Therapeutic Opportunities of GPBAR1 in Cholestatic Diseases

GPBAR1, a transmembrane G protein-coupled receptor for bile acids, is widely expressed in multiple tissues in humans and rodents. In recent years, GPBAR1 has been thought to play an important role in bile homeostasis, metabolism and inflammation. This review specifically focuses on the function of GPBAR1 in cholestatic liver disease and summarizes the various pathways through which GPBAR1 acts in cholestatic models. GPBAR1 mainly regulates cholestasis in a holistic system of liver-gallbladder-gut formation. In the state of cholestasis, the activation of GPBAR1 could regulate liver inflammation, induce cholangiocyte regeneration to maintain the integrity of the biliary tree, control the hydrophobicity of the bile acid pool and promote the secretion of bile HCO3−. All these functions of GPBAR1 might be clear ways to protect against cholestatic diseases and liver injury. However, the characteristic of GPBAR1-mediated proliferation increases the risk of proliferation of cholangiocarcinoma in malignant transformed cholangiocytes. This dichotomous function of GPBAR1 limits its use in cholestasis. During disease treatment, simultaneous activation of GPBAR1 and FXR receptors often results in improved outcomes, and this strategy may become a crucial direction in the development of bile acid-activated receptors in the future.

Cardioprotection of Immature Heart by Simultaneous Activation of PKA and EPAC : A Role for the Mitochondrial Permeability Transition Pore

Metabolic and ionic changes during ischaemia predispose the heart to the damaging effects of reperfusion. Such changes and the resulting injury differ between immature and adult heart. Therefore, cardioprotective strategies for adults need to be tested in immature heart. We have recently shown that simultaneous activation of PKA and EPAC confers marked cardioprotection in adult hearts. The aim of this study is to investigate the efficacy of this intervention in immature hearts and determine whether the mitochondrial permeability transition pore (MPTP) is involved. Isolated perfused Langendorff hearts from both adult and immature rats were exposed to global ischaemia and reperfusion injury (I/R) following control perfusion or perfusion after an equilibra-tion period with activators of PKA and/or EPAC. Functional outcome and reperfusion injury were measured and in parallel, mitochondria were isolated following 5 min reperfusion to determine whether cardioprotective interventions involved changes in MPTP opening behaviour. Perfusion for 5 minutes preceding ischaemia of injury- matched adult and immature hearts with 5 µM 8-Br (8-Br-cAMP-AM), an activator of both PKA and EPAC, led to significant reduction in post-reperfusion CK release and infarct size. Perfusion with this agent also led to a reduction in MPTP opening propensity in both adult and immature hearts. These data show that immature hearts are innately more resistant to I/R injury than adults, and that this is due to a reduced ten-dency to MPTP opening following reperfusion. Further, simultaneous stimulation of PKA & EPAC causes cardioprotection which is additive to the innate resistance.

Waste-honeycomb-derived in situ N-doped Hierarchical porous carbon as sulfur host in lithium–sulfur battery

Waste honeycomb derived porous carbon with a high specific surface area of 1683.6 m2 g−1 are prepared via a facile simultaneous activation/carbonization. The corresponding porous carbon/sulfur composite cathode exhibits a durable stable performance up to 500 cycles at 1 C.

Multiplexed CRISPR-mediated engineering of protein secretory pathway genes in the thermotolerant methylotrophic yeast Ogataea thermomethanolica

CRISPR multiplex gRNA systems have been employed in genome engineering in various industrially relevant yeast species. The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 is an alternative host for heterologous protein production. However, the limited secretory capability of this yeast is a bottleneck for protein production. Here, we refined CRISPR-based genome engineering tools for simultaneous mutagenesis and activation of multiple protein secretory pathway genes to improve heterologous protein secretion. We demonstrated that multiplexed CRISPR-Cas9 mutation of up to four genes (SOD1, VPS1, YPT7 and YPT35) in one single cell is practicable. We also developed a multiplexed CRISPR-dCas9 system which allows simultaneous activation of multiple genes in this yeast. 27 multiplexed gRNA combinations were tested for activation of three genes (SOD1, VPS1 and YPT7), three of which were demonstrated to increase the secretion of fungal xylanase and phytase up to 29% and 41%, respectively. Altogether, our study provided a toolkit for mutagenesis and activation of multiple genes in O. thermomethanolica, which could be useful for future strain engineering to improve heterologous protein production in this yeast.

Synergy of epidermal growth factor (EGFR) and angiotensin II (AT1R) receptor determines composition and temporal pattern of transcriptome variation

The tyrosine kinase receptor EGFR and the G-protein-coupled receptor AT1R induce essential cellular responses, in part via receptor crosstalk with an unknown role in nuclear information transfer and transcription regulation. We investigated whether this crosstalk results in linear, EGFR-mediated nuclear signalling or in parallel, synergistic information transfer leading to qualitative and temporal variations, relevant for gene expression and environment interaction. AT1R and EGFR synergistically activate SRF via the ERK1/2-TCF and actin-MRTF pathways. Synergism, comprised of switch-like and graded single cell response, converges on the transcription factors AP1 and EGR, resulting in synergistic transcriptome alterations, in qualitative (over-additive number of genes), quantitative (over-additive expression changes of individual genes) and temporal (more late onset and prolonged expressed genes) terms. Gene ontology and IPA® pathway analysis indicate prolonged cell stress (e.g. hypoxia-like) and dysregulated vascular biology. Synergism occurs during separate but simultaneous activation of both receptors and during AT1R-induced EGFR transactivation. EGFR and AT1R synergistically regulate gene expression in qualitative, quantitative and temporal terms with (patho)physiological relevance, extending the importance of EGFR-AT1R crosstalk beyond cytoplasmic signalling.

Clinical characteristics and cellular immunity in children with rotavirus infection

Objective. To determine clinical course and state of cellular immunity in young children with rotavirus infection.Children characteristics and research methods. The scientists examined children without infectious pathology and with rotavirus infection (20 patients in each group) using general clinical methods. Rotavirus infection was diagnosed by polymerase chain reaction and immunochromatography. Cellular immunity parameters were determined by flow cytometry.Results. All the children under observation had a moderate form of the disease with symptoms of exsicosis of the II degree. Changes in the immune status were mainly of a regulatory, adaptive nature, which contributed to the favorable course of rotavirus infection in children, however, the dynamics of the number of cells expressing Toll-like receptors indicates the immunosuppressive properties of rotavirus.Conclusion. Currently, rotavirus infection in young children is typical with watery diarrhea as the most pronounced and long-lasting clinical symptom. Shifts in immunogram indices in general indicate a deficiency of the cellular link of immunity and a violation of its regulation with simultaneous activation of the immune system in an effort to achieve the eradication of the rotavirus with immunosuppressive properties.

AF Inducibility Is Related to Conduction Abnormalities at Bachmann’s Bundle

We investigated whether patterns of activation at Bachmann’s bundle are related to AF inducibility. Epicardial mapping of Bachmann’s bundle during sinus rhythm was performed prior to cardiac surgery (192 electrodes, interelectrode distances: 2 mm). Compared to non-inducible patients (N = 20), patients with inducible AF (N = 34) had longer lines of conduction block (18(2–164) mm vs. 6(2–28) mm, p = 0.048), prolonged total activation time (55(28–143) ms vs. 46(24–73) ms, p = 0.012), multiple wavefronts entering Bachmann’s bundle more frequently (64% vs. 37%, p = 0.046) and more often areas of simultaneous activation (conduction velocity > 1.7 m/s, 45% vs. 16%, p = 0.038). These observations further support a relation between conduction abnormalities at Bachmann’s bundle and AF inducibility. The next step is to examine whether Bachmann’s bundle activation patterns can also be used to identify patients who will develop AF after cardiac surgery during both short- and long-term follow-up.

Crosstalk between calcineurin and the cell wall integrity pathways prevents chitin overexpression in Candida albicans

Echinocandins such as caspofungin are front line antifungal drugs that compromise β-1,3 glucan synthesis in the cell wall. Recent reports have shown that fungal cells can resist killing by caspofungin by up-regulation of chitin synthesis, thereby sustaining cell wall integrity. When echinocandins are removed, the chitin content of cells quickly returns to basal levels, suggesting that there is a fitness cost associated with having elevated levels of chitin in the cell wall. We show here that simultaneous activation of the calcineurin and CWI pathways generates a sub-population of Candida albicans yeast cells that have supra-normal chitin levels interspersed throughout the inner and outer cell wall, and that these cells are non-viable, perhaps due to loss of wall elasticity required for cell expansion and growth. Mutations in the Ca2+-calcineurin pathway prevented the formation of these non-viable super high chitin cells by negatively regulating chitin synthesis driven by the CWI pathway. The Ca2+-calcineurin pathway may therefore act as an attenuator that prevents the overproduction of chitin by coordinating both chitin upregulation and negative regulation of the CWI signaling pathway.

BH3-only proteins Puma and Beclin1 regulate autophagic death in neurons in response to Amyloid-β

Alzheimer’s disease (AD) is characterized by accumulation of senile amyloid-β (Aβ) plaques and hyperphosphorylated tau tangles causing progressive loss of synapse and neuronal death. Out of the various neuron death modalities, autophagy and apoptosis are reported to be the major death paradigms in AD. However, how these two processes lead to neuronal loss is still inconspicuous. Here we report that under Aβ toxicity, aberrant autophagy is induced with inefficient autophagic flux in neurons. Simultaneous activation of both autophagy and apoptosis are seen in primary cortical neurons as well as in transgenic mice brains. We found that induction of autophagy by rapamycin is detrimental for neurons; whereas downregulation of Beclin1, an important autophagy inducing protein, provides significant protection in Aβ treated neuronal cells by blocking cytochrome-c release from the mitochondria. We further report that downregulation of Puma, a BH3-only pro-apoptotic protein, inhibits the induction of aberrant autophagy and also ameliorates the autophagy flux under the influence of Aβ. Notably, stereotactic administration of shRNAs against Puma and Beclin1 in adult Aβ-infused rat brains inhibits both apoptotic and autophagic pathways. The regulation of both of the death processes is brought about by the direct interaction between Puma and Beclin1 upon Aβ treatment. We conclude that both Beclin1 and Puma play essential roles in the neuronal death caused by the induction of aberrant autophagy in AD and targeting their interaction could be vital to understand the crosstalk of autophagy and apoptosis as well as to develop a potential therapeutic strategy in AD.

Cardiovascular Responses to Simultaneous Diving and Muscle Metaboreflex Activation

Background: The aim of study was to assess hemodynamic changes during the simultaneous activation of muscle metaboreflex (MM) and diving reflex (DR) in a laboratory setting. We hypothesized that as long as the exercise intensity is mild DR can overwhelm the MM.Methods: Ten trained divers underwent all four phases (randomly assigned) of the following protocol. (A) Postexercise muscle ischemia session (PEMI): 3 min of resting followed by 3 min of handgrip at 30% of maximum force, followed immediately by 3 min of PEMI on the same arm induced by inflating a sphygmomanometer. Three minutes of recovery was further allowed after the cuff was deflated for a total of 6 min of recovery. (B) Control exercise recovery session: the same rest-exercise protocol used for A followed by 6 min of recovery without inflation. (C) DR session: the same rest-exercise protocol used for A followed by 1 min of breath-hold (BH) with face immersion in cold water. (D) PEMI-DR session: the same protocol used for A with 60 s of BH with face immersion in cold water during the first minute of PEMI. Stroke volume (SV), heart rate (HR), and cardiac output (CO) were collected by means of an impedance method.Results: At the end of apnea, HR was decreased in condition C and D with respect to A (−40.8 and −40.3%, respectively vs. −9.1%; p < 0.05). Since SV increase was less pronounced at the same time point (C = +32.4 and D = +21.7% vs. A = +6.0; p < 0.05), CO significantly decreased during C and D with respect to A (−23 and −29.0 vs. −1.4%, respectively; p < 0.05).Conclusion: Results addressed the hypothesis that DR overcame the MM in our setting.