Linker histone H1 is essential for Drosophila development, the establishment of pericentric heterochromatin, and a normal polytene chromosome structure.

We generated mutant alleles of Drosophila melanogaster in which expression of the linker histone H1 can be down-regulated over a wide range by RNAi. When the H1 protein level is reduced to approximately 20% of the level in wild-type larvae, lethality occurs in the late larval - pupal stages of development. Here we show that H1 has an important function in gene regulation within or near heterochromatin. It is a strong dominant suppressor of position effect variegation (PEV). Similar to other suppressors of PEV, H1 is simultaneously involved in both the repression of euchromatic genes brought to the vicinity of pericentric heterochromatin and the activation of heterochromatic genes that depend on their pericentric localization for maximal transcriptional activity. Studies of H1-depleted salivary gland polytene chromosomes show that H1 participates in several fundamental aspects of chromosome structure and function. First, H1 is required for heterochromatin structural integrity and the deposition or maintenance of major pericentric heterochromatin-associated histone marks, including H3K9Me(2) and H4K20Me(2). Second, H1 also plays an unexpected role in the alignment of endoreplicated sister chromatids. Finally, H1 is essential for organization of pericentric regions of all polytene chromosomes into a single chromocenter. Thus, linker histone H1 is essential in Drosophila and plays a fundamental role in the architecture and activity of chromosomes in vivo.

Directional X Chromosome Skewing of White Blood Cells from Subjects with Heterozygous Mosaicism for the Variant IRAK1 Haplotype.

Random X chromosome (ChrX) inactivation and consequent cellular mosaicism for the active ChrXs in white blood cells (WBCs) is unique to females and may contribute to sex-biased modulation of the innate immune response. Polymorphic differences between the two parental ChrXs may result in ChrX skewing of circulating WBCs (ChrX inactivation-ratio (XCI) > 3) driven by differences in stem cell selection and activity in the bone marrow or WBC trafficking at the periphery. Independent of the mechanism, ChrX skewing may result in genotype-phenotype discrepancies. This study aimed to develop an allele-specific assay and test its applicability in clinical samples to determine the "direction" of ChrX skewing in the variant IRAK1 haplotype, a common X-linked polymorphism with major clinical impacts. Because alternative splice variants of IRAK1 are also produced in the region surrounding the critical single-nucleotide polymorphism (SNP, rs1059703), we also tested the abundance of alternative splice variant IRAK1 transcripts. The expression of splice variants IRAK1-B and IRAK1-C was about 30 and 50% of the full-length (IRAK1-A) in WBCs from healthy subjects (n = 53). IRAK1-A, B, and C showed about 30% lower expression level in males (n = 25) than females (n = 28). By contrast, the expression levels of IRAK1-A, B, and C were not affected by the variant IRAK1 haplotype in either sex. Allele-specific primers generated WT and variant-IRAK1 amplicons with high selectivity, and on average produced about half the expression levels of each transcript in heterozygous IRAK1-mosaic females. Because injury was shown to induce de novo ChrX skewing of WBCs, we tested the directional XCI ratio changes in WBC in a sample of trauma patients heterozygous for the variant IRAK1 haplotype (n = 18). Using the allele-specific assay in combination with the DNA methylation status at the polymorphic HUMARA locus, we found that at admission, about 60% the patients presented XCI ratios skewed toward WBCs with active ChrXs carrying the variant-IRAK1 similar to healthy controls. De novo, trauma-induced XCI ratio changes showed increased extravasation of the more abundant mosaic WBC subset without reversal in the direction of ChrX skewing during the injury course.

X-Linked IRAK1 Polymorphism is Associated with Sex-Related Differences in Polymorphonuclear Granulocyte and Monocyte Activation and Response Variabilities.

Common X-linked genetic polymorphisms are expected to alter cellular responses affecting males and females differently through sex-linked inheritance pattern as well as X chromosome (ChrX) mosaicism and associated ChrX skewing, which is unique to females. We tested this hypothesis in ex vivo lipopolysaccharide and phorbol ester-stimulated polymorphonuclear granulocytes (PMNs) and monocytes from healthy volunteers (n = 51). Observations were analyzed after stratification by sex alone or the presence of variant IRAK1 haplotype a common X-linked polymorphism with previously demonstrated major clinical impacts. Upon cell activation, CD11b, CD45, CD66b, CD63, and CD14 expression was markedly and similarly elevated in healthy males and females. By contrast, PMN and monocyte activation measured by CD11b, CD66b, and CD63 was increased in variant-IRAK1 subjects as compared with WT. Stratification by IRAK1 genotype and sex showed similar cell activation effect on variant-IRAK1 subjects and an intermediate degree of cell activation in heterozygous mosaic females. The increased membrane expression of these proteins in variant-IRAK1 subjects was associated with similar or increased intersubject but uniformly decreased intrasubject cell response variabilities as compared with WT. We also tested white blood cell ChrX skewing in the healthy cohort as well as in a sample of female trauma patients (n = 201). ChrX inactivation ratios were similar in IRAK1 WT, variant, and heterozygous healthy subjects. Trauma patients showed a trend of blunted ChrX skewing at admission in homozygous variant-IRAK1 and heterozygous mosaic-IRAK1 female subjects as compared with WT. Trauma-induced de novo ChrX skewing was also depressed in variant-IRAK1 and mosaic-IRAK1 female trauma patients as compared with WT. Our study indicates that augmented PMN and monocyte activation in variant-IRAK1 subjects is accompanied by decreased intrasubject cellular variability and blunted de novo ChrX skewing in response to trauma. A more pronounced cell activation of PMNs and monocytes accompanied by decreased response variabilities in variant-IRAK1 subjects may be a contributing mechanism affecting the course of sepsis and trauma and may also impact sex-based outcome differences due to its X-linked inheritance pattern and high prevalence.

Transmission of olfactory information between three populations of neurons in the antennal lobe of the fly.

Three classes of neurons form synapses in the antennal lobe of Drosophila, the insect counterpart of the vertebrate olfactory bulb: olfactory receptor neurons, projection neurons, and inhibitory local interneurons. We have targeted a genetically encoded optical reporter of synaptic transmission to each of these classes of neurons and visualized population responses to natural odors. The activation of an odor-specific ensemble of olfactory receptor neurons leads to the activation of a symmetric ensemble of projection neurons across the glomerular synaptic relay. Virtually all excited glomeruli receive inhibitory input from local interneurons. The extent, odor specificity, and partly interglomerular origin of this input suggest that inhibitory circuits assemble combinatorially during odor presentations. These circuits may serve as dynamic templates that extract higher order features from afferent activity patterns.

Differential effects of chronic intermittent and chronic constant hypoxia on postnatal growth and development.

Exposure to chronic constant or intermittent hypoxia (CCH or CIH) may have different effects on growth and development in early life. In this work, we exposed postnatal day 2 (P2) CD1 mice to CCH or CIH (11% O2) for 4 weeks and examined the effect of hypoxia on body and organ growth until P30. Regression analysis showed that weight increased in control, CCH and CIH cohorts with age with r2 values of 0.99, 0.97, and 0.94, respectively. Between days 2 and 30, slopes were 0.93+/-0.057, 0.76+/-0.108, and 0.63+/-0.061 (g/day, means+/-SEM) for control, CIH, and CCH, respectively and significantly different from each other (P<0.001). The slopes between P2 and P16 were 0.78+/-0.012, 0.46+/-0.002, and 0.47+/-0.019 for control, CCH and CIH, respectively. From P16 to 30, slopes were 1.12+/-0.033, 1.09+/-0.143, and 0.82+/-0.08 for control, CIH, and CCH, respectively with no significant difference from each other, suggesting a catch-up growth in the latter part of the hypoxic period. Slower weight gain resulted in a 12% and 23% lower body weight in CIH and CCH mice (P<0.001) by P30. Lung/body ratios were 0.010, 0.015, 0.015 for control, CIH, and CCH at P30, respectively. The decrease in liver, kidney, and brain weight were greater in CCH than CIH. Smaller liver weight was shown to be due to a reduction in cell size and cell number. Liver in CIH and CCH mice showed a 5% and 10% reduction in cell size (P<0.05) and a reduction of 28% in cell number (P<0.001) at P30. In contrast, CCH and CIH heart weight was 13% and 33% greater than control at P30 (P<0.05), respectively. This increase in the heart weight was due to an increase in the size of cardiomyocytes which showed an increase of 12% and 14% (P<0.001) for CIH and CCH, respectively as compared to control. Brain weight was 0.48 and 0.46 g for CIH and CCH, respectively (95% and 92% of normal). We concluded that (a) CIH and CCH follow different body and organ growth patterns; (b) mostly with CCH, the liver and kidneys are reduced in size in a proportionate way to body size but heart, lung, and brain are either spared or increased in size compared to body weight; and (c) the decrease in liver is secondary mostly to a decrease in cell number.

Intraoperative Low-frequency Electroacupuncture under General Anesthesia Improves Postoperative Recovery in a Randomized Trial.

Neuronal stimulation improves physiological responses to infection and trauma, but the clinical potential of this strategy is unknown. We hypothesized that transdermal neural stimulation through low-frequency electroacupuncture might control the immune responses to surgical trauma and expedite the postoperative recovery. However, the efficiency of electroacupuncture is questioned due to the placebo effect. Here, electroacupuncture was performed on anesthetized patients to avoid any placebo. This is a prospective double-blinded pilot trial to determine whether intraoperative electroacupuncture on anesthetized patients improves postoperative recovery. Patients with electroacupuncture required 60% less postoperative analgesic, even they had pain scores similar to those in the control patients. Electroacupuncture prevented postoperative hyperglycemia and attenuated serum adrenocorticotropic hormone in the older and heavier group of patients. From an immunological perspective, electroacupuncture did not affect the protective immune responses to surgical trauma, including the induction of interleukin-6 and interleukin-10. The most significant immunological effect of electroacupuncture was enhancing transforming growth factor-ß1 production during surgery in the older and lighter group of patients. These results suggest that intraoperative electroacupuncture on anesthetized patients can reduce postoperative use of analgesics and improve immune and stress responses to surgery.

Experimental selection for Drosophila survival in extremely low O(2) environment.

BACKGROUND: Cellular hypoxia, if severe enough, results usually in injury or cell death. Our research in this area has focused on the molecular mechanisms underlying hypoxic tissue injury to explore strategies to prevent injury or enhance tolerance. The current experiments were designed to determine the genetic basis for adaptation to long term low O(2) environments. METHODOLOGY/PRINCIPAL FINDINGS: With long term experimental selection over many generations, we obtained a Drosophila melanogaster strain that can live perpetually in extremely low, normally lethal, O(2) condition (as low as 4% O(2)). This strain shows a dramatic phenotypic divergence from controls, including a decreased recovery time from anoxic stupor, a higher rate of O(2 )consumption in hypoxic conditions, and a decreased body size and mass due to decreased cell number and size. Expression arrays showed that about 4% of the Drosophila genome altered in expression and about half of the alteration was down-regulation. The contribution of some altered transcripts to hypoxia tolerance was examined by testing the survival of available corresponding P-element insertions (and their excisions) under extremely low O(2) conditions. We found that down-regulation of several candidate genes including Best1, broad, CG7102, dunce, lin19-like and sec6 conferred severe hypoxia tolerance in Drosophila. CONCLUSIONS/SIGNIFICANCE: We have identified a number of genes that play an important role in the survival of a selected Drosophila strain in extremely low O(2) conditions, selected by decreasing O(2) availability over many generations. Because of conservation of pathways, we believe that such genes are critical in hypoxia adaptation in physiological or pathological conditions not only in Drosophila but also in mammals.