Bicarbonate signalling via G protein-coupled receptor regulates ischaemia-reperfusion injury.

Homoeostatic regulation of the acid-base balance is essential for cellular functional integrity. However, little is known about the molecular mechanism through which the acid-base balance regulates cellular responses. Here, we report that bicarbonate ions activate a G protein-coupled receptor (GPCR), i.e., GPR30, which leads to Gq-coupled calcium responses. Gpr30-Venus knock-in mice reveal predominant expression of GPR30 in brain mural cells. Primary culture and fresh isolation of brain mural cells demonstrate bicarbonate-induced, GPR30-dependent calcium responses. GPR30-deficient male mice are protected against ischemia-reperfusion injury by a rapid blood flow recovery. Collectively, we identify a bicarbonate-sensing GPCR in brain mural cells that regulates blood flow and ischemia-reperfusion injury. Our results provide a perspective on the modulation of GPR30 signalling in the development of innovative therapies for ischaemic stroke. Moreover, our findings provide perspectives on acid/base sensing GPCRs, concomitantly modulating cellular responses depending on fluctuating ion concentrations under the acid-base homoeostasis.

Incomplete accumulation of perilesional reactive astrocytes exacerbates wound healing after closed-head injury by increasing inflammation and BBB disruption.

Wound healing after closed-head injury is a significant medical issue. However, conventional models of focal traumatic brain injury, such as fluid percussion injury and controlled cortical impact, employ mechanical impacts on the exposed cerebral cortex after craniotomy. These animal models are inappropriate for studying gliosis, as craniotomy itself induces gliosis. To address this, we developed a closed-head injury model and named "photo injury", which employs intense light illumination through a thinned-skull cranial window. Our prior work demonstrated that the gliosis of focal cerebral lesion after the photo injury does not encompass artificial gliosis and comprises two distinct reactive astrocyte subpopulations. The reactive astrocytes accumulated in the perilesional recovery area actively proliferate and express Nestin, a neural stem cell marker, while those in distal regions do not exhibit these traits. The present study investigated the role of perilesional reactive astrocytes (PRAs) in wound healing using the ablation of reactive astrocytes by the conditional knockout of Stat3. The extensive and non-selective ablation of reactive astrocytes in Nestin-Cre:Stat3f/f mice resulted in an exacerbation of injury, marked by increased inflammation and BBB disruption. On the other hand, GFAP-CreERT2:Stat3f/f mice exhibited the partial and selective ablation of the PRAs, while their exacerbation of injury was at the same extent as in Nestin-Cre:Stat3f/f mice. The comparison of these two mouse strains indicates that the PRAs are an essential astrocyte component for wound healing after closed-head injury, and their anti-inflammatory and regenerative functions are significantly affected even by incomplete accumulation. In addition, the reporter gene expression in the PRAs by GFAP-CreERT2 indicated a substantial elimination of these cells and an absence of differentiation into other cell types, despite Nestin expression, after wound healing. Thus, the accumulation and subsequent elimination of PRA are proposed as promising diagnostic and therapeutic avenues to bolster wound healing after closed-head injury.

A pilot study of skin stretching and blood circulation effects of dermal suctioning in dogs.

Dermal suctioning has been reported to yield effects similar to those of cupping therapy in humans and horses, including pain reduction, increased blood circulation, improved flexibility, and healing. However, there is a dearth of reported outcomes concerning cupping or dermal suctioning in dogs. In this study, we examined the physiological effects of dermal suctioning in dogs. Employing the Medicell MINI pro8 device, dermal suctioning was applied to the dorsal surfaces of eight healthy beagle dogs for 20 min. Metrics such as body surface temperature, rectal temperature, pulse rate, respiratory rate, and skin pinch test results were gauged. Seven healthy beagle dogs were used as controls, and the same measurements were performed without dermal suctioning. The results showed a significant increase in the body surface temperature and skin pinch test results after dermal suctioning. We believe that the elimination of torsion in the blood vessels and nerves in the shallow fascia positively affected the thermoregulatory mechanism, resulting in an increase in body surface temperature, and also improving skin flexibility. Thus, dermal suctioning promotes subcutaneous blood circulation and improves skin flexibility in dogs. Further research is needed to identify the mechanisms underlying the effects of dermal suctioning and evaluate the stress in dogs caused by the implementation of the process.

The developmental profile of visual cortex astrocytes.

We investigated how astrocytes in layer 5 mouse visual cortex mature over postnatal days (P) 3-50. Across this age range, resting membrane potential increased, input resistance decreased, and membrane responses became more passive with age. Two-photon (2p) and confocal imaging of dye-loaded cells revealed that gap-junction coupling increased starting ∼P7. Morphological reconstructions revealed increased branch density but shorter branches after P20, suggesting that astrocyte branches may get pruned as tiling is established. Finally, we visualized spontaneous Ca2+ transients with 2p microscopy and found that Ca2+ events decorrelated, became more frequent and briefer with age. As astrocytes mature, spontaneous Ca2+ activity thus changes from relatively cell-wide, synchronous waves to local transients. Several astrocyte properties were stably mature from ∼P15, coinciding with eye opening, although morphology continued to develop. Our findings provide a descriptive foundation of astrocyte maturation, useful for the study of astrocytic impact on visual cortex critical period plasticity.

Streptocyanine as an activation mode of amine catalysis for the conversion of pyridine rings to benzene rings.

Amine catalysts have emerged as an invaluable tool in organic synthesis. Iminium, enamine, and enamine radical cation species are representative activation modes of amine catalysis. However, the development of new amine catalysis activation modes that enable novel synthetic strategies remains highly desirable. Herein, we report streptocyanine as a new amine catalysis activation mode, which enables the skeletal editing of pyridine rings to benzene rings. N-Arylation of pyridines bearing an alkenyl substituent at the 3-position generates the corresponding N-arylpyridiniums. The resulting pyridinum reacts with a catalytic amount of piperidine to afford a streptocyanine intermediate. Catalytically generated streptocyanine forms a benzene ring via a ring-closing reaction, thereby releasing the amine catalyst. Consequently, the alkene moiety in the starting pyridines is incorporated into the benzene ring of the products. Pyridiniums bearing various alkene moieties were efficiently converted to formyl-substituted benzene derivatives. Mechanistic studies support the postulation that the present catalytic process was intermediated by streptocyanine. In this reaction system, streptocyanine could be regarded as a new activation mode of amine catalysis.

The leukotriene B4 /BLT1-dependent neutrophil accumulation exacerbates immune complex-mediated glomerulonephritis.

Crescent formation is the most important pathological finding that defines the prognosis of nephritis. Although neutrophils are known to play an important role in the progression of crescentic glomerulonephritis, such as anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis, the key chemoattractant for neutrophils in ANCA-associated glomerulonephritis has not been identified. Here, we demonstrate that a lipid chemoattractant, leukotriene B4 (LTB4 ), and its receptor BLT1 are primarily involved in disease pathogenesis in a mouse model of immune complex-mediated crescentic glomerulonephritis. Circulating neutrophils accumulated into glomeruli within 1 h after disease onset, which was accompanied by LTB4 accumulation in the kidney cortex, leading to kidney injury. LTB4 was produced by cross-linking of Fc gamma receptors on neutrophils. Mice deficient in BLT1 or LTB4 biosynthesis exhibited suppressed initial neutrophil infiltration and subsequent thrombotic glomerulonephritis and renal fibrosis. Depletion of neutrophils before, but not after, disease onset prevented proteinuria and kidney injury, indicating the essential role of neutrophils in the early phase of glomerulonephritis. Administration of a BLT1 antagonist before and after disease onset almost completely suppressed induction of glomerulonephritis. Finally, we found that the glomeruli from patients with ANCA-associated glomerulonephritis contained more BLT1-positive cells than glomeruli from patients with other etiologies. Taken together, the LTB4 -BLT1 axis is the key driver of neutrophilic glomerular inflammation, and will be a novel therapeutic target for the crescentic glomerulonephritis.

The plasticitome of cortical interneurons.

Hebb postulated that, to store information in the brain, assemblies of excitatory neurons coding for a percept are bound together via associative long-term synaptic plasticity. In this view, it is unclear what role, if any, is carried out by inhibitory interneurons. Indeed, some have argued that inhibitory interneurons are not plastic. Yet numerous recent studies have demonstrated that, similar to excitatory neurons, inhibitory interneurons also undergo long-term plasticity. Here, we discuss the many diverse forms of long-term plasticity that are found at inputs to and outputs from several types of cortical inhibitory interneuron, including their plasticity of intrinsic excitability and their homeostatic plasticity. We explain key plasticity terminology, highlight key interneuron plasticity mechanisms, extract overarching principles and point out implications for healthy brain functionality as well as for neuropathology. We introduce the concept of the plasticitome - the synaptic plasticity counterpart to the genome or the connectome - as well as nomenclature and definitions for dealing with this rich diversity of plasticity. We argue that the great diversity of interneuron plasticity rules is best understood at the circuit level, for example as a way of elucidating how the credit-assignment problem is solved in deep biological neural networks.

Dietary intake of n-3 polyunsaturated fatty acids alters the lipid mediator profile of the kidney but does not attenuate renal insufficiency.

The efficacy of n-3 polyunsaturated fatty acids (PUFAs) in improving outcomes in a renal ischemia-reperfusion injury (IRI) model has previously been reported. However, the underlying mechanisms remain poorly understood and few reports demonstrate how dietary n-3 PUFAs influence the composition of membrane phospholipids in the kidney. Additionally, it has not been elucidated whether perilla oil (PO), which is mainly composed of the n-3 alpha-linolenic acid, mitigates renal IRI. In this study, we investigated the effect of dietary n-3 PUFAs (PO), compared with an n-6 PUFA-rich soybean oil (SO) diet, on IRI-induced renal insufficiency in a rat model. Levels of membrane phospholipids containing n-3 PUFAs were higher in the kidney of PO-rich diet-fed rats than the SO-rich diet-fed rats. Levels of blood urea nitrogen and serum creatinine were significantly higher in the ischemia-reperfusion group than the sham group under both dietary conditions. However, no significant differences were observed in blood urea nitrogen, serum creatinine, or histological damage between PO-rich diet-fed rats and SO-rich diet-fed rats. In the kidney of PO-rich diet-fed rats, levels of arachidonic acid and arachidonic acid-derived pro-inflammatory lipid mediators were lower than SO-rich diet-fed rats. Eicosapentaenoic acid and eicosapentaenoic acid-derived lipid mediators were significantly higher in the kidney of PO-rich than SO-rich diet-fed rats. These results suggest that dietary n-3 PUFAs alter the fatty acid composition of membrane phospholipids and lipid mediators in the kidney; however, this does not attenuate renal insufficiency or histological damage in a renal IRI model.

Leukotriene A4 hydrolase deficiency protects mice from diet-induced obesity by increasing energy expenditure through neuroendocrine axis.

Obesity is a health problem worldwide, and brown adipose tissue (BAT) is important for energy expenditure. Here, we explored the role of leukotriene A4 hydrolase (LTA4 H), a key enzyme in the synthesis of the lipid mediator leukotriene B4 (LTB4 ), in diet-induced obesity. LTA4 H-deficient (LTA4 H-KO) mice fed a high-fat diet (HFD) showed a lean phenotype, and bone-marrow transplantation studies revealed that LTA4 H-deficiency in non-hematopoietic cells was responsible for this lean phenotype. LTA4 H-KO mice exhibited greater energy expenditure, but similar food intake and fecal energy loss. LTA4 H-KO BAT showed higher expression of thermogenesis-related genes. In addition, the plasma thyroid-stimulating hormone and thyroid hormone concentrations, as well as HFD-induced catecholamine secretion, were higher in LTA4 H-KO mice. In contrast, LTB4 receptor (BLT1)-deficient mice did not show a lean phenotype, implying that the phenotype of LTA4 H-KO mice is independent of the LTB4 /BLT1 axis. These results indicate that LTA4 H mediates the diet-induced obesity by reducing catecholamine and thyroid hormone secretion.

A Practical Guide to Using CV Analysis for Determining the Locus of Synaptic Plasticity.

Long-term synaptic plasticity is widely believed to underlie learning and memory in the brain. Whether plasticity is primarily expressed pre- or postsynaptically has been the subject of considerable debate for many decades. More recently, it is generally agreed that the locus of plasticity depends on a number of factors, such as developmental stage, induction protocol, and synapse type. Since presynaptic expression alters not just the gain but also the short-term dynamics of a synapse, whereas postsynaptic expression only modifies the gain, the locus has fundamental implications for circuits dynamics and computations in the brain. It therefore remains crucial for our understanding of neuronal circuits to know the locus of expression of long-term plasticity. One classical method for elucidating whether plasticity is pre- or postsynaptically expressed is based on analysis of the coefficient of variation (CV), which serves as a measure of noise levels of synaptic neurotransmission. Here, we provide a practical guide to using CV analysis for the purposes of exploring the locus of expression of long-term plasticity, primarily aimed at beginners in the field. We provide relatively simple intuitive background to an otherwise theoretically complex approach as well as simple mathematical derivations for key parametric relationships. We list important pitfalls of the method, accompanied by accessible computer simulations to better illustrate the problems (downloadable from GitHub), and we provide straightforward solutions for these issues.

Trialkylborane-Mediated Propargylation of Aldehydes Using γ-Stannylated Propargyl Acetates.

A transition-metal-free three-component process that combines aldehydes, 3-(tributylstannyl)propargyl acetates formed in situ from readily available propargyl acetates, and trialkylboranes provides access to a range of 1,2,4-trisubstituted homopropargylic alcohols. The addition of diisopropylamine plays a crucial role in the selective formation of homopropargylic alcohols. Importantly, this methodology can be extended to a single-flask reaction sequence starting from propargyl acetates.

The Role of Leukotrienes as Potential Therapeutic Targets in Allergic Disorders.

Leukotrienes (LTs) are lipid mediators that play pivotal roles in acute and chronic inflammation and allergic diseases. They exert their biological effects by binding to specific G-protein-coupled receptors. Each LT receptor subtype exhibits unique functions and expression patterns. LTs play roles in various allergic diseases, including asthma (neutrophilic asthma and aspirin-sensitive asthma), allergic rhinitis, atopic dermatitis, allergic conjunctivitis, and anaphylaxis. This review summarizes the biology of LTs and their receptors, recent developments in the area of anti-LT strategies (in settings such as ongoing clinical studies), and prospects for future therapeutic applications.

Histone deacetylase inhibitors sensitize 5-fluorouracil-resistant MDA-MB-468 breast cancer cells to 5-fluorouracil.

Resistance to 5-fluorouracil (5-FU) is a serious problem in cancer therapy and overcoming it is required in order to improve the efficacy of cancer chemotherapy. Histone deacetylase (HDAC) inhibitors are used in cancer treatments and, recently, it has been reported that HDAC inhibitors can overcome resistance to various anti-cancer drugs in vitro. In the present study, a 5-FU-resistant breast cancer cell line was established, and the effects of HDAC inhibitors in these cells were examined. The 5-FU-resistant cell line MDA-MB-468 (MDA468/FU) was established by continuous exposure of the parental cells to 5-FU. This subline was characterized by high resistance to 5-FU, higher mRNA expression levels of thymidylate synthetase and dihydropyrimidine dehydrogenase (DPD), and lower mRNA expression levels of uridine monophosphate synthetase (UMPS) than the parental cells. Gimeracil, a DPD inhibitor, did not affect the sensitivity of MDA468/FU cells to 5-FU. Oteracil, a UMPS inhibitor, decreased the cytotoxicity of 5-FU in MDA468 cells, but not in MDA468/FU cells. The HDAC inhibitors, valproic acid and suberanilohydroxamic acid sensitized the two cell lines to 5-FU in a concentration-dependent manner. In conclusion, the results of the present study revealed that HDAC inhibitors increase the sensitivity to 5-FU in 5-FU-sensitive and -resistant cells.

Leukotriene B4 receptor type 2 protects against pneumolysin-dependent acute lung injury.

Although pneumococcal infection is a serious problem worldwide and has a high mortality rate, the molecular mechanisms underlying the lethality caused by pneumococcus remain elusive. Here, we show that BLT2, a G protein-coupled receptor for leukotriene B4 and 12(S)-hydroxyheptadecatrienoic acid (12-HHT), protects mice from lung injury caused by a pneumococcal toxin, pneumolysin (PLY). Intratracheal injection of PLY caused lethal acute lung injury (ALI) in BLT2-deficient mice, with evident vascular leakage and bronchoconstriction. Large amounts of cysteinyl leukotrienes (cysLTs), classically known as a slow reactive substance of anaphylaxis, were detected in PLY-treated lungs. PLY-dependent vascular leakage, bronchoconstriction, and death were markedly ameliorated by treatment with a CysLT1 receptor antagonist. Upon stimulation by PLY, mast cells produced cysLTs that activated CysLT1 expressed in vascular endothelial cells and bronchial smooth muscle cells, leading to lethal vascular leakage and bronchoconstriction. Treatment of mice with aspirin or loxoprofen inhibited the production of 12-HHT and increased the sensitivity toward PLY, which was also ameliorated by the CysLT1 antagonist. Thus, the present study identifies the molecular mechanism underlying PLY-dependent ALI and suggests the possible use of CysLT1 antagonists as a therapeutic tool to protect against ALI caused by pneumococcal infection.

Galacto-oligosaccharides attenuate renal injury with microbiota modification.

Tubulointerstitial injury is central to the progression of end-stage renal disease. Recent studies have revealed that one of the most investigated uremic toxins, indoxyl sulfate (IS), caused tubulointerstitial injury through oxidative stress and endoplasmic reticulum (ER) stress. Because indole, the precursor of IS, is synthesized from dietary tryptophan by the gut microbiota, we hypothesized that the intervention targeting the gut microbiota in kidney disease with galacto-oligosaccharides (GOS) would attenuate renal injury. After 2 weeks of GOS administration for 5/6 nephrectomized (Nx) or sham-operated (Sham) rats, cecal indole and serum IS were measured, renal injury was evaluated, and the effects of GOS on the gut microbiota were examined using pyrosequencing methods. Cecal indole and serum IS were significantly decreased and renal injury was improved with decreased infiltrating macrophages in GOS-treated Nx rats. The expression levels of ER stress markers and apoptosis were significantly increased in the Nx rats and decreased with GOS. The microbiota analysis indicated that GOS significantly increased three bacterial families and decreased five families in the Nx rats. In addition, the analysis also revealed that the bacterial family Clostridiaceae was significantly increased in the Nx rats compared with the Sham rats and decreased with GOS. Taken altogether, our data show that GOS decreased cecal indole and serum IS, attenuated renal injury, and modified the gut microbiota in the Nx rats, and that the gut microbiota were altered in kidney disease. GOS could be a novel therapeutic agent to protect against renal injury.

Glyoxalase I reduces glycative and oxidative stress and prevents age-related endothelial dysfunction through modulation of endothelial nitric oxide synthase phosphorylation.

Endothelial dysfunction is a major contributor to cardiovascular disease (CVD), particularly in elderly people. Studies have demonstrated the role of glycation in endothelial dysfunction in nonphysiological models, but the physiological role of glycation in age-related endothelial dysfunction has been poorly addressed. Here, to investigate how vascular glycation affects age-related endothelial function, we employed rats systemically overexpressing glyoxalase I (GLO1), which detoxifies methylglyoxal (MG), a representative precursor of glycation. Four groups of rats were examined, namely young (13 weeks old), mid-age (53 weeks old) wild-type, and GLO1 transgenic (WT/GLO1 Tg) rats. Age-related acceleration in glycation was attenuated in GLO1 Tg rats, together with lower aortic carboxymethyllysine (CML) and urinary 8-hydroxydeoxyguanosine (8-OHdG) levels. Age-related impairment of endothelium-dependent vasorelaxation was attenuated in GLO1 Tg rats, whereas endothelium-independent vasorelaxation was not different between WT and GLO1 Tg rats. Nitric oxide (NO) production was decreased in mid-age WT rats, but not in mid-age GLO1 Tg rats. Age-related inactivation of endothelial NO synthase (eNOS) due to phosphorylation of eNOS on Thr495 and dephosphorylation on Ser1177 was ameliorated in GLO1 Tg rats. In vitro, MG increased phosphorylation of eNOS (Thr495) in primary human aortic endothelial cells (HAECs), and overexpression of GLO1 decreased glycative stress and phosphorylation of eNOS (Thr495). Together, GLO1 reduced age-related endothelial glycative and oxidative stress, altered phohphorylation of eNOS, and attenuated endothelial dysfunction. As a molecular mechanism, GLO1 lessened inhibitory phosphorylation of eNOS (Thr495) by reducing glycative stress. Our study demonstrates that blunting glycative stress prevents the long-term impact of endothelial dysfunction on vascular aging.

Retroperitoneal fibrosis with periaortic and pericardial involvement.

A 66-year old man, who had been diagnosed with dilated cardiomyopathy and felt a progressive shortness of breath and fatigability, was admitted to hospital. Computed tomography showed a thickening of the aortic wall from the aortic arch to the aortic bifurcation, as well as mild pleural and pericardial effusion. Intravenous pyelography showed severe ureteral stenosis, along with hydronephrosis, of the left side. There was a marked increase in C-reactive protein and the erythrocyte sedimentation rate, but the serology for connective tissue disease and perinuclear antineutrophil cytoplasmic antibodies was negative. Retroperitoneal fibrosis (RPF) with intrathoracic extension was diagnosed. After confirming the absence of malignant disease, an oral predonisolone treatment of 30 mg/day was started, and this ameliorated the ureteral obstruction, aortic wall thickening and pericardial effusion. The patient had been taking 300 mg of loxoprofen sodium for headaches every day for 16 years. The relationship between loxoprofen, cardiomyopathy and RPF remains unclear. There is a possibility of RPF in the patients with a thickening of thoracic aortic wall, as in this case.

Effects of creatine on mental fatigue and cerebral hemoglobin oxygenation.

While the role of creatine in preventing muscle (peripheral) fatigue for high performance athletes is well understood, its biochemical role in prevention of mental (central) fatigue is not. Creatine is abundant in muscles and the brain and after phosphorylation used as an energy source for adenosine triphosphate synthesis. Using double-blind placebo-controlled paradigm, we demonstrated that dietary supplement of creatine (8 g/day for 5 days) reduces mental fatigue when subjects repeatedly perform a simple mathematical calculation. After taking the creatine supplement, task-evoked increase of cerebral oxygenated hemoglobin in the brains of subjects measured by near infrared spectroscopy was significantly reduced, which is compatible with increased oxygen utilization in the brain.