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Stroke is a major public health problem and ranks third most common cause of death in adults worldwide. Thrombolysis with recombinant tissue plasminogen activator and endovascular thrombectomy are the main revascularization therapies for acute ischemic stroke. However, ischemia-reperfusion injury, mainly caused by oxidative/nitrosative stress injury, after revascularization therapy can result in worsening outcomes. For better clinical prognosis, more and more studies have focused on the pharmaceutical neuroprotective therapies against free radical damage. The impact of vitamin C (ascorbic acid) on oxidative stress-related diseases is moderate because of its limited oral bioavailability and rapid clearance. However, recent evidence of the clinical benefit of parenteral vitamin C administration has emerged, especially in critical care. In this study we demonstrated that parenteral administration of vitamin C significantly improved neurological deficits and reduced brain infarction and brain edema by attenuating the transient middle cerebral artery occlusion (tMCAO)-induced nitrosative stress, inflammatory responses, and the resultant disruptions of blood brain barrier and cerebral neuronal apoptosis. These results suggest that parenteral administration of vitamin C has potential as an adjuvant agent with intravenous thrombolysis or endovascular thrombectomy in acute treatment of ischemic stroke.

Objectives Virus infection is underevaluated in older adults with severe acute respiratory infections (SARIs). We aimed to evaluate the clinical impact of combining point-of-care molecular viral test and serum procalcitonin (PCT) level for antibiotic stewardship in the emergency department (ED). Design A prospective twin-center cohort study was conducted between January 2017 and March 2018. Setting and Participants Older adult patients who presented to the ED with SARIs received a rapid molecular test for 17 respiratory viruses and a PCT test. Measures To evaluate the clinical impact, we compared the outcomes of SARI patients between the experimental cohort and a propensity score–matched historical cohort. The primary outcome was the proportion of antibiotics discontinuation or de-escalation in the ED. The secondary outcomes included duration of intravenous antibiotics, length of hospital stay, and mortality. Results A total of 676 patients were included, of which 169 patients were in the experimental group and 507 patients were in the control group. More than one-fourth (27.9%) of the patients in the experimental group tested positive for virus. Compared with controls, the experimental group had a significantly higher proportion of antibiotics discontinuation or de-escalation in the ED (26.0% vs 16.1%, P = .007), neuraminidase inhibitor uses (8.9% vs 0.6%, P

Nanodiamonds (NDs) are considered to be relatively safe carbon nanomaterials used for the transmission of DNA, proteins and drugs. The feasibility of utilizing the NDs to deliver CRISPR-Cas9 system for gene editing has not been clearly studied. Therefore, in this study, we aimed to use NDs as the carriers of CRISPR-Cas9 components designed to introduce the mutation in RS1 gene associated with X-linked retinoschisis (XLRS). ND particles with a diameter of 3 nm were functionalized by carboxylation of the surface and covalently conjugated with fluorescent mCherry protein. Two linear DNA constructs were attached to the conjugated mCherry: one encoded Cas9 endonuclease and GFP reporter, another encoded sgRNA and contained insert of HDR template designed to introduce RS1 c.625C>T mutation. Such nanoparticles were successfully delivered and internalized by human iPSCs and mouse retinas, the efficiency of internalization was significantly improved by mixing with BSA. The delivery of ND particles led to introduction of RS1 c.625C>T mutation in both human iPSCs and mouse retinas. Rs1 gene editing in mouse retinas resulted in several pathological features typical for XLRS, such as aberrant photoreceptor structure. To conclude, our ND-based CRISPR-Cas9 delivery system can be utilized as a tool for creating in vitro and in vivo disease models of XLRS. Statement of significance X-linked retinoschisis (XLRS) is a prevalent hereditary retinal disease, which is caused by mutations in RS1 gene, whose product is important for structural organization of the retina. The recent development of genome editing techniques such as CRISPR-Cas9 significantly improved the prospects for better understanding the pathology and development of treatment for this disease. Firstly, gene editing can allow development of appropriate in vitro and in vivo disease models; secondly, CRISPR-Cas9 can be applied for gene therapy by removing the disease-causative mutation in vivo. The major prerequisite for these approaches is to develop safe and efficient CRISPR-Cas9 delivery system. In this study, we tested specifically modified nanodiamonds for such a delivery system. We were able to introduce Rs1 mutation into the mouse retina and, importantly, observed several XLRS-specific effects.

Increasing evidence has supported that transplantation of human stem cells induces neuroprotective and reparative effects in animal models of Parkinson’s disease (PD). However, without systemic immunosuppressive therapy, most of these grafted cells are rejected by the hosts. Long term and systemic injection of cyclosporine-A (CsA) is required to maintain the survival of grafted cells. The purpose this study is to examine a new treatment strategy to suppress the immunorejection by locally co-grafting of polylactic/glycolic acid nanoparticles containing CsA (NanoCsA) with differentiated human induced pluripotent stem cells (iPSCs). In the in vitro media, NanoCsA provided sustained release of CsA for >6 weeks. The differentiated human iPSCs were co-grafted with NanoCsA or NanoVeh (nanoparticle without CsA) to the striatum of unilaterally 6-hydroxydopamine -lesioned rats. NanoCsA/iPSCs co-graft significantly improved locomotor activity compared to NanoVeh/iPSCs co-grafts or iPSC grafts + sytemic CsA at 1 month after transplantation. Brain tissues were collected for measurements of tyrosine hydroxylase (TH) and human marker Stem121 immunoreactivity. Cografting with NanoCsA/iPSCs, compared to NanoVeh/iPSCs, significantly increased TH and Stem121 immunoreactivity as well as tumor formation in the lesioned striatum. Taken together, our study supports that NanoCsA provides long-lasting CsA release and reduces immunorejection of human iPSCs xenograft in a 6-hydroxydopamine rat model of PD.

Background The aim of this study is to evaluate the feasibility of single-incision laparoscopic surgery (SILS) for idiopathic intussusception in children and compare the outcomes with conventional laparoscopy (CLS). Methods Between January 2011 and December 2012, patients who underwent CLS for idiopathic intussusception were assigned into the group of CLS. Between January 2013 and March 2017, patients who underwent SILS were assigned to the group of SILS. For patients who failed to reduce by SILS, bimanual transabdominal approach was conducted. Results A total of 23 patients were enrolled, including 7 and 16 patients in SILS and CLS, respectively. The mean age was similar in both group (22.4 ± 18.7 vs. 24.6 ± 18.6 months, p = 0.80). There is no difference in gender distribution. The main indication was radiological reduction failure in both groups (85.7% vs. 75%, p = 0.58). Ileocolic intussusception was found in 6 (85.7%) and 15 (93.8%) patients of SILS and CLS, respectively (p = 0.25). The level of intussusception was at ascending colon in 3 (42.9%) and 12 (75.0%) patients, respectively (p = 0.11). The operation time was similar in both groups (64.9 ± 53.7 and 70.9 ± 26.1 min, p = 0.79). There were 2 (28.6%) and 1 (6.2%) conversions, respectively (p = 0.15). For the two patients in SILS, the intussusception was successfully reduced by bimanual transabdominal approach. There was no significant difference in time to feeding (1.9 ± 1.1 vs. 1.4 ± 0.7 days, p = 0.21). The mean length of postoperative hospital stay was 3.9 ± 1.6 and 3.1 ± 1.1 days, respectively (p = 0.17). Conclusions SILS for pediatric intussusception is technically feasible and has comparable results to CLS. Transabdominal bimanual reduction is applicable in cases of failed laparoscopic reduction. Level of evidence III

Background: The purpose of this study is to examine the outcome of transumbilical minilaparotomy for infants and compare the results between normal birthweight (NBW) and low birthweight (LBW). Methods: Between July 2010 and March 2017, infants who underwent abdominal surgery through transumbilical minilaparotomy were enrolled. Medical records were retrospectively reviewed. Patients were divided into two groups, NBW and LBW. Complexity was defined as complicated conditions other than intestinal atresia and malrotation. Results: Totally, 16 patients were included. The diagnosis included intestinal atresia (n = 3), meconium peritonitis (n = 4), bowel infarction/necrosis (n = 4), spontaneous intestinal perforation (n = 2), segmental volvulus and necrosis (n = 1), necrotizing enterocolitis (n = 1), and malrotation (n = 1). The median gestational age and body weight were 32 (24–40) weeks and 1731 (560–4200) grams respectively. The median age at operation was 3 (1–41) days. The surgical procedure included primary repair of the intestine (n = 14), ileostomy (n = 1) and Ladd's procedure (n = 1). Postoperative complications included anastomotic leakage (n = 2), adhesion ileus (n = 1), and missed rectal atresia (n = 1). There was one mortality due to extremely low birthweight and poor lung maturation. Re-operation was required in 3 patients for anastomotic leakage (n = 2) and missed rectal atresia (n = 1). Mean birthweight was 2932 ± 97 and 1263 ± 667 g in NBW (n = 5) and LBW (n = 11), respectively (p

Background Whether or not non-vitamin K antagonist oral anticoagulants (NOACs) are associated with a lower risk of acute kidney injury (AKI) in patients with non-valvular atrial fibrillation (NVAF) remains unknown in real world practice. Methods In this nationwide retrospective cohort study, 1507, 3200, 5765 and 4227 NVAF patients with chronic kidney disease (CKD) and 4368, 16,945, 22,301, and 16,908 NVAF patients without CKD taking apixaban, dabigatran, rivaroxaban, and warfarin, respectively, from June 1, 2012 to December 31, 2016 were enrolled from the Taiwan National Health Insurance Program. Propensity-score weighted method was used to balance covariates across study groups. Patients were followed until occurrence of AKI or end date of study. Results Three NOACs were all associated with a significantly lower risk of AKI compared with warfarin for both CKD-free (hazard ratio, [95% confidential interval]; 0.65, [0.60–0.72] for apixaban; 0.68, [0.64–0.74] for dabigatran; 0.73, [0.68–0.79] for rivaroxaban) and CKD cohorts (0.50, [0.45–0.56] for apixaban; 0.54, [0.49–0.59] for dabigatran; 0.53, [0.49–0.58] for rivaroxaban). The annual incidence of AKI for all NOACs and warfarin increased gradually as the increment of CHA2DS2-VASc for both CKD-free and CKD cohorts after propensity score weighting. The reduced risk of AKI for three NOACs persisted in most subgroups in either CKD-free or CKD cohort. Multivariate analysis indicated that all three NOACs were all associated with lower risk of AKI than warfarin in either CKD-free or CKD cohort. Conclusions All three NOACs are associated with a lower risk of AKI than warfarin among Asians with NVAF in real-world practice.

Background and aims Renal dysfunction is a potent risk factor for cardiovascular diseases, including stroke. This study aimed to evaluate the impact of admission estimated glomerular filtration rate (eGFR) levels on short-term (1-month) and long-term (1-year) mortality in patients with acute ischemic stroke. Methods From the Taiwan Stroke Registry data, we classified ischemic stroke patients, identified from April 2006 to December 2015, into 5 groups by eGFR at admission: ≥ 90, 60–89, 30–59, 15–29, and Results Among 52,732 ischemic stroke patients, 1480 died within one month. The 1-month mortality rate was over 5-fold greater in patients with eGFR Conclusions After acute ischemic stroke, patients with reduced eGFR are at elevated risks of short-term and long-term deaths in a graded relationship.

MicroRNA (miRNA) sensing plays an essential role in the diagnosis of several diseases, especially cancers, for appropriate intervention and treatment. However, quantifying miRNA demands highly sensitive and selective assays which can distinguish analogous sequences with low abundance in bio-samples and determine wide range of concentrations. In this report, we present a novel technique satisfying all those requirements by modifying silicon nanowire field-effect transistors (SiNWFETs) with 2-component mixed self-assembled monolayers (mSAMs) of polyethylene glycol (PEG) at different ratios (silane-PEG-NH2:silane-PEG-OH = 1:1, 1:3, and 1:5) and glutaraldehyde to immobilize DNA probes for miRNA-21 detection, a biomarker in several types of cancers. Empirical results reveal that all the fabricated PEG-SiNWFET DNA biosensors could quantify miRNA-21 within 1 fM – 10 pM. Especially, the ones modified with silane-PEG-NH2:silane-PEG-OH = 1:3 exhibited an outstanding performance to recognize miRNA-21 at an ultra-low concentration of 10 aM in the dynamic range up to 6 orders of magnitude (10 aM – 10 pM). This approach is more convenient, analytical competitive, and cost-effective in comparison with currently used methods for nucleic acid testing because of label- and amplification-free characteristics. It is therefore not only feasible for miRNA detection by SiNWFET-based biosensors but also potential for clinical applications of disease diagnosis with oligonucleotide biomarkers.

Single-incision laparoscopic surgery has been widely popularized for pediatric appendicitis. Various techniques have been proposed with two main approaches: extracorporeal and intracorporeal. The purpose of this study is to compare the result of different approaches in single-incision laparoscopic appendectomy (SILA) in children.With IRB approval, patients less than 18years of age who underwent SILA were enrolled from July 2012 to December 2015. The patients were divided into three groups based on surgical approach: extracorporeal (Extra), mixed (Mix), and intracorporeal (Intra) approaches. Parameters were retrospectively reviewed, including age, gender, white blood cell (WBC), operation time, operative findings, time to diet, length of hospital stay (LOS), and complications. Statistical analysis was performed separately for simple and complicated appendicitis.There were 32, 32, and 24 patients with simple appendicitis in Extra, Mix, and Intra respectively. There were 27, 15, and 31 patients with complicated appendicitis in the three groups, respectively. No significant difference was noted in the mean age, gender distribution, or WBCs between the different groups. A higher percentage of patients with complicated appendicitis received intracorporeal approach than those with simple appendicitis (42.5% vs. 27.3%, p=0.044). In simple appendicitis, the LOS was significantly longer in Extra as compared to Mix (p=0.043). Otherwise, the mean LOS, time to diet, and complications were not significantly different. The mean operation time was similar between groups of simple appendicitis (56.5±19.5, 63.6±23.5, and 70.1±23.1 min, p=0.08), whereas it was significantly shorter in Extra of complicated appendicitis (67.6±16.4, 86.6±19.0, and 89.9±23.4 min, p0.001). Multivariate analysis showed that intracorporeal approach is an independent factor for prolonged operation time in both simple and complicated appendicitis.Different approaches of SILA in children have similar outcomes for both simple and complicated appendicitis. Extracorporeal is the most time efficient; however, intracorporeal can be helpful to deal with complex situations.III.

In this paper we address the support of wearable mHealth applications in LTE and future 5G networks following a holistic approach that spans across the elements of a mobile network. The communication requirements change from one application to another so we propose a measurement methodology to facilitate the selection of the user equipment to fulfil these requirements. We also discuss a new network architecture to support traffic prioritization, RAN programmability, low latency and group communications to over-the-top applications. Our proposal is validated using several realistic experimentation platforms and the results show that mHealth systems can benefit from our approach.

We investigated the impact of serum cholesterol levels on 30-day mortality after ischemic stroke in dialysis patients.From the Taiwan Stroke Registry data, we identified 46,770 ischemic stroke cases, including 1101 dialysis patients and 45,669 nondialysis patients from 2006 to 2013.Overall, the 30-day mortality was 1.46-fold greater in the dialysis group than in the nondialysis group (1.75 versus 1.20 per 1000 person-days). The mortality rates were 1.64, .62, 2.82, and 2.23 per 1000 person-days in dialysis patients with serum total cholesterol levels of 120 mg/dL, 120-159 mg/dL, 160-199 mg/dL, and ≥200 mg/dL, respectively. Compared to dialysis patients with serum total cholesterol levels of 120-159 mg/dL, the corresponding adjusted hazard ratios of mortality were 4.20 (95% confidence interval [CI] = 1.01-17.4), 8.06 (95% CI = 2.02-32.2), and 6.89 (95% CI = 1.59-29.8) for those with cholesterol levels of 120 mg/dL, 160-199 mg/dL, and ≥200 mg/dL, respectively.Dialysis patients with serum total cholesterol levels of ≥160 mg/dL or 120 mg/dL on admission are at an elevated hazard of 30-day mortality after ischemic stroke.

Paraquat, an herbicide used extensively worldwide, can cause severe toxicity in humans and animals, leading to irreversible, lethal lung fibrosis. The potential of CO-releasing molecules (CORMs), substances that release CO (Carbon monoxide) within animal tissues, for treating paraquat-induced ROS generation and inflammation is investigated here. Our results show that the fast CO releaser CORM-3 (4-20 μM) acts as a potential scavenger of free radicals and decreases fibrosis progression by inhibiting paraquat-induced overexpression of connective tissue growth factor and angiotensin II in MRC-5 cells. The slow CO releaser CORM-A1 (5 mg/kg) clearly decreased expression of the lung profibrogenic cytokines COX-2, TNF-α, and α-SMA and serum hydroxyproline, resulting in a lower mortality rate in paraquat-treated mice. Mice treated with higher-dose CORM-A1 (10 mg/kg) had relatively intact lung lobes and fewer fibrotic patches by gross observation, with less collagen deposition, mesangial matrix accumulation, and pulmonary fibrosis resulting from the mitigation of TGF-β overexpression. In conclusion, our data demonstrate for the first time that CORM-A1 alleviated the development of the fibrotic process and improved survival rate in mice exposed to PQ, would be an attractive therapeutic approach to attenuate the progression of pulmonary fibrosis following PQ exposure.

A simple and effective method for determining short chain chlorinated paraffins (SCCPs) in indoor dust is presented. The method employed a modified vortex-homogenized matrix solid-phase dispersion (VH-MSPD) prior to its detection by gas chromatography - electron-capture negative-ion mass spectrometry (GC-ECNI-MS) operating in the selected-ion-monitoring (SIM) mode. Under the best extraction conditions, 0.1-g of dust sample was dispersed with 0.1-g of silica gel by using vortex (2min) instead of using a mortar and pestle (3min). After that step, the blend was transferred to a glass column containing 3-g acidic silica gel, 2-g basic silica gel, and 2-g of deactivated silica gel, used as clean-up co-sorbents. Then, target analytes were eluted with 5mL of n-hexane/dichloromethane (2:1, v/v) mixture. The extract was evaporated to dryness under a gentle stream of nitrogen. The residue was then re-dissolved in n-hexane (10μL), and subjected to GC-ECNI-MS analysis. The limits of quantitation (LOQs) ranged from 0.06 to 0.25μg/g for each SCCP congener. Precision was less than 7% for both intra- and inter-day analysis. Trueness was above 89%, which was calculated by mean extraction recovery. The VH-MSPD combined with GC-ECNI-MS was successfully applied to quantitatively detect SCCPs from various indoor dust samples, and the concentrations ranged from 1.2 to 31.2μg/g.

The impact of ascorbate on oxidative stress-related diseases is moderate because of its limited oral bioavailability and rapid clearance. However, recent evidence of the clinical benefit of parenteral vitamin C administration has emerged, especially in critical care. Heatstroke is defined as a form of excessive hyperthermia associated with a systemic inflammatory response that results in multiple organ dysfunctions in which central nervous system disorders such as delirium, convulsions, and coma are predominant. The thermoregulatory, immune, coagulation and tissue injury responses of heatstroke closely resemble those observed during sepsis and are likely mediated by similar cellular mechanisms. This study was performed by using the characteristic high lethality rate and sepsis-mimic systemic inflammatory response of a murine model of heat stroke to test our hypothesis that supra-physiological doses of ascorbate may have therapeutic use in critical care. We demonstrated that parenteral administration of ascorbate abrogated the lethality and thermoregulatory dysfunction in murine model of heat stroke by attenuating heat stroke-induced accelerated systemic inflammatory, coagulation responses and the resultant multiple organ injury, especially in hypothalamus. Overall, our findings support the hypothesis and notion that supra-physiological doses of ascorbate may have therapeutic use in critical care.

Composite Fe 3 O 4 /α-Fe 2 O 3 nanorods (180N) with a specific surface area of 16.2 m 2 /g were successfully synthesized via a facile hydrothermal reaction at 180 °C. To increase the specific surface area, a mesoporous carbon aerogel (CA) with a large specific surface area was added to the hydrothermal reaction to form mesoporous Fe 3 O 4 /α-Fe 2 O 3 /CA structures (CA-180) with a specific surface area of 267.7 m 2 /g instead of nanorods. The as-prepared 180N and CA-180 samples were further annealed at 400 °C to oxidize the Fe 3 O 4 phase, resulting in the formation of composite γ-Fe 2 O 3 /α-Fe 2 O 3 nanorods (400N) with a specific surface area of 27.7 m 2 /g and mesoporous γ-Fe 2 O 3 /α-Fe 2 O 3 /CA structures (CA-400) with a specific surface area of 551 m 2 /g. The CA-400 samples were further used to remove the dye Rhodamine B (RhB) from aqueous solution, and 98.2% of the RhB dye was removed using the CA-400 sample when the initial RhB concentration was 8 ppm. The as-prepared CA-400 sample exhibits strong potential for use in wastewater treatment applications, such as organic dye removal.

Summary Intraluminal duodenal diverticulum (IDD) is a rare congenital anomaly. We present the report of an 8-year-old girl who had an entrapped coin in an IDD for 3 years that was associated with recurrent pancreatitis. Besides, a duodenoduodenal intussusception was found during the course of investigation and it seemed that a concomitant duodenal polyp contributed to the development of the intussusception. In view of the rarity of each of the aforementioned situations and the improbability of these conditions occurring together, this unusual and possibly unique case is therefore reported here.

Metal atoms on surface edges or with specific coordinates possess high surface potentials, and serve as core atoms for organic molecule bonding. However, these active metal atoms may lose their activity by self-diffusion on the surface. A polyethylene terephthalate (PET) substrate provided a stable rough surface in nanoscale for metal atoms absorption and formation of a stable and active nano-thin-film (NTF) surface. However, the NTF surface roughness decreased with increasing thickness of palladium (Pd) granules, indicating that the surface growth of the Pd film followed a Stranski-Krastanov-like growth model. Therefore, the proper thickness of Pd-NTF on PET substrate (Pd NTF-PET) was developed for electrochemical impedance spectroscopy (EIS). Protein was immobilized on the Pd NTF-PET electrodes within 15 min and its biosensing sensitivity was as low as 0.1 ng per-test in 1 μl. We expect that the developed Pd NTF-PET electrode can be used for other biosensing applications as well.

Over the years, culturable cellulase-producing microorganisms have been isolated from a variety of sources and genes of cellulolytic enzymes have been cloned. Then again, the “great plate count anomaly” phenomenon necessitates a culture-independent metagenomic approach for the isolation of cellulolytic genes from microorganisms in their natural environment. We have constructed a metagenomic library derived from rice straw composts. Of 2739 clones screened, a lambda clone carrying a 12 kb genomic fragment was able to yield a clear zone on an agar plate containing carboxymethyl cellulose (CMC). A 4.7 kb subclone, generated by restriction enzyme digestion, was found to harbor a GH12 cellulase gene, RSC-EG1, encoding 464 amino acids along with two other ORFs. The identified cellulolytic gene showed more than 70% similarity on the amino acid level with cellulase from Micromonospora aurantiaca and Thermobispora sp. Interestingly, RSC-EG1 contains a stretch of approximately 86 amino acids not present in either of these close relatives. Our results demonstrated that RSC-EG1, stable over a wide temperature and pH range, is a novel endoglucanase, and provided the first example of metagenomics approach to isolate cellulolytic gene from rice straw composts.

Methamphetamine (METH) is an abused drug that may cause psychiatric and neurotoxic damage, including degeneration of monoaminergic terminals and apoptosis of non-monoaminergic cells in the brain. The cellular and molecular mechanisms underlying these METH-induced neurotoxic effects remain to be clarified. In this study, we performed a time course assessment to investigate the effects of METH on intracellular oxidative stress and mitochondrial alterations in a human dopaminergic neuroblastoma SH-SY5Y cell line. We characterized that METH induces a temporal sequence of several cellular events including, firstly, a decrease in mitochondrial membrane potential within 1 h of the METH treatment, secondly, an extensive decline in mitochondrial membrane potential and increase in the level of reactive oxygen species (ROS) after 8 h of the treatment, thirdly, an increase in mitochondrial mass after the drug treatment for 24 h, and finally, a decrease in mtDNA copy number and mitochondrial proteins per mitochondrion as well as the occurrence of apoptosis after 48 h of the treatment. Importantly, vitamin E attenuated the METH-induced increases in intracellular ROS level and mitochondrial mass, and prevented METH-induced cell death. Our observations suggest that enhanced oxidative stress and aberrant mitochondrial biogenesis may play critical roles in METH-induced neurotoxic effects.

Using a prospective hospital-based registry, 146 patients with transient ischaemic attack (TIA) were compared with 376 patients with minor first-ever ischaemic stroke with respect to the 3-month risk of subsequent vascular events, in order to clarify the distinctions between the disease entities. All patients were enrolled within 48 h of onset. The risk factor distribution for the two groups was comparable, except that the TIA patients had more previous TIAs. Large artery atherosclerosis (34%) and small vessel occlusion (32%) were the main aetiologies in the TIA group, whereas small vessel occlusion (49%) was the major cause in the stroke group. The 3-month risk of combined endpoints of stroke, myocardial infarction, and vascular death for TIA patients was higher than that for the minor stroke group (15.1% vs. 3.2%; hazard ratio 4.6, 95% confidence interval 2.3-9.3 in multivariate analysis). Large artery atherosclerosis and male sex were the other significant predictors. TIA may demand more urgent management than minor stroke. The fact that aetiology is a predictor, highlights the need for rapid diagnostic tests to establish pathogenesis.

Nucleoside diphosphate kinase (NDK) is a ubiquitous enzyme found in all organisms and cell types, and catalyzes the transfer of the phosphoryl group from a nucleoside triphosphate to a nucleoside diphosphate. The enzyme is involved in and required for coleoptile elongation in rice as the level of the rice NDK (rNDK) changes during seed germination and the early stages of seedling growth. The expression of rice NDK gene is up-regulated in the growing coleoptiles when the anaerobic stress persists. The rNDK structure determined at 2.5 A resolution consists of a four-stranded anti-parallel beta-sheet, of which the surfaces are partially covered with six alpha-helices; its overall and active site structures are similar to those of homologous enzymes except the major conformation variations of residue 132-138 regions, involving significant structural contacts. The model contains 148 residues of 149 residues in total and averaged 19 water molecules per monomer for 12 molecules in an asymmetric unit. A mold of 12 superimposed molecules shows that the alphaA-alpha2 area has greater variations and higher temperature factors, indicating the flexibility for a substrate entrance. Hexameric molecular packing in both crystal and solution implies that rNDK functions as hexamers. This rNDK structure, which is the first NDK structure from a higher plant system, provides the structural information essential to understand the functional significance of this enzyme during growth and development in both rice and other plants.

Prolonged activation of opioid receptors leads to their phosphorylation, desensitization, internalization, and down-regulation. To elucidate the relationship between mu-opioid receptor (MOR) phosphorylation and the regulation of receptor activity, a series of receptor mutants was constructed in which the 12 Ser/Thr residues of the COOH-terminal portion of the receptor were substituted to Ala, either individually or in combination. All these mutant constructs were stably expressed in human embryonic kidney 293 cells and exhibited similar expression levels and ligand binding properties. Among those 12 Ser/Thr residues, Ser(363), Thr(370), and Ser(375) have been identified as phosphorylation sites. In the absence of the agonist, a basal phosphorylation of Ser(363) and Thr(370) was observed, whereas [d-Ala(2),Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO)-induced receptor phosphorylation occurs at Thr(370) and Ser(375) residues. Furthermore, the role of these phosphorylation sites in regulating the internalization of MOR was investigated. The mutation of Ser(375) to Ala reduced the rate and extent of receptor internalization, whereas mutation of Ser(363) and Thr(370) to Ala accelerated MOR internalization kinetics. The present data show that the basal phosphorylation of MOR could play a role in modulating agonist-induced receptor internalization kinetics. Furthermore, even though mu-receptors and delta-opioid receptors have the same motif encompassing agonist-induced phosphorylation sites, the different agonist-induced internalization properties controlled by these sites suggest differential cellular regulation of these two receptor subtypes.

Objectives: In determining the plasma malondialdehyde MDA levels in some Taiwanese college students, we found rather different results by using different thiobarbituric acid TBA tests, even by the high-performance liquid chromatography HPLC-based methods. Here, we re-evaluated four commonly used TBA tests and improved the HPLC-based test. Design and Methods: We used the blood plasma of 16 college volunteers to determine plasma MDA by using four methods: a spectrophotometric measurement of thiobarbituric acid-reactive substances (TBARS) in the TCA-supernatant of plasma (Method A); a fluorescence measurement of plasma lipid peroxides (Method B); and two different HPLC-based measurements of MDA with either 532-nm measurement (Method C, HPLC/532 nm) or fluorescence measurement (Method D, HPLC/fluor.). Results: The levels of MDA or TBA reactive substances obtained from the four methods differed substantially (0.39 ± 0.15; 2.14 ± 0.73; 0.75 ± 0.22; and 0.38 ± 0.15 μM for Methods A, B, C, and D, respectively). The results were positively correlated between Methods A and B (r = 0.740, p < 0.02) and between Methods C and D (r = 0.516, p < 0.05). However, results were negatively correlated between Methods B and D (r = −0.548, p < 0.05). Because most plasma MDA is bound to proteins, we modified the HPLC-based methods (C and D) by adding an alkaline hydrolysis step, and the plasma TBA-MDA adduct detected by HPLC/532 nm was referred to as total MDA. Results show that alkaline hydrolysis was a critical step for measurement of total MDA in plasma because this treatment led to release of MDA from plasma proteins. We also adapted the potassium iodide (KI) treatment of plasma from Method D to reduce lipid hydroperoxides. Our modified method gave a total MDA level in the 16 volunteers of ∼1.5 μM, which was equal to protein-bound MDA plus free MDA. This total MDA level was positively (p < 0.05) correlated with the level of TBA reactive substances obtained from Methods C (r = 0.63, p < 0.05) and D (r = 0.48, p < 0.07), but was not correlated with those from Methods A and B. The recovery (84∼105%), precision (within-assay coefficient of variation: 2.4%, between-assay coefficient of variation: 4∼8%) and sensitivity of the modified procedure were comparable to other HPLC-based methods. Conclusion: By using a validated modification of HPLC-based TBA method, the total plasma MDA in 16 Taiwanese college students was found to be 1.54 μM, which was relatively high compared to those obtained by other HPLC-based method, primarily due to the release of protein-bound MDA by alkaline hydrolysis. This level equaled the sum of protein-bound MDA and free MDA in plasma, confirming that this level represents total plasma MDA.

Both chronic and acute ethanol exposure have been shown to be cytotoxic and also to disrupt normal cell function or responses in a variety of cell types. Macrophage function has specifically been shown to be disrupted by chronic ethanol exposure by mechanisms that have not been elucidated. It is known that exposure of macrophages to lipopolysaccharide (LPS) from gram-negative bacteria will decrease the number of cells. Since increased exposure to endotoxin is often associated with chronic alcoholism, this may be one mechanism to account for loss of macrophages in alcoholic patients. The loss of macrophages, as a consequence of endotoxin treatment, appears to be linked to cell activation and, in particular, LPS-stimulated synthesis of nitric oxide which has been suggested to cause an increase in apoptosis. Ethanol also increases apoptosis in some cell types but, in general, ethanol inhibits activation of macrophages. Thus, the overall effect on cell numbers and cell proliferation elicited by treating macrophages concomitantly with ethanol and LPS depends on the balance between inhibiting LPS-mediated activation and the actions of ethanol. The interaction between ethanol and LPS was investigated in a macrophage cell line (RAW 264.7 cells) by measuring nitric oxide production and cell proliferation. A 24-h exposure to ethanol (100 mM) decreased [3H]-thymidine incorporation significantly. LPS treatment elicited a concentration-dependent decrease in [3H]-thymidine incorporation at LPS concentrations of 0.1 ng/ml to 1000 ng/ml and stimulated nitric oxide production at concentrations above 1 ng/ml. LPS-stimulated nitric oxide production was inhibited by ethanol (20 to 100 mM) and the nitric oxide synthesis inhibitor, N(G)Nitro-L-arginine methyl L-NAME) ester (100 and 500 microM). However, LPS-inhibited [3H]-thymidine incorporation was not be totally reversed by ethanol- or L-NAME-treatment. A direct correlation between nitric oxide production and inhibition of cell proliferation could not be demonstrated. However, it appears that ethanol and LPS do affect some common mechanism(s) in this cell line.

Presentation of acute ST segment elevation myocardial infarction in the setting of acute subclavian artery thrombosis in a patient who underwent coronary artery bypass grafting with a left internal mammary artery graft, which is not believed to have been previously described. We report a 75-year-old woman with presentations of dizziness, nausea, left-arm numbness, and a cold left hand, who later had chest pain develop. Acute ST segment elevation myocardial infarction was diagnosed, and both a computed tomography and an angiography disclosed a thrombus extending from the proximal portion of the left subclavian artery to the orifice of the left internal mammary artery. The patient was free from the previously listed symptoms after undergoing emergent thrombectomy, with complete extraction of the long thrombus from the subclavian artery. Unfortunately, she died of pneumonia and septic shock 1 1/2 months later.