Direct and Indirect Control of the Initiation of Meiotic Recombination by DNA Damage Checkpoint Mechanisms in Budding Yeast

Meiotic recombination plays an essential role in the proper segregation of chromosomes at meiosis I in many sexually reproducing organisms. Meiotic recombination is initiated by the scheduled formation of genome-wide DNA double-strand breaks (DSBs). The timing of DSB formation is strictly controlled because unscheduled DSB formation is detrimental to genome integrity. Here, we investigated the role of DNA damage checkpoint mechanisms in the control of meiotic DSB formation using budding yeast. By using recombination defective mutants in which meiotic DSBs are not repaired, the effect of DNA damage checkpoint mutations on DSB formation was evaluated. The Tel1 (ATM) pathway mainly responds to unresected DSB ends, thus the sae2 mutant background in which DSB ends remain intact was employed. On the other hand, the Mec1 (ATR) pathway is primarily used when DSB ends are resected, thus the rad51 dmc1 double mutant background was employed in which highly resected DSBs accumulate. In order to separate the effect caused by unscheduled cell cycle progression, which is often associated with DNA damage checkpoint defects, we also employed the ndt80 mutation which permanently arrests the meiotic cell cycle at prophase I. In the absence of Tel1, DSB formation was reduced in larger chromosomes (IV, VII, II and XI) whereas no significant reduction was found in smaller chromosomes (III and VI). On the other hand, the absence of Rad17 (a critical component of the ATR pathway) lead to an increase in DSB formation (chromosomes VII and II were tested). We propose that, within prophase I, the Tel1 pathway facilitates DSB formation, especially in bigger chromosomes, while the Mec1 pathway negatively regulates DSB formation. We also identified prophase I exit, which is under the control of the DNA damage checkpoint machinery, to be a critical event associated with down-regulating meiotic DSB formation.     

The Landscape of Candidate Driver Genes Differs between Male and Female Breast Cancer

The rapidly growing collection of diverse genome-scale data from multiple tumor types sheds light on various aspects of the underlying tumor biology. With the objective to identify genes of importance for breast tumorigenesis in men and to enable comparisons with genes important for breast cancer development in women, we applied the computational framework COpy Number and EXpression In Cancer (CONEXIC) to detect candidate driver genes among all altered passenger genes. Unique to this approach is that each driver gene is associated with several gene modules that are believed to be altered by the driver. Thirty candidate drivers were found in the male breast cancers and 67 in the female breast cancers. We identified many known drivers of breast cancer and other types of cancer, in the female dataset (e.g. GATA3, CCNE1, GRB7, CDK4). In contrast, only three known cancer genes were found among male breast cancers; MAP2K4, LHP, and ZNF217. Many of the candidate drivers identified are known to be involved in processes associated with tumorigenesis, including proliferation, invasion and differentiation. One of the modules identified in male breast cancer was regulated by THY1, a gene involved in invasion and related to epithelial-mesenchymal transition. Furthermore, men with THY1 positive breast cancers had significantly inferior survival. THY1 may thus be a promising novel prognostic marker for male breast cancer. Another module identified among male breast cancers, regulated by SPAG5, was closely associated with proliferation. Our data indicate that male and female breast cancers display highly different landscapes of candidate driver genes, as only a few genes were found in common between the two. Consequently, the pathobiology of male breast cancer may differ from that of female breast cancer and can be associated with differences in prognosis; men diagnosed with breast cancer may consequently require different management and treatment strategies than women.     

Chronic Alterations in Monoaminergic Cells in the Locus Coeruleus in Orexin Neuron-Ablated Narcoleptic Mice

Narcolepsy patients often suffer from insomnia in addition to excessive daytime sleepiness. Narcoleptic animals also show behavioral instability characterized by frequent transitions between all vigilance states, exhibiting very short bouts of NREM sleep as well as wakefulness. The instability of wakefulness states in narcolepsy is thought to be due to deficiency of orexins, neuropeptides produced in the lateral hypothalamic neurons, which play a highly important role in maintaining wakefulness. However, the mechanism responsible for sleep instability in this disorder remains to be elucidated. Because firing of orexin neurons ceases during sleep in healthy animals, deficiency of orexins does not explain the abnormality of sleep. We hypothesized that chronic compensatory changes in the neurophysiologica activity of the locus coeruleus (LC) and dorsal raphe (DR) nucleus in response to the progressive loss of endogenous orexin tone underlie the pathological regulation of sleep/wake states. To evaluate this hypothesis, we examined firing patterns of serotonergic (5-HT) neurons and noradrenergic (NA) neurons in the brain stem, two important neuronal populations in the regulation of sleep/wakefulness states. We recorded single-unit activities of 5-HT neurons and NA neurons in the DR nucleus and LC of orexin neuron-ablated narcoleptic mice. We found that while the firing pattern of 5-HT neurons in narcoleptic mice was similar to that in wildtype mice, that of NA neurons was significantly different from that in wildtype mice. In narcoleptic mice, NA neurons showed a higher firing frequency during both wakefulness and NREM sleep as compared with wildtype mice. In vitro patch-clamp study of NA neurons of narcoleptic mice suggested a functional decrease of GABAergic input to these neurons. These alterations might play roles in the sleep abnormality in narcolepsy.     

Localization of Microfibrillar-Associated Protein 4 (MFAP4) in Human Tissues: Clinical Evaluation of Serum MFAP4 and Its Association with Various Cardiovascular Conditions

Microfibrillar-associated protein 4 (MFAP4) is located in the extracellular matrix (ECM). We sought to identify tissues with high levels of MFAP4 mRNA and MFAP4 protein expression. Moreover, we aimed to evaluate the significance of MFAP4 as a marker of cardiovascular disease (CVD) and to correlate MFAP4 with other known ECM markers, such as fibulin-1, osteoprotegerin (OPG), and osteopontin (OPN). Quantitative real-time PCR demonstrated that MFAP4 mRNA was more highly expressed in the heart, lung, and intestine than in other elastic tissues. Immunohistochemical studies demonstrated high levels of MFAP4 protein mainly at sites rich in elastic fibers and within blood vessels in all tissues investigated. The AlphaLISA technique was used to determine serum MFAP4 levels in a clinical cohort of 172 patients consisting of 5 matched groups with varying degrees of CVD: 1: patients with ST elevation myocardial infarction (STEMI), 2: patients with non-STEMI, 3: patients destined for vascular surgery because of various atherosclerotic diseases (stable atherosclerotic disease), 4: apparently healthy individuals with documented coronary artery calcification (CAC-positive), and 5: apparently healthy individuals without signs of coronary artery calcification (CAC-negative). Serum MFAP4 levels were significantly lower in patients with stable atherosclerotic disease than CAC-negative individuals (p<0.05). Furthermore, lower serum MFAP4 levels were present in patients with stable atherosclerotic disease compared with STEMI and non-STEMI patients (p<0.05). In patients with stable atherosclerotic disease, positive correlations between MFAP4 and both fibulin-1 (ρ = 0.50; p = 0.0244) and OPG (ρ = 0.62; p = 0.0014) were found. Together, these results indicate that MFAP4 is mainly located in elastic fibers and is highly expressed in blood vessels. The present study suggests that serum MFAP4 varies in groups of patients with different cardiovascular conditions. Further studies are warranted to describe the role of serum MFAP4 as a biomarker of stable atherosclerotic disease.     

Characterization of Rice Black-Streaked Dwarf Virus- and Rice Stripe Virus-Derived siRNAs in Singly and Doubly Infected Insect Vector Laodelphax striatellus

Replication of RNA viruses in insect cells triggers an antiviral defense that is mediated by RNA interference (RNAi) which generates viral-derived small interfering RNAs (siRNAs). However, it is not known whether an antiviral RNAi response is also induced in insects by reoviruses, whose double-stranded RNA genome replication is thought to occur within core particles. Deep sequencing of small RNAs showed that when the small brown planthopper (Laodelphax striatellus) was infected by Rice black-streaked dwarf virus (RBSDV) (Reoviridae; Fijivirus), more viral-derived siRNAs accumulated than when the vector insect was infected by Rice stripe virus (RSV), a negative single-stranded RNA virus. RBSDV siRNAs were predominantly 21 and 22 nucleotides long and there were almost equal numbers of positive and negative sense. RBSDV siRNAs were frequently generated from hotspots in the 5′- and 3′-terminal regions of viral genome segments but these hotspots were not associated with any predicted RNA secondary structures. Under laboratory condition, L. striatellus can be infected simultaneously with RBSDV and RSV. Double infection enhanced the accumulation of particular genome segments but not viral coat protein of RBSDV and correlated with an increase in the abundance of siRNAs derived from RBSDV. The results of this study suggest that reovirus replication in its insect vector potentially induces an RNAi-mediated antiviral response.     

Characteristic Cerebrospinal Fluid Cytokine/Chemokine Profiles in Neuromyelitis Optica,Relapsing Remitting or Primary Progressive Multiple Sclerosis

Background

Differences in cytokine/chemokine profiles among patients with neuromyelitis optica (NMO), relapsing remitting multiple sclerosis (RRMS), and primary progressive MS (PPMS), and the relationships of these profiles with clinical and neuroimaging features are unclear. A greater understanding of these profiles may help in differential diagnosis.

Methods/Principal Findings

We measured 27 cytokines/chemokines and growth factors in CSF collected from 20 patients with NMO, 26 with RRMS, nine with PPMS, and 18 with other non-inflammatory neurological diseases (OND) by multiplexed fluorescent bead-based immunoassay. Interleukin (IL)-17A, IL-6, CXCL8 and CXCL10 levels were significantly higher in NMO patients than in OND and RRMS patients at relapse, while granulocyte-colony stimulating factor (G-CSF) and CCL4 levels were significantly higher in NMO patients than in OND patients. In NMO patients, IL-6 and CXCL8 levels were positively correlated with disability and CSF protein concentration while IL-6, CXCL8, G-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IFN-γ were positively correlated with CSF neutrophil counts at the time of sample collection. In RRMS patients, IL-6 levels were significantly higher than in OND patients at the relapse phase while CSF cell counts were negatively correlated with the levels of CCL2. Correlation coefficients of cytokines/chemokines in the relapse phase were significantly different in three combinations, IL-6 and GM-CSF, G-CSF and GM-CSF, and GM-CSF and IFN-γ, between RRMS and NMO/NMOSD patients. In PPMS patients, CCL4 and CXCL10 levels were significantly higher than in OND patients.

Conclusions

Our findings suggest distinct cytokine/chemokine alterations in CSF exist among NMO, RRMS and PPMS. In NMO, over-expression of a cluster of Th17- and Th1-related proinflammatory cytokines/chemokines is characteristic, while in PPMS, increased CCL4 and CXCL10 levels may reflect on-going low grade T cell and macrophage/microglia inflammation in the central nervous system. In RRMS, only a mild elevation of proinflammatory cytokines/chemokines was detectable at relapse.     

Effects of Zinc Acetate on Serum Zinc Concentrations in Chronic Liver Diseases: a Multicenter,Double-Blind,Randomized, Placebo-Controlled Trial and a Dose Adjustment Trial

Biological Trace Element Research - The essential trace element zinc maintains liver functions. Liver diseases can alter overall zinc concentrations, and hypozincemia is associated with various...     

α-Amylase expressed in human small intestinal epithelial cells is essential for cell proliferation and differentiation

α-Amylase, which plays an essential role in starch degradation, is expressed mainly in the pancreas and salivary glands. Human α-amylase is also detected in other tissues, but it is unclear whether the α-amylase is endogenously expressed in each tissue or mixed exogenously with one expressed by the pancreas or salivary glands. Furthermore, the biological significance of these α-amylases detected in tissues other than the pancreas and salivary glands has not been elucidated. We discovered that human α-amylase is expressed in intestinal epithelial cells and analyzed the effects of suppressing α-amylase expression. α-Amylase was found to be expressed at the second-highest messenger RNA level in the duodenum in human normal tissues after the pancreas. α-Amylase was detected in the cell extract of Caco-2 intestinal epithelial cells but not secreted into the culture medium. The amount of α-amylase expressed increased depending on the length of the culture of Caco-2 cells, suggesting that α-amylase is expressed in small intestine epithelial cells rather than the colon because the cells differentiate spontaneously upon reaching confluence in culture to exhibit the characteristics of small intestinal epithelial cells rather than colon cells. The α-amylase expressed in Caco-2 cells had enzymatic activity and was identified as AMY2B, one of the two isoforms of pancreatic α-amylase. The suppression of α-amylase expression by small interfering RNA inhibited cell differentiation and proliferation. These results demonstrate for the first time that α-amylase is expressed in human intestinal epithelial cells and affects cell proliferation and differentiation. This α-amylase may induce the proliferation and differentiation of small intestine epithelial cells, supporting a rapid turnover of cells to maintain a healthy intestinal lumen.