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    N-Methyl-D-Aspartate Receptor Hypofunction in Meg-01 Cells Reveals a Role for Intracellular Calcium Homeostasis in Balancing Megakaryocytic-Erythroid Differentiation
    (Thieme Gruppe, 2020-04-14) Hearn JI; Green TN; Chopra M; Nursalim YNS; Ladvanszky L; Knowlton N; Blenkiron C; Poulsen RC; Singleton DC; Bohlander SK; Kalev-Zylinska ML
    The release of calcium ions (Ca2+) from the endoplasmic reticulum (ER) and related store-operated calcium entry (SOCE) regulate maturation of normal megakaryocytes. The N-methyl-D-aspartate (NMDA) receptor (NMDAR) provides an additional mechanism for Ca2+ influx in megakaryocytic cells, but its role remains unclear. We created a model of NMDAR hypofunction in Meg-01 cells using CRISPR-Cas9 mediated knockout of the GRIN1 gene, which encodes an obligate, GluN1 subunit of the NMDAR. We found that compared with unmodified Meg-01 cells, Meg-01-GRIN1 -/- cells underwent atypical differentiation biased toward erythropoiesis, associated with increased basal ER stress and cell death. Resting cytoplasmic Ca2+ levels were higher in Meg-01-GRIN1 -/- cells, but ER Ca2+ release and SOCE were lower after activation. Lysosome-related organelles accumulated including immature dense granules that may have contributed an alternative source of intracellular Ca2+. Microarray analysis revealed that Meg-01-GRIN1 -/- cells had deregulated expression of transcripts involved in Ca2+ metabolism, together with a shift in the pattern of hematopoietic transcription factors toward erythropoiesis. In keeping with the observed pro-cell death phenotype induced by GRIN1 deletion, memantine (NMDAR inhibitor) increased cytotoxic effects of cytarabine in unmodified Meg-01 cells. In conclusion, NMDARs comprise an integral component of the Ca2+ regulatory network in Meg-01 cells that help balance ER stress and megakaryocytic-erythroid differentiation. We also provide the first evidence that megakaryocytic NMDARs regulate biogenesis of lysosome-related organelles, including dense granules. Our results argue that intracellular Ca2+ homeostasis may be more important for normal megakaryocytic and erythroid differentiation than currently recognized; thus, modulation may offer therapeutic opportunities.
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    Cerebrovascular and cardiovascular responses to the Valsalva manoeuvre during hyperthermia.
    (John Wiley & Sons Ltd on behalf of Scandinavian Society of Clinical Physiology and Nuclear Medicine, 2023-06-18) Perry BG; Korad S; Mündel T
    BACKGROUND: During hyperthermia, the perturbations in mean arterial blood pressure (MAP) produced by the Valsalva manoeuvre (VM) are more severe. However, whether these more severe VM-induced changes in MAP are translated to the cerebral circulation during hyperthermia is unclear. METHODS: Healthy participants (n = 12, 1 female, mean ± SD: age 24 ± 3 years) completed a 30 mmHg (mouth pressure) VM for 15 s whilst supine during normothermia and mild hyperthermia. Hyperthermia was induced passively using a liquid conditioning garment with core temperature measured via ingested temperature sensor. Middle cerebral artery blood velocity (MCAv) and MAP were recorded continuously during and post-VM. Tieck's autoregulatory index was calculated from the VM responses, with pulsatility index, an index of pulse velocity (pulse time) and mean MCAv (MCAvmean ) also calculated. RESULTS: Passive heating significantly raised core temperature from baseline (37.9 ± 0.2 vs. 37.1 ± 0.1°C at rest, p < 0.01). MAP during phases I through III of the VM was lower during hyperthermia (interaction effect p < 0.01). Although an interaction effect was observed for MCAvmean (p = 0.02), post-hoc differences indicated only phase IIa was lower during hyperthermia (55 ± 12 vs. 49.3 ± 8 cm s- 1 for normothermia and hyperthermia, respectively, p = 0.03). Pulsatility index was increased 1-min post-VM in both conditions (0.71 ± 0.11 vs. 0.76 ± 0.11 for pre- and post-VM during normothermia, respectively, p = 0.02, and 0.86 ± 0.11 vs. 0.99 ± 0.09 for hyperthermia p < 0.01), although for pulse time only main effects of time (p < 0.01), and condition (p < 0.01) were apparent. CONCLUSION: These data indicate that the cerebrovascular response to the VM is largely unchanged by mild hyperthermia.
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    Role of a local transcription factor in governing cellular carbon/nitrogen homeostasis in Pseudomonas fluorescens
    (Oxford University Press on behalf of Nucleic Acids Research, 2021-04-06) Naren N; Zhang X-X
    Autoactivation of two-component systems (TCSs) can increase the sensitivity to signals but inherently cause a delayed response. Here, we describe a unique negative feedback mechanism enabling the global NtrB/NtrC regulator to rapidly respond to nitrogen starvation over the course of histidine utilization (hut) in Pseudomonas fluorescens. NtrBC directly activates transcription of hut genes, but overexpression will produce excess ammonium leading to NtrBC inactivation. To prevent this from occurring, the histidine-responsive repressor HutC fine-tunes ntrBC autoactivation: HutC and NtrC bind to the same operator site in the ntrBC promoter. This newly discovered low-affinity binding site shows little sequence similarity with the consensus sequence that HutC recognizes for substrate-specific induction of hut operons. A combination of genetic and transcriptomic analysis indicated that both ntrBC and hut promoter activities cannot be stably maintained in the ΔhutC background when histidine fluctuates at high concentrations. Moreover, the global carbon regulator CbrA/CbrB is involved in directly activating hut transcription while de-repressing hut translation via the CbrAB-CrcYZ-Crc/Hfq regulatory cascade. Together, our data reveal that the local transcription factor HutC plays a crucial role in governing NtrBC to maintain carbon/nitrogen homeostasis through the complex interactions between two TCSs (NtrBC and CbrAB) at the hut promoter.
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    Cerebral autoregulation across the menstrual cycle in eumenorrheic women
    (Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society, 2022-05-06) Korad S; Mündel T; Fan J-L; Perry BG
    There is emerging evidence that ovarian hormones play a significant role in the lower stroke incidence observed in pre-menopausal women compared with men. However, the role of ovarian hormones in cerebrovascular regulation remains to be elucidated. We examined the blood pressure-cerebral blood flow relationship (cerebral autoregulation) across the menstrual cycle in eumenorrheic women (n = 12; mean ± SD: age, 31 ± 7 years). Participants completed sit-to-stand and Valsalva maneuvers (VM, mouth pressure of 40 mmHg for 15 s) during the early follicular (EF), late follicular (LF), and mid-luteal (ML) menstrual cycle phases, confirmed by serum measurement of progesterone and 17β-estradiol. Middle cerebral artery blood velocity (MCAv), arterial blood pressure and partial pressure of end-tidal carbon dioxide were measured. Cerebral autoregulation was assessed by transfer function analysis during spontaneous blood pressure oscillations, rate of regulation (RoR) during sit-to-stand maneuvers, and Tieck's autoregulatory index during VM phases II and IV (AI-II and AI-IV, respectively). Resting mean MCAv (MCAvmean ), blood pressure, and cerebral autoregulation were unchanged across the menstrual cycle (all p > 0.12). RoR tended to be different (EF, 0.25 ± 0.06; LF; 0.19 ± 0.04; ML, 0.18 ± 0.12 sec-1 ; p = 0.07) and demonstrated a negative relationship with 17β-estradiol (R2  = 0.26, p = 0.02). No changes in AI-II (EF, 1.95 ± 1.20; LF, 1.67 ± 0.77 and ML, 1.20 ± 0.55) or AI-IV (EF, 1.35 ± 0.21; LF, 1.27 ± 0.26 and ML, 1.20 ± 0.2) were observed (p = 0.25 and 0.37, respectively). Although, a significant interaction effect (p = 0.02) was observed for the VM MCAvmean response. These data indicate that the menstrual cycle has limited impact on cerebrovascular autoregulation, but individual differences should be considered.
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    Effect of Faecalibacterium prausnitzii on intestinal barrier function and immune homeostasis : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Science, Massey University, Manawatū, New Zealand
    (Massey University, 2017) Maier, Eva
    Various gastrointestinal (GI) diseases, for example inflammatory bowel disease, are linked to impaired barrier function, chronic inflammation and dysbiosis of the resident microbiota. Faecalibacterium prausnitzii, an abundant obligate anaerobe of the healthy human microbiota, has reduced abundance in the GI tract of people with these diseases, and has been suggested to exert beneficial effects. Only a few studies have investigated its mechanisms of action, partly due to the difficulty of co-culturing live obligate anaerobes with oxygen-requiring human cells. The novel apical anaerobic co-culture model used in this study allows this co-culture through the separation of anaerobic and aerobic compartments. This model was used to investigate the effects of live F. prausnitzii (strains A2-165, ATCC 27768 and HTF-F) on intestinal barrier integrity, measured by transepithelial electrical resistance (TEER) of the intestinal epithelial cell line Caco-2, and on immune homeostasis, specifically on Toll-like receptor (TLR) activation. Method development was required to adapt these assays to the novel model and to optimise the growth of F. prausnitzii co-cultured with Caco-2 cells and TLR-expressing cell lines while maintaining their viabilities. Firstly, the optimised co-culture conditions were used to determine the effect of the three F. prausnitzii strains on barrier integrity of healthy and tumour necrosis factor alpha (TNF-α) treated Caco-2 cells. Live and growing F. prausnitzii did not alter the TEER across healthy Caco-2 cells. However, under TNF-α mediated inflammatory conditions, dead F. prausnitzii decreased TEER, whereas live bacteria maintained TEER. Secondly, the TLR activation assay was adapted to be carried out in the novel model. Using the adapted assay conditions it was determined that live F. prausnitzii induced greater TLR2 and TLR2/6 activation than dead F. prausnitzii. Collectively, these results indicate greater immuno-stimulatory effects of live F. prausnitzii, via TLR2 activation, and this effect is potentially linked to its barrier maintaining properties, because previous research showed enhancement of barrier integrity induced by TLR2 signalling. This new knowledge contributes to the understanding of how F. prausnitzii may maintain immune homeostasis in the GI tract. Unravelling the biological mechanisms used by prevalent species of the human microbiota, such as F. prausnitzii, will ultimately allow better comprehension of microbial regulation of GI function.