CO2-dependent migration and relocation of LCIB, a pyrenoid-peripheral protein in Chlamydomonas reinhardtii.

Most microalgae overcome the difficulty of acquiring inorganic carbon (Ci) in aquatic environments by inducing a CO2-concentrating mechanism (CCM). In the green alga Chlamydomonas reinhardtii, two distinct photosynthetic acclimation states have been described under CO2-limiting conditions (low-CO2 [LC] and very low-CO2 [VLC]). LC-inducible protein B (LCIB), structurally characterized as carbonic anhydrase, localizes in the chloroplast stroma under CO2-supplied and LC conditions. In VLC conditions, it migrates to aggregate around the pyrenoid, where the CO2-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase is enriched. Although the physiological importance of LCIB localization changes in the chloroplast has been shown, factors necessary for the localization changes remain uncertain. Here, we examined the effect of pH, light availability, photosynthetic electron flow, and protein synthesis on the localization changes, along with measuring Ci concentrations. LCIB dispersed or localized in the basal region of the chloroplast stroma at 8.3-15 µM CO2, whereas LCIB migrated toward the pyrenoid at 6.5 µM CO2. Furthermore, LCIB relocated toward the pyrenoid at 2.6-3.4 µM CO2, even in cells in the dark or treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea and cycloheximide in light. In contrast, in the mutant lacking CCM1, a master regulator of CCM, LCIB remained dispersed even at 4.3 µM CO2. Meanwhile, a simultaneous expression of LCIC, an interacting protein of LCIB, induced the localization of several speckled structures at the pyrenoid periphery. These results suggest that the localization changes of LCIB require LCIC and are controlled by CO2 concentration with ∼7 µM as the boundary.

Effects of glucokinase activator, DS-7309, on embryo-fetal developmental toxicity in rats and rabbits.

The toxicological effects of DS-7309, a glucokinase activator, on pregnancy and embryo-fetal development in rats and rabbits and maternal blood glucose levels were examined. DS-7309 was administered at 3, 10, or 100 mg/kg to rats from Days 7-17 of pregnancy or at 10, 30, or 100 mg/kg to rabbits from Days 6-18 of pregnancy. In rats, maternal hypoglycemia (approximately 50 mg/dL) was seen at 3 and 10 mg/kg, but it recovered 7 h after dosing, leading to no toxic changes. In contrast, continuous severe maternal hypoglycemia (approximately 40 mg/dL, ≥7 h), fetal eye anomalies, and decreased fetal body weight were noted at 100 mg/kg. In rabbits, no fetal anomalies were seen at 10 and 30 mg/kg where maternal blood glucose level dropped to approximately 60-90 mg/dL, but recovered by 7 h after dosing at the latest. In contrast, at 100 mg/kg, severe hypoglycemia (around 60 mg/dL) was maintained and did not recover until 24 h after dosing; it resulted in decreased fetal viability and increased fetal skeleton anomalies. These findings indicate that DS-7309 could lead to embryo-fetal toxicity in rats and rabbits, with such toxicity considered to be related to continuous severe maternal hypoglycemia.

Safety and Efficacy of Intravenous Low-Dose Alteplase in Relative Contraindication Patients with Acute Ischemic Stroke.

OBJECT: The purpose of this study was to investigate the safety and efficacy of intravenous low-dose alteplase for acute ischemic stroke patients with relative contraindications. METHODS: The consecutive series of patients admitted within 4.5 hours of ischemic stroke onset between September 2012 and April 2017 were retrospectively evaluated. A good outcome at 90 days and symptomatic intracerebral hemorrhage were evaluated to determine the association with intravenous low-dose alteplase, especially in the presence of relative contraindications. RESULTS: Intravenous low-dose alteplase was administered to 219 of 315 patients (70%). A significantly higher number of patients treated with intravenous low-dose alteplase achieved a good outcome compared with those treated without alteplase (60% versus 44%; P = .014). The incidence of symptomatic intracerebral hemorrhage was not significantly different between the treatment groups. Multivariable logistic regression analysis of good outcome revealed that the significant independent factors were age of 81 years or older (odds ratio, .33; 95% confidence interval, .18-.60), National Institutes of Health Stroke Scale (NIHSS) of 4 or less (compared with NIHSS, 5-25; odds ratio, 3.3; 95% confidence interval, 1.8-6.4), modified Rankin scale score of 1 before stroke (odds ratio, .32; 95% confidence interval, .14-.73), and large changes on first brain imaging (odds ratio, .16; 95% confidence interval, .058-.44). Even with these relative contraindications, intravenous low-dose alteplase was still associated with good outcome (odds ratio, 3.1; 95% confidence interval, 1.6-5.8). CONCLUSIONS: Intravenous low-dose alteplase treatment can be safe and effective in relative contraindication patients with acute ischemic stroke.

Fracture-based fabrication of normally closed, adjustable, and fully reversible microscale fluidic channels.

Adjustable fluidic structures play an important role in microfluidic systems. Fracture of multilayered materials under applied tension has been previously demonstrated as a convenient, simple, and inexpensive approach to fabricate nanoscale adjustable structures; here, it is demonstrated how to extend this concept to the microscale. This is achieved by a novel pairing of materials that leverages fracture mechanics to limit crack formation to a specified region, allowing to create size-controllable and adjustable microfluidic structures. This technique can be used to fabricate "normally closed" microfluidic channels that are completely reversible, a feature that is challenging to achieve in conventional systems without careful engineering controls. The adjustable microfluidic channels are then applied to mechanically lyse single cells, and subsequently manipulate the released nuclear chromatin, creating new possibilities for epigenetic analysis of single cells. This simple, versatile, and robust technology provides an easily accessible pathway to construct adjustable microfluidic structures, which will be useful in developing complex assays and experiments even in resource-limited settings.

Retrograde thrombectomy of acute common carotid artery occlusion with mobile thrombus: illustrative cases.

BACKGROUND: Acute embolic occlusion of the common carotid artery (CCA) alone is rare. However, once it occurs, recanalization is challenging due to the large volume of the clot, larger diameter of the CCA, and risk of procedure-related distal embolism into the intracranial arteries. OBSERVATIONS: The authors report two cases of acute embolic occlusion of CCA alone, caused by a cardiac embolus trapped at the proximal end of a preexisting atherosclerotic plaque at the cervical carotid bifurcation. In both cases, the CCA was successfully recanalized using retrograde thrombectomy in a hybrid operating room. In case 1, a 78-year-old male with acute right CCA occlusion underwent retrograde thrombectomy, where the cervical carotid bifurcation was exposed and incised, and the entire embolus was retrieved with forceps. Despite successful revascularization, massive bleeding from the CCA just after the retrieval remained a concern. In case 2, a 79-year-old female with acute right CCA occlusion underwent retrograde thrombectomy in the same manner. Because manual retrieval failed, a Fogarty balloon catheter inserted from the arteriotomy successfully retrieved the entire thrombus with minimal blood loss. LESSONS: Retrograde thrombectomy through the arteriotomy of the cervical carotid bifurcation safely and effectively recanalizes acute embolic occlusion of the CCA alone.

Nanoscale squeezing in elastomeric nanochannels for single chromatin linearization.

This paper describes a novel nanofluidic phenomenon where untethered DNA and chromatin are linearized by rapidly narrowing an elastomeric nanochannel filled with solutions of the biopolymers. This nanoscale squeezing procedure generates hydrodynamic flows while also confining the biopolymers into smaller and smaller volumes. The unique features of this technique enable full linearization then trapping of biopolymers such as DNA. The versatility of the method is also demonstrated by analysis of chromatin stretchability and mapping of histone states using single strands of chromatin.

Disruption of fetal eye development caused by insulin-induced maternal hypoglycemia in rats.

We previously revealed that insulin-induced severe and long-lasting maternal hypoglycemia in rats caused anophthalmia and microphthalmia in fetuses; however, it remained unclear whether hypoglycemia-induced eye anomalies were developmental retardation or disruption, and when and how they developed. Hence, we induced hypoglycemia in pregnant Sprague-Dawley rats by injecting insulin from Days 6 to 11 of pregnancy and performed periodical histopathological examination of fetal eyes from embryonic days (E)10 to 20. On E10, optic vesicle had developed normally both in the control and insulin-treated group; however, on E11, optic cup (OC) had developed in the control group but not in the insulin-treated group. On E12, neural retina (NR), retinal pigmented epithelium (RPE), lens, and presumptive cornea had been observed in the control group. In contrast, lens pit and OC with remaining space between RPE and NR had developed in the insulin-treated group. From E13 to E15, developmental disruption characterized by defects, hypoplasia, and degeneration in the retina, lens, and cornea was observed in the insulin-treated group, resulting in anophthalmia or microphthalmia on E20. Moreover, the expression of MITF and chx10, which are essential for early eye development by expressing in the presumptive retina and lens and regulating each other's expression level, was ectopic and suppressed on E11. In conclusion, insulin-induced maternal hypoglycemia caused developmental disruption, but not simple developmental retardation of fetal eyes, and its trigger might be a failure of presumptive retina and lens to interact on E11.

Activation of the prefrontal cortex is associated with exertional dyspnea in chronic obstructive pulmonary disease.

BACKGROUND: Exertional dyspnea is the primary symptom that limits exercise in patients with chronic obstructive pulmonary disease (COPD). It is unknown which activated brain area is associated with this symptom in COPD patients. OBJECTIVES: To investigate the activation of cortical areas associated with dyspnea during exercise in COPD patients. METHODS: COPD patients (n = 10) and age-matched controls (n = 10) performed mild-intensity constant work rate cycle exercise (40% of their symptom-limited peak work rates) for 10 min, while cerebral hemodynamics and oxygenation were measured by near-infrared spectroscopy (NIRS). Ventilatory responses (breathing pattern and pulmonary gas exchange) and Borg scale ratings of dyspnea and leg fatigue were measured during exercise. Three NIRS probes were placed over the prefrontal and temporoparietal cortical regions of the subjects' heads. Changes in cortical oxyhemoglobin (oxy-Hb), deoxyhemoglobin (deoxy-Hb), and total hemoglobin (total Hb) concentrations from baseline recordings were measured. Increased oxy-Hb (oxygenation) was assumed to reflect cortical activation. RESULTS: Oxy-Hb concentration was significantly increased in the prefrontal region during exercise in both groups but not in the temporoparietal regions. The change in prefrontal oxy-Hb concentration of COPD patients was not different from that of controls. Dyspnea scores were positively correlated with changes in oxy-Hb concentrations of the prefrontal regions in both groups. Multivariate analysis showed that oxy-Hb concentration in the prefrontal region was the best predictor of dyspnea in both groups. CONCLUSIONS: Exertional dyspnea was related to activation (oxygenation) of the prefrontal cortex in COPD patients and control subjects.

Involvement of neurokinin receptors in the control of pulsatile luteinizing hormone secretion in rats.

It has recently been shown that neurokinin B, a tachykinin, is associated with GnRH pulse generation in sheep and goats. The aim of the present study was to clarify the role of tachykinin receptors in the control of LH secretion in rats. To this end, we evaluated the effect of CS-003, an antagonist for all three neurokinin receptors (NK1, NK2 and NK3 receptors), on pulsatile LH secretion in both sexes of rats with different routes of administration. Both oral and third ventricular administration of CS-003 suppressed LH secretion in both sexes of gonadectomized animals. Furthermore, intact male rats with oral administration of CS-003 showed decreased serum testosterone levels, which might be due to suppressed LH secretion. None of the three subtype-specific neurokinin receptor antagonists showed a significant effect on LH secretion in ovariectomized rats when each antagonist was singly administered. The present results suggest that neurokinins play a role in the control of pulsatile GnRH/LH secretion via multiple neurokinin receptors in both male and female rats.

Hyaluronan reversed proteoglycan synthesis inhibited by mechanical stress: possible involvement of antioxidant effect.

INTRODUCTION: Abnormal mechanical stress loaded on the cartilage leads to the osteoarthritis (OA). Although intraarticular hyaluronan (HA) injection is an effective treatment for OA, the underlying mechanism has not been made clear. METHODS: Mechanical compression was loaded on the bovine cartilage using the Biopress system. Proteoglycan (PG) and reactive oxygen species (ROS) synthesis were measured with [(35)S] incorporation and fluorescent dye, respectively. Accumulation of peroxynitrite was determined with western blotting using nitrotyrosine antibody. RESULTS: Mechanical compression inhibited PG synthesis and enhanced ROS. Externally added HA reversed stress-inhibited PG synthesis and attenuated ROS synthesis. HA also significantly decreased the generation of nitrotyrosine. CONCLUSIONS: HA neutralized stress-enhanced ROS synthesis and resulted in the reversing of PG synthesis. These data suggest that HA plays an anabolic effect as an antioxidant.

Three-dimensional Visualization of Aortic Arch Morphology via Rearrangement of Non-enhanced Thoracic CT Images.

Objective: The objective of this study was to evaluate the reproducibility of three-dimensional (3D) images of the aortic arch reconstructed using a novel image processing algorithm for non-enhanced computed tomography (CT) images of the cervicothorax and abdomen obtained before emergency endovascular surgery. Case Presentations: In all, 46 patients who underwent acute mechanical thrombectomy between January and December 2018 were examined. The anatomical variations of the aortic arch were reproduced in all cases; however, the reproduction of the carotid arteries was difficult. Conclusion: Our novel 3D analysis system enables obtaining information on the aortic arch easily from plain CT data that may be useful in acute endovascular treatment.

Acute Proximal Anterior Circulation Occlusion after Pulmonary Lobectomy Treated by Endovascular Therapy: Two Case Reports.

Objective: We report two cases of acute proximal anterior circulation occlusion after pulmonary lobectomy. Case Presentation: Case 1 was a 64-year-old male who presented with occlusion of the right middle cerebral artery (MCA) one day after left lower lobectomy. Case 2 was a 68-year-old male who presented with occlusion of the right internal carotid artery (ICA). In both cases, mechanical thrombectomy was performed for complete recanalization and symptoms were improved. Conclusion: Prompt mechanical thrombectomy in the acute phase after pulmonary lobectomy improved the prognosis of patients with acute proximal anterior circulation occlusion. It is important to share information about ischemic complications with medical staff engaged in thoracic surgery.

Abnormal DNA methylation of the Oct-4 enhancer region in cloned mouse embryos.

Oct-4 is essential for normal embryonic development, and abnormal Oct-4 expression in cloned embryos contributes to cloning inefficiency. However, the causes of abnormal Oct-4 expression in cloned embryos are not well understood. As DNA methylation in regulatory regions is known to control transcriptional activity, we investigated the methylation status of three transcriptional regulatory regions of the Oct-4 gene in cloned mouse embryos--the distal enhancer (DE), the proximal enhancer (PE), and the promoter regions. We also investigated the level of Oct-4 gene expression in cloned embryos. Immunochemistry revealed that 85% of cloned blastocysts expressed Oct-4 in both trophectoderm and inner cell mass cells. DNA methylation analysis revealed that the PE region methylation was greater in cloned morulae than in normal morulae. However, the same region was less methylated in cloned blastocysts than in normal blastocysts. We found abnormal expression of de novo methyltransferase 3b in cloned blastocysts. These results indicate that cloned embryos have aberrant DNA methylation in the CpG sites of the PE region of Oct-4, and this may contribute directly to abnormal expression of this gene in cloned embryos.

Optimizing in-hospital triage for large vessel occlusion using a novel clinical scale (GAI2AA).

OBJECTIVE: To identify a proximal anterior circulation occlusion for effectively administering immediate mechanical thrombectomy by developing a novel, simple diagnostic scale to predict the occlusion, to compare its validity with available scales, and to assess its utility. METHODS: To develop a novel clinical scale, we retrospectively analyzed a cohort of 429 patients with acute ischemic stroke from a single center. The novel scale GAI2AA was applied to a prospective cohort of 259 patients from 3 stroke centers for external validation. The utility of the scale as an in-hospital triage was compared for the temporal factors of 158 patients with the occlusion. RESULTS: In a scale-developmental phase, those with a proximal anterior circulation occlusion had significantly more frequent signs of hemispheric symptoms, including gaze palsy, aphasia, inattention, arm paresis, and atrial fibrillation. The GAI2AA scale was developed using consolidated hemispheric symptoms and was scored as follows: score = 2, arm paresis score = 1, and atrial fibrillation score = 1. A cutoff value ≥3 was optimal for the correlation between sensitivity (88%) and specificity (81%), with a C statistic of 0.90 (95% confidence interval 0.87-0.93). External validation indicated that discrimination was significantly better than or not different from that of available complex scales. Door-to-puncture time was significantly reduced (91 [82-111] vs 52 [32-75] minutes, p < 0.001). CONCLUSION: The GAI2AA scale showed high sensitivity and specificity when an optimal cutoff score was used and was useful as an in-hospital triage tool.

Historical control data on developmental toxicity studies in rats.

Historical control data from prenatal developmental toxicity studies in rats have been used to evaluate whether toxicology outcomes were induced by exposure to a chemical or were within the range of spontaneous variation. These data are also important for monitoring animal characteristics. As a follow-up to historical control data from 1998 to 2010, this study analyzed control data from prenatal developmental studies performed in rats from 2011 to 2015. Data were collected from studies performed by 24 Japanese laboratories, including 15 pharmaceutical and chemical companies and nine contract research organizations, in Sprague-Dawley and two-sub-strains of Wistar Hannover rats. The data included maternal reproductive findings at terminal cesarean section and fetal findings, including incidences of spontaneous external, visceral, and skeletal anomalies. No noticeable differences in maternal reproductive data were observed among laboratories. The inter-laboratory variations in the incidences of fetal anomalies seemed to be due to differences in the selection of observation parameters, observation criteria, and classification of the findings, as well as to differences in terminology of fetal alterations. These historical control data may be helpful for adequate interpretation of experimental results and for evaluating the reproductive and developmental toxicities of various chemicals.

Cis-acting elements (E-box and NBE) in the promoter region of three maternal genes (Histone H1oo, Nucleoplasmin 2, and Zygote Arrest 1) are required for oocyte-specific gene expression in the mouse.

We examined the promoter activities of three mouse maternal genes (H1oo, Npm2, and Zar1) in oocytes and pre-implantation embryos, and examined the promoters for cis-acting elements of 5'-flanking region to obtain the best promoter for inducing oocyte-specific gene expression. For the assay, we injected firefly luciferase gene constructs under the control of the promoters into the oocytes and embryos. Each promoter region showed transcriptional activity in oocytes, but not in fertilized embryos. Deletion analysis showed that a putative E-box region at position -72 of the H1oo promoter and at the -180 of the Npm2 promoter were required for basal transcriptional activity in oocytes. Moreover, a putative NBE motif (NOBOX DNA binding elements) (-1796) was shown to enhance basal transcriptional activity of the Npm2 promoter. Thus, the E-box and/or NBE may be key regulatory regions for the expression of the examined maternal genes (H1oo and Npm2) in growing mouse oocytes.

Mechanical Thrombectomy in Anterior Circulation Occlusion Could Be More Effective than Medical Management Even in Low DWI-ASPECTS Patients.

The purpose of this study was to investigate whether patients with low preoperative Diffusion-weighted Imaging Alberta Stroke Program Early Computed Tomography Score (DWI-ASPECTS) could benefit from mechanical thrombectomy for acute anterior circulation occlusion. This was a retrospective, non-blinded, cohort study. From September 2012 to August 2016, 83 consecutive patients of acute anterior circulation occlusion were treated with thrombectomy using second-generation devices or medical management. The DWI-ASPECTS was scored after the first MRI. Patient characteristics and clinical outcomes were compared between the treatment groups. Significant dependence was defined as a modified Rankin scale score ≥3 at 90 days. As a result, 33 patients underwent mechanical thrombectomy and 50 received medical management. In the mechanical thrombectomy group, the variable of lower DWI-ASPECTS (5, 4-6 vs. 8, 7-8, P < 0.001), especially ≤6, was significantly associated with poor prognosis. However, compared with patients of DWI-ASPECTS ≤ 6 who received medical management, there were significantly fewer patients with poor outcomes in thrombectomy (dependent in 11 of 15 vs. 23 of 23, respectively; P = 0.019). Although patients with lower pretreatment DWI-ASPECTS could benefit less from thrombectomy, their outcomes were still better than medical management. Therefore, mechanical thrombectomy could be considered in some patients with low pretreatment DWI-ASPECTS.

Effects of maternal hypoglycemia on fetal eye and skeleton development in rats.

The relationship between insulin-induced maternal hypoglycemia and teratogenicity was investigated in detail. We injected 4 different forms of insulin (insulin human, aspart, glargine, and detemir) subcutaneously at 1 or 2 dose levels to Sprague-Dawley rats from Days 6 to 11 of pregnancy, measured blood glucose levels, and conducted fetal examination. In the insulin human and aspart (low dose) groups, while severe hypoglycemia (approximately 50mg/dL) was seen, it lasted only 6h and no fetal anomalies were observed. Fetal axial skeleton anomalies were observed in the aspart (high dose) group, which exhibited intermediate-duration of severe hypoglycemia (9h). Eye and axial skeleton anomalies were observed in the glargine and detemir groups, which exhibited continuous severe hypoglycemia (≥9h). These results revealed that insulin-induced maternal hypoglycemia caused fetal eye and skeleton anomalies and the causative key factors were duration of maternal severe hypoglycemia.

Enhanced erythropoiesis mediated by activation of the renin-angiotensin system via angiotensin II type 1a receptor.

Although clinical and experimental studies have long suggested a role for the renin-angiotensin system (RAS) in the regulation of erythropoiesis, the molecular basis of this role has not been well understood. We report here that transgenic mice carrying both the human renin and human angiotensinogen genes displayed persistent erythrocytosis as well as hypertension. To identify the receptor molecule responsible for this phenotype, we introduced both transgenes into the AT1a receptor null background and found that the hematocrit level in the compound mice was restored to the normal level. Angiotensin II has been shown to influence erythropoiesis by two means, up-regulation of erythropoietin levels and direct stimulation of erythroid progenitor cells. Thus, we conducted bone marrow transplantation experiments and clarified that AT1a receptors on bone marrow-derived cells were dispensable for RAS-dependent erythrocytosis. Plasma erythropoietin levels and kidney erythropoietin mRNA expression in the double transgenic mice were significantly increased compared with those of the wild-type control, while the elevated plasma erythropoietin levels were significantly attenuated in the compound mice. These results provide clear genetic evidence that activated RAS enhances erythropoiesis through the AT1a receptor of kidney cells and that this effect is mediated by the elevation of plasma erythropoietin levels in vivo.

Dynamic simulations show repeated narrowing maximizes DNA linearization in elastomeric nanochannels.

This paper uses computer simulations to reveal unprecedented details about linearization of deoxyribonucleic acid (DNA) inside dynamic nanochannels that can be repeatedly widened and narrowed. We first analyze the effect of rate of channel narrowing on DNA linearization dynamics. Quick (∼0.1 s) narrowing of nanoscale channels results in rapid overstretching of the semi-flexible chain followed by a slower (∼0.1-10 s) relaxation to an equilibrium extension. Two phenomena that induce linearization during channel narrowing, namely, elongational-flow and confinement, occur simultaneously, regardless of narrowing speed. Interestingly, although elongational flow is a minimum at the mid-point of the channel and increases towards the two ends, neither the linearization dynamics nor the degree of DNA extension varies significantly with the center-of-mass of the polymer projected on the channel axis. We also noticed that there was a significant difference in time to reach the equilibrium length, as well as the degree of DNA linearization at short times, depending on the initial conformation of the biopolymer. Based on these observations, we tested a novel linearization protocol where the channels are narrowed and widened repeatedly, allowing DNA to explore multiple conformations. Repeated narrowing and widening, something uniquely enabled by the elastomeric nanochannels, significantly decrease the time to reach the equilibrium-level of stretch when performed within periods comparable to the chain relaxation time and more effectively untangle chains into more linearized biopolymers.