using a mouse model that reproduces the clinical progression of human NAFLD, including the development of simple steatosis, nonalcoholic steatohepatitis (NASH), and advanced "burnt-out" NASH with hepatocellular carcinoma, we demonstrate that FGF19 as well as an engineered nontumorigenic FGF19 analogue, M70, ameliorate bile acid toxicity and lipotoxicity to restore liver health. Mass spectrometry-based lipidomics analysis of livers from mice treated with FGF19 or M70 revealed significant reductions in the levels of toxic lipid species (i.e., diacylglycerols, ceramides and free cholesterol) and an increase in levels of unoxidized cardiolipins, an important component of the inner mitochondrial membrane. Furthermore, treatment with FGF19 or M70 rapidly and profoundly reduced levels of liver enzymes, resolved the histologic features of NASH, and enhanced insulin sensitivity, energy homeostasis, and lipid metabolism. Whereas FGF19 induced hepatocellular carcinoma formation following prolonged exposure in these mice, animals expressing M70 showed no evidence of liver tumorigenesis in this model. Conclusion: We have engineered an FGF19 hormone that is capable of regulating multiple pathways to deliver antisteatotic, anti-inflammatory, and antifibrotic activities and that represents a potentially promising therapeutic for patients with NASH. (Hepatology Communications 2017;1:1024-1042).

title: Aberrant luminal progenitors as the candidate target population for basal tumor development in BRCA1 mutation carriers

text: Basal-like breast cancers arising in women carrying mutations in the BRCA1 gene, encoding the tumor suppressor protein BRCA1, are thought to develop from the mammary stem cell. To explore early cellular changes that occur in BRCA1 mutation carriers, we have prospectively isolated distinct epithelial subpopulations from normal mammary tissue and preneoplastic specimens from individuals heterozygous for a BRCA1 mutation. We describe three epithelial subsets including basal stem/progenitor, luminal progenitor and mature luminal cells. Unexpectedly, we found that breast tissue from BRCA1 mutation carriers harbors an expanded luminal progenitor population that shows factor-independent growth in vitro. Moreover, gene expression profiling revealed that breast tissue heterozygous for a BRCA1 mutation and basal breast tumors were more similar to normal luminal progenitor cells than any other subset, including the stem cell–enriched population. The c-KIT tyrosine kinase receptor (encoded by KIT) emerged as a key marker of luminal progenitor cells and was more highly expressed in BRCA1-associated preneoplastic tissue and tumors. Our findings suggest that an aberrant luminal progenitor population is a target for transformation in BRCA1-associated basal tumors .

title: Novel roles for KLF1 in erythropoiesis revealed by mRNA-seq.

text: KLF1 (formerly known as EKLF) regulates the development of erythroid cells from bi-potent progenitor cells via the transcriptional activation of a diverse set of genes. Mice lacking Klf1 die in utero prior to E15 from severe anemia due to the inadequate expression of genes controlling hemoglobin production, cell membrane and cytoskeletal integrity, and the cell cycle. We have recently described the full repertoire of KLF1 binding sites in vivo by performing KLF1 ChIP-seq in primary erythroid tissue (E14.5 fetal liver). Here we describe the KLF1-dependent erythroid transcriptome by comparing mRNA-seq from Klf1(+/+) and Klf1(-/-) erythroid tissue. This has revealed novel target genes not previously obtainable by traditional microarray technology, and provided novel insights into the function of KLF1 as a transcriptional activator. We define a cis-regulatory module bound by KLF1, GATA1, TAL1, and EP300 that coordinates a core set of erythroid genes. We also describe a novel set of erythroid-specific promoters that drive high-level expression of otherwise ubiquitously expressed genes in erythroid cells. Our study has identified two novel lncRNAs that are dynamically expressed during erythroid differentiation, and discovered a role for KLF1 in directing apoptotic gene expression to drive the terminal stages of erythroid maturation.