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Urinary extracellular vesicles (uEVs) are emerging as non-invasive biomarkers for various kidney diseases, but it is unknown how differences in nephron mass impact uEV excretion. To address this, uEV excretion was measured before and after human kidney donor nephrectomy and rat nephrectomy. In male and female donors, uEVs were quantified in cell-free spot and 24-h urine samples using nanoparticle tracking analysis (NTA), EVQuant, and CD9-time-resolved fluorescence immunoassay. Female donors had significantly lower total kidney volume (TKV) and excreted 49% fewer uEVs than male donors. uEV excretion correlated positively with estimated glomerular filtration rate (eGFR), creatinine clearance, and TKV (R's between 0.6 and 0.7). uEV excretion rate could also be predicted from spot urines after multiplying spot uEV/creatinine by 24-h urine creatinine. Donor nephrectomy reduced eGFR by 36% ± 10%, but the excretion of uEVs by only 16% (CD9+ uEVs -37%, CD9- uEVs no decrease). Donor nephrectomy increased the podocyte marker WT-1 and the proximal tubule markers NHE3, NaPi-IIa, and cubilin in uEVs two- to four-fold when correcting for the nephrectomy. In rats, the changes in GFR and kidney weight correlated with the changes in uEV excretion rate (R = 0.46 and 0.60, P th nephrectomy). Taken together, our data show that uEV excretion depends on nephron mass, and that nephrectomy reduces uEV excretion less than expected based on nephron loss due to compensatory hypertrophy. The major implication of our findings is that a measure for nephron mass or uEV excretion rate should be included when comparing uEV biomarkers between individuals.

Targeted color-dots with varying shapes and sizes in images are first exhaustively identified, and then their multiscale 2D geometric patterns are extracted for testing spatial uniformness in a progressive fashion. Based on color theory in physics, we develop a new color-identification algorithm relying on highly associative relations among the three color-coordinates: RGB or HSV. Such high associations critically imply low color-complexity of a color image, and renders potentials of exhaustive identification of targeted color-dots of all shapes and sizes. Via heterogeneous shaded regions and lighting conditions, our algorithm is shown being robust, practical and efficient comparing with the popular Contour and OpenCV approaches. Upon all identified color-pixels, we form color-dots as individually connected networks with shapes and sizes. We construct minimum spanning trees (MST) as spatial geometries of dot-collectives of various size-scales. Given a size-scale, the distribution of distances between immediate neighbors in the observed MST is extracted, so do many simulated MSTs under the spatial uniformness assumption. We devise a new algorithm for testing 2D spatial uniformness based on a Hierarchical clustering tree upon all involving MSTs. Our developments are illustrated on images obtained by mimicking chemical spraying via drone in Precision Agriculture., 21 pages, 21 figures

Background Triage plays a crucial role in the emergency department (ED) management of mass casualty incidents (MCIs) when resources are limited. This study aimed to compare the performance of simple triage and rapid treatment (START) with that of the Taiwan Triage and Acuity Scale (TTAS) for the ED triage of victims following an earthquake-related MCI. Methods We retrospectively reviewed the records of victims presenting at our ED with earthquake-related injuries within 24 h of a large-scale earthquake. TTAS was initially used at our ED for this event, and START was performed by retrospectively reviewing the patient records in a blinded manner. Area under the receiver operating characteristic curve (AUC), sensitivity, and specificity of START and TTAS were determined for predicting ED discharge. Results We enrolled 105 patients (predominantly women, 60.0%; median age, 45.0 years) in this study; most of them presented with traumatic injuries and were initially triaged as TTAS level III (78.1%), followed by TTAS level II (11.4%). Although the majority of the victims (81.0%) were discharged, four deaths occurred. A moderate agreement in differentiating emergency from nonemergency patients was observed between START and TTAS. Furthermore, both the triage systems showed similar predictions for ED disposition (START AUC/sensitivity/specificity: 0.709/82.35%/55.00%; TTAS AUC/sensitivity/specificity: 0.709/90.59%/45.00%). Conclusions The present study demonstrated that START and TTAS have similar triage accuracy and ability to predict ED disposition. Our findings demonstrate that START may be used as an alternative to TTAS for the ED triage of victims following earthquake-related MCIs.

Acute respiratory distress syndrome (ARDS) is an inflammatory lung disorder that frequently complicates critical illness and commonly occurs in sepsis. Although numerous clinical and environmental risk factors exist, not all patients with risk factors develop ARDS, raising the possibility of genetic underpinnings for ARDS susceptibility. We have previously reported that circulating cell-free hemoglobin (CFH) is elevated during sepsis, and higher levels predict worse outcomes. Excess CFH is rapidly scavenged by haptoglobin (Hp). A common HP genetic variant, HP2, is unique to humans and is common in many populations worldwide. HP2 haptoglobin has reduced ability to inhibit CFH-mediated inflammation and oxidative stress compared with the alternative HP1. We hypothesized that HP2 increases ARDS susceptibility during sepsis when plasma CFH levels are elevated. In a murine model of sepsis with elevated CFH, transgenic mice homozygous for Hp2 had increased lung inflammation, pulmonary vascular permeability, lung apoptosis, and mortality compared with wild-type mice. We then tested the clinical relevance of our findings in 496 septic critically ill adults, finding that HP2 increased ARDS susceptibility after controlling for clinical risk factors and plasma CFH. These observations identify HP2 as a potentially novel genetic ARDS risk factor during sepsis and may have important implications in the study and treatment of ARDS.

The influenza A virus RNA-dependent RNA polymerase complex consists in three subunits, PB2, PB1 and PA, that perform transcription and replication of the viral genome through very distinct mechanisms. Biochemical and structural studies have revealed that the polymerase can adopt multiple conformations and form oligomers. However so far it remained unclear whether the available oligomeric crystal structures represent a functional state of the polymerase. Here we gained new insights into this question, by investigating the incompatibility between non-cognate subunits of influenza polymerase brought together through genetic reassortment. We observed that a 7:1 reassortant virus whose PB2 segment derives from the A/WSN/33 (WSN) virus in an otherwise A/PR/8/34 (PR8) backbone is attenuated, despite a 97% identity between the PR8-PB2 and WSN-PB2 proteins. Independent serial passages led to the selection of phenotypic revertants bearing distinct second-site mutations on PA, PB1 and/or PB2. The constellation of mutations present on one revertant virus was studied extensively using reverse genetics and cell-based reconstitution of the viral polymerase. The PA-E349K mutation appeared to play a major role in correcting the initial defect in replication (cRNA -> vRNA) of the PR8xWSN-PB2 reassortant. Strikingly the PA-E349K mutation, and also the PB2-G74R and PB1-K577G mutations present on other revertants, are located at a dimerization interface of the polymerase. All three restore wild-type-like polymerase activity in a minigenome assay while decreasing the level of polymerase dimerization. Overall, our data show that the polymerase subunits co-evolve to ensure not only optimal inter-subunit interactions within the heterotrimer, but also proper levels of dimerization of the heterotrimer which appears to be essential for efficient viral RNA replication. Our findings point to influenza polymerase dimerization as a feature that is controlled by a complex interplay of genetic determinants, can restrict genetic reassortment, and could become a target for antiviral drug development., Author summary Influenza viruses, because of their yearly recurrence and the occasional emergence of pandemics, represent a worldwide major public health threat. Enhancing the fundamental knowledge on the influenza RNA-dependent RNA-polymerase, a PB1-PB2-PA heterotrimer which ensures transcription and replication of the viral genome, is essential to reach the goal of better prevention and treatment of the disease. Here we gained new insights into the viral polymerase function by investigating the incompatibility between polymerase subunits derived from distinct parental influenza viruses and brought together through genetic reassortment. Our data show that the polymerase subunits co-evolve to ensure not only optimal inter-subunit cooperation within the heterotrimer, but also proper levels of dimerization of the heterotrimer which appears to be essential for efficient viral RNA replication. Our findings point to influenza polymerase dimerization as a feature that can restrict genetic reassortment, a major evolutionary mechanism for influenza viruses, and could become an attractive target for antiviral drug development.

Introduction Few studies have reported outcomes of lung cancer patients with acute respiratory failure (RF) using noninvasive positive pressure ventilation (NIPPV). The aim of this study was to investigate the prognostic factors in these patients. Materials and methods This retrospective observational study included all hospitalized lung cancer patients who received NIPPV for acute RF. It was conducted at a tertiary medical center in Taiwan from 2005 to 2010. The primary outcome was all cause mortality at 28 days after the initiation of NIPPV. Secondary outcomes included all-cause in-hospital mortality, weaning from NIPPV, intubation rate, tracheostomy rate, duration of NIPPV, hospital stay and intensive care unit stay. Results The all-cause mortality rate at day 28 of the enrolled 58 patients was 39.66%. The 90-day and 1-year mortality rates were 63.79% and 86.21%, respectively. NIPPV as the first line therapy for RF had higher 28-day mortality rate than it used for post-extubation RF (57.6% versus 16.0%, p

Object Traumatic intracranial hemorrhage (TICH) patients with acute kidney injury (AKI) were reported to have a high mortality rate. Renal replacement therapy (RRT) is indicated for patients with a severe kidney injury. This study aimed to compare the effects of different RRT modalities regarding chronic dialysis rate among adult TICH patients with AKI. Methods A retrospective search of computerized hospital records from 2000 to 2010 for patients with a discharge diagnosis of TICH was conducted to identify the index cases. We collected the data of TICH patients with increased intracranial pressure combined with severe AKI who received intermittent hemodialysis (IHD) or continuous veno-venous hemofiltration (CVVH) as RRT. The outcome was dialysis dependence between 2000 and 2010. Results From a total of 310 patients who were enrolled in the study, 134 (43%) received CVVH and 176 (57%) received IHD. The risk of dialysis dependency was significantly lower in the CVVH group than in the IHD group (adjusted hazard ratio: 0.368, 95% CI, 0.158–0.858, P = 0.034). Diabetes mellitus and coronary artery disease were risk factors for dialysis dependency. CVVH compared with IHD modality was associated with lower dialysis dependency rate in TICH patients combined with AKI and diabetes mellitus and those with an injury severity score (ISS) ≥16. Conclusion CVVH may yield better renal outcomes than IHD among TICH patients with AKI, especially those with diabetes mellitus and an ISS ≥16. The beneficial impact of CVVH on TICH patients needs to be clarified in a large cohort study in future.

Exposure to thirdhand smoke (THS) is a recently described health concern that arises in many indoor environments. However, the carcinogenic potential of THS, a critical consideration in risk assessment, remains untested. Here we investigated the effects of short-term early exposure to THS on lung carcinogenesis in A/J mice. Forty weeks after THS exposure from 4 to 7 weeks of age, the mice had increased incidence of lung adenocarcinoma, tumor size and, multiplicity, compared with controls. In vitro studies using cultured human lung cancer cells showed that THS exposure induced DNA double-strand breaks and increased cell proliferation and colony formation. RNA sequencing analysis revealed that THS exposure induced endoplasmic reticulum stress and activated p53 signaling. Activation of the p53 pathway was confirmed by an increase in its targets p21 and BAX. These data indicate that early exposure to THS is associated with increased lung cancer risk.

Background We evaluated the risk of osteoporosis in patients with primary biliary cirrhosis (PBC) using a nationwide population-based dataset. Methods In a cohort study of 986,713 individuals, we selected 2,493 PBC patients who were aged 18 years or older and had been diagnosed with PBC, based on the International Classification of Disease (ICD-9-CM) codes 571.6, during 20002010. The control cohort comprised 9,972 randomly selected, propensity matched patients (by age, gender, and index date), without PBC. Using this adjusted data, a possible association between PBC and the risk of developing osteoporosis was estimated using a Cox proportional hazard regression model. Results During the follow-up period, osteoporosis was diagnosed in 150 (6.02%) patients in the PBC cohort and in 539 (5.41%) patients in the non-PBC cohort. After adjusting for covariates, osteoporosis risk was found to be 3.333 times greater in the PBC cohort than in the non-PBC cohort when measured over 6 years after PBC diagnosis. Stratification revealed that the use of ursodeoxycholic acid (UDCA) had no significance in decreasing the risk of osteoporosis when comparing the PBC cohorts with the non-PBC cohorts (P = 0.124). Additionally, osteoporosis risk was significantly higher in PBC patients with steroid use (aHR: 6.899 vs 3.333). Moreover, when comparing the PBC cohorts to the non-PBC cohorts, the non-cirrhotic patients were prone to osteoporosis at a younger age compared to those in the cirrhotic cohorts. We also found that the associated risk of fractures is only prominent for vertebral and wrist fractures in the PBC cohort compared to that in the non-PBC cohort. Conclusion A significant association exists between PBC and subsequent risk for osteoporosis. Therefore, PBC patients, particularly those treated with steroids, should be evaluated for subsequent risk of osteoporosis.

Proteome analysis of serum from type 2 diabetics with complications may lead to the discovery of diagnostic or prognostic biomarkers. To circumvent the principal barrier of serum proteomics, our investigation aimed to evaluate whether a study of post-translational modification enriched serum proteins could be valuable for the discovery of biomarkers or metabolic pathways related to type 2 diabetes pathogenesis. Type 2 diabetes was induced from high-fat diet fed Sprague Dawley rats with streptozotocin injection. Once diabetic status was confirmed, serum samples from either fasted healthy or diabetic rats were pooled and profiled by two-dimensional difference gel electrophoresis or comparative 2D electrophoresis after protein enrichments using immobilized metal ion, concanavalin A, and lentil affinity chromatography, respectively. Differential expressed proteins were identified and the associated networks were established by an Ingenuity Pathway Analysis. As a result, induced rats became severe diabetic and accompanied by hyperlipidemia, fatty liver, and glomerular hypertrophy. There were 3 total, 14 phosphorylated and 23 glycosylated protein targets differentially expressed. Proteins could be linked to HNF4A, HNF1A, and NFκB transcriptional factors and antigen presentation, humoral immune response, and inflammatory response pathways. Predicted organ toxicity in kidney, heart, and liver matched with our histopathological results. In conclusion, post-translational modification based serum protein enrichment could be a valuable approach to enhance the resolution of serum proteomics without depleting potentially valuable abundant proteins. Our results also indicated the potential association of the hepatic secretome and hepatocyte nuclear factors in the pathogenesis of type 2 diabetes and its complications.

Objectives. To demonstrate the use of acupuncture in the lower limbs to treat myofascial pain of the upper trapezius muscles via a remote effect.Methods. Five adults with latent myofascial trigger points (MTrPs) of bilateral upper trapezius muscles received acupuncture at Weizhong (UB40) and Yanglingquan (GB34) points in the lower limbs. Modified acupuncture was applied at these points on a randomly selected ipsilateral lower limb (experimental side) versus sham needling on the contralateral lower limb (control side) in each subject. Each subject received two treatments within a one-week interval. To evaluate the remote effect of acupuncture, the range of motion (ROM) upon bending the contralateral side of the cervical spine was assessed before and after each treatment.Results. There was significant improvement in cervical ROM after the second treatment (P=0.03) in the experimental group, and the increased ROM on the modified acupuncture side was greater compared to the sham needling side (P=0.036).Conclusions. A remote effect of acupuncture was demonstrated in this pilot study. Using modified acupuncture needling at remote acupuncture points in the ipsilateral lower limb, our treatments released tightness due to latent MTrPs of the upper trapezius muscle.

Benign prostatic hyperplasia (BPH), an imbalance between androgen/estrogen, overexpression of stromal, and epithelial growth factors associated with chronic inflammation, has become an atypical direct cause of mortality of aged male diseases.Ginkgopossesses anti-inflammatory, blood flow-enhancing, and free radical scavenging effects. Considering strenuous exercise can reduce BPH risks, we hypothesizeGinkgo+ exercise (Ginkgo+ Ex) could be beneficial to BPH. To verify this, rat BPH model was induced by s.c. 3.5 mg testosterone (T) and 0.1 mg estradiol (E2) per head per day successively for 8 weeks, using mineral oil as placebo. Cerenin®8.33 μL/100 g was applied s.c. from the 10th to the 13th week, and simultaneously, Ex was applied (30 m/min, 3 times/week). In BPH,Ginkgoalone had no effect on T, 5α-reductase, and dihydrotestosterone (DHT), but suppressed androgen receptor (AR), aromatase, E2 and estrogen receptor (ER), and the proliferating cell nuclear antigen (PCNA); Ex alone significantly reduced T, aromatase, E2, ER, AR, and PCNA, but highly raised DHT. WhileGinkgo+ Ex androgenically downregulated T, aromatase, E2, and ER, but upregulated DHT, AR, and PCNA, implyingGinkgo+ Ex tended to worsen BPH. Conclusively,Ginkgoor Ex alone may be more beneficial thanGinkgo+ Ex for treatment of BPH.

Eukaryotic initiation factor 5A (eIF-5A) is the only protein in nature that contains hypusine, an unusual amino acid formed post-translationally by deoxyhypusine synthase and deoxyhypusine hydroxylase. Genetic and pharmacological evidence suggests that eIF-5A is essential for cell survival and proliferation. However, the precise function and interacting partners of eIF-5A remain unclear. We have shown previously that eIF-5A can bind to RRE (Rev-response element) and U6 RNA in vitro. Using SELEX (systematic evolution of ligands by exponential enrichment), we have also shown that eIF-5A is capable of binding to RNA in a sequence-specific manner [Xu and Chen (2001) J. Biol. Chem. 276, 2555–2561]. In the present paper, we show that the identification of mRNA species that bind to eIF-5A can be achieved by affinity co-purification and PCR differential display. Using this approach with three sets of anchoring and arbitrary primers, we have found 20 RNA sequences that co-purified specifically with eIF-5A. Five of them contained AAAUGU, the putative eIF-5A-interacting element that we identified previously using the SELEX method. Direct binding of the cloned RNA to eIF-5A could be demonstrated by electrophoretic mobility-shift assay. BLAST analysis revealed that the eIF-5A-interacting RNAs encode proteins such as ribosomal L35a, plasminogen activation inhibitor mRNA-binding protein, NADH dehydrogenase subunit and ADP-ribose pyrophosphatase. Some, however, encode hypothetical proteins. All the cloned RNAs have the potential to form extensive stem-loop structures.

Background and Purpose Timely intravenous (IV) thrombolysis for acute ischemic stroke is associated with better clinical outcomes. Acute stroke care implemented with “Stroke Code” (SC) may increase IV tissue plasminogen activator (tPA) administration. The present study aimed to investigate the impact of SC on thrombolysis. Methods The study period was divided into the “pre-SC era” (January 2006 to July 2010) and “SC era” (August 2010 to July 2013). Demographics, critical times (stroke symptom onset, presentation to the emergency department, neuroimaging, thrombolysis), stroke severity, and clinical outcomes were recorded and compared between the two eras. Results During the study period, 5957 patients with acute ischemic stroke were admitted; of these, 1301 (21.8%) arrived at the emergency department within 3 h of stroke onset and 307 (5.2%) received IV-tPA. The number and frequency of IV-tPA treatments for patients with an onset-to-door time of

Background Microarray technology can acquire information about thousands of genes simultaneously. We analyzed published breast cancer microarray databases to predict five-year recurrence and compared the performance of three data mining algorithms of artificial neural networks (ANN), decision trees (DT) and logistic regression (LR) and two composite models of DT-ANN and DT-LR. The collection of microarray datasets from the Gene Expression Omnibus, four breast cancer datasets were pooled for predicting five-year breast cancer relapse. After data compilation, 757 subjects, 5 clinical variables and 13,452 genetic variables were aggregated. The bootstrap method, Mann-Whitney U test and 20-fold cross-validation were performed to investigate candidate genes with 100 most-significant p-values. The predictive powers of DT, LR and ANN models were assessed using accuracy and the area under ROC curve. The associated genes were evaluated using Cox regression. Results The DT models exhibited the lowest predictive power and the poorest extrapolation when applied to the test samples. The ANN models displayed the best predictive power and showed the best extrapolation. The 21 most-associated genes, as determined by integration of each model, were analyzed using Cox regression with a 3.53-fold (95% CI: 2.24-5.58) increased risk of breast cancer five-year recurrence… Conclusions The 21 selected genes can predict breast cancer recurrence. Among these genes, CCNB1, PLK1 and TOP2A are in the cell cycle G2/M DNA damage checkpoint pathway. Oncologists can offer the genetic information for patients when understanding the gene expression profiles on breast cancer recurrence.

Ferulic acid (4-hydroxy-3-methoxycinnamic acid) (FA) is a ubiquitous health beneficial phenolic acid. Although FA has shown a diversity of biological activities including anti-inflammatory, antihypercholesterolemic and anticancer bioactivities, studies revealing its adverse effects are accumulating. Recently, 3D-cultures are shown to exhibit uniquely biological behaviors different from that of 2D cultures. To understand whether the cytotoxicity of FA against the T24 cell line (a bladder cancer cell line) in 2D-culture could consistently retain similar bioactivity if cultured in the 3D-systems, we conducted this experiment with 2 mM FA. Much higher cytotoxicity was found for 3D- than 2D-culture, showing (2D vs. 3D): apoptotic rates, 64% and 76%; cell killing rates,3.00×105 cells mmol−1·h−1and2.63×106cells mmol−1·h−1, attaining a 8.77-fold. FA upregulated the activities at 72 h (2D vs. 3D in folds that of control): SOD, 1.73-folds (P<0.05) versus 3.18 folds (P<0.001); and catalase, 2.58 versus 1.33-folds. Comparing to the control (without FA), Bcl-2 was prominently downregulated while Bax, caspase-3 and cleaved caspase-9 were more upregulated in 3D-cultures (P<0.05). Conclusively, different microenvironments could elicit different biological significance which in part can be ascribed to different mass transport rate.

Deoxyhypusine synthase is an NAD(+)-dependent enzyme that catalyses the formation of a deoxyhypusine residue on the eukaryotic initiation factor 5A (eIF-5A) precursor by transferring an aminobutyl moiety from spermidine to the epsilon-amino group of a unique lysine residue. We have recently cloned and characterized the Neurospora crassa deoxyhypusine synthase cDNA using a reverse genetics approach. A GenBank search showed that a stretch of the deduced amino acid sequence (96 amino acids) of Neurospora deoxyhypusine synthase matches a short human expressed sequence tag (EST), {"type":"entrez-nucleotide","attrs":{"text":"Z25337","term_id":"396018","term_text":"Z25337"}}Z25337, with greater than 70% amino acid identity. Gene-specific primers based on this EST were used together with universal primers to obtain 1219 bp and 1078 bp cDNAs from a human cDNA library. The 1219 bp and 1078 bp sequences, each containing an open reading frame, encode polypeptides of respectively 368 and 321 amino acids. The short sequence is identical to the long one except that it is missing a stretch of 47 amino acids spanning residues 261-307. The 368-amino-acid sequence of human deoxyhypusine synthase shares a high degree of identity ( > 50%) and similarity ( > 60%) with that of the Neurospora and yeast deoxyhypusine synthases. After cloning into an expression vector, the 368-amino-acid recombinant protein exhibits high deoxyhypusine synthase activity. In contrast, the 321-amino-acid recombinant protein shows no detectable activity.

The developmental stage- and erythroid lineage-specific activation of the human embryonic zeta- and fetal/adult alpha-globin genes is controlled by an upstream regulatory element [hypersensitive site (HS)-40] with locus control region properties, a process mediated by multiple nuclear factor-DNA complexes. In vitro DNase I protection experiments of the two G+C-rich, adult alpha-globin promoters have revealed a number of binding sites for nuclear factors that are common to HeLa and K-562 extracts. However, genomic footprinting analysis has demonstrated that only a subset of these sites, clustered between -130 and +1, is occupied in an erythroid tissue-specific manner. The function of these in vivo-occupied motifs of the alpha-globin promoters, as well as those previously mapped in the HS-40 region, is assayed by site-directed mutagenesis and transient expression in embryonic/fetal erythroid K-562 cells. These studies, together with our expression data on the human embryonic zeta-globin promoter, provide a comprehensive view of the functional roles of individual nuclear factor-DNA complexes in the final stages of transcriptional activation of the human alpha-like globin promoters by the HS-40 element.

Life-threatening refractory metabolic acidosis due to starvation ketoacidosis is rarely reported, even among nondiabetic pregnant women, and may be overlooked. Furthermore, stressful situations may increase the acidosis severity. In the present case, a nondiabetic multiparous woman was admitted for a near-fatal asthma attack and vomiting during the third trimester of pregnancy. She was intubated and rapidly developed high anion gap metabolic acidosis. We diagnosed the patient with starvation ketoacidosis based on vomiting with concomitant periods of stress during pregnancy and the absence of other causes of high anion gap metabolic acidosis. She responded poorly to standard treatment, although the ketoacidosis and asthma promptly resolved after an emergency caesarean section. The patient and her baby were safely discharged. Short-term starvation, if it occurs during periods of stress and medication, can result in life-threatening ketoacidosis, even among nondiabetic women during the third trimester of pregnancy. Awareness of this condition may facilitate prompt recognition and proactive treatment for dietary and stress control, and emergent interventions may also improve outcomes.

Osmoregulation is important to living organisms for survival in responding to environmental changes of water and ionic strength. We demonstrated here for the first time that exposure of HeLa cells to a hypotonic medium (30% growth medium and 70% water) prominently induced the binding activity of the heat shock transcription factor (HSF). Pretreatment of cells with cycloheximide did not inhibit the induction of HSF-binding activity, indicating that the mechanisms of induction are independent of new protein synthesis. The magnitude of hypo-osmotic stress-induced HSF-binding activity was comparable with that induced by heat shock. The induction, as monitored by gel-mobility-shift assay, occurred within 5 min of hypo-osmotic stress and persisted at least up to 4 h in HeLa cells under the hypotonic conditions. Addition of sorbitol to the hypotonic medium abolished HSF activation. Hypo-osmotic stress-induced HSF binding could also be demonstrated in HeLa cells maintained in simple sorbitol solution by decreasing the sorbitol concentration from 300 mM to 200 mM or less. Competition analysis suggests that the effects of hypo-osmotic stress on HSF-binding activity was specific. Cross-linking experiments and Western-blot analysis demonstrated that hypo-osmotic stress induced trimerization of human heat shock factor 1 (HSF1) in intact HeLa cells, suggesting that trimer formation of HSF1 was responsible for inducing HSF-binding activity in hypo-osmotically stressed cells. However, unlike heat shock response, the activation of HSF by hypo-osmotic stress did not lead to accumulation of hsp70 mRNA in HeLa cells.

Eukaryotic initiation factor 5A (eIF-5A) is the only cellular protein known to contain a hypusine residue that is formed by transferring the aminobutyl moiety from spermidine to a specific lysine residue, followed by hydroxylation at the aminobutyl group. A simple PCR-based strategy was developed to obtain a full-length cDNA of Neurospora crassa eIF-5A. The strategy consists of (i) the design of a pair of key primers (21-mer) based on the highly conserved eIF-5A cDNA domains known in other species, (ii) PCR amplification of Neurospora cDNA using the two key primers to obtain the core sequence for the design of core primers, and (iii) combined use of the key primers, core primers and the universal primers, T3 and T7, to amplify the target sequence in a Neurospora cDNA library. The longest cDNA obtained was cloned into pBlueScript phagemid, and sequence analysis indicated that it encodes a polypeptide of 163 amino acid residues with a codon usage preference characteristic of abundant Neurospora genes. The Neurospora polypeptide showed 59% and 67% identity with human and yeast eIF-5A precursor protein respectively. We subcloned the Neurospora eIF-5A cDNA into pQE-30, which introduces six adjacent histidine residues to the N-terminus of the recombinant protein. The resulting plasmid, pQTy21, was overexpressed in Escherichia coli, and the soluble polyhistidine-tagged protein was purified by metal chelation chromatography. We obtained about 60 mg of purified eIF-5A precursor from 1 litre of culture in a single step using a Ni(II)-nitrilotriacetic acid (NTA)-agarose column. The histidine-tagged eIF-5A precursor protein could be recognized by anti-Neurospora crassa 21 kDa protein serum raised against wild-type eIF-5A precursor and could serve as the substrate protein for deoxyhypusine synthase. Using the histidine-tagged recombinant protein and the Ni(II)-NTA-agarose column, we constructed a protein affinity column and demonstrated an affinity binding between eIF-5A precursor and deoxyhypusine synthase in the presence of NAD+. One-step eIF-5A precursor affinity-column chromatography could lead to a 30-fold purification of deoxyhypusine synthase.

Purpose A retrospective study to evaluate the effect of hyperbaric oxygen (HBO2) therapy on sternal infection and osteomyelitis following median sternotomy. Materials and methods A retrospective analysis of patients who received sternotomy and cardiothoracic surgery which developed sternal infection and osteomyelitis between 2002 and 2009. Twelve patients who received debridement and antibiotic treatment were selected, and six of them received additional HBO2 therapy. Demographic, clinical characteristics and outcome were compared between patients with and without HBO2 therapy. Results HBO2 therapy did not cause any treatment-related complication in patients receiving this additional treatment. Comparisons of the data between two study groups revealed that the length of stay in ICU (8.7 ± 2.7 days vs. 48.8 ± 10.5 days, p < 0.05), duration of invasive (4 ± 1.5 days vs. 34.8 ± 8.3 days, p < 0.05) and non-invasive (4 ± 1.9 days vs. 22.3 ± 6.2 days, p < 0.05) positive pressure ventilation were all significantly lower in patients with additional HBO2 therapy, as compared to patients without HBO2 therapy. Hospital mortality was also significantly lower in patients who received HBO2 therapy (0 case vs. 3 cases, p < 0.05), as compared to patients without the HBO2 therapy. Conclusions In addition to primary treatment with debridement and antibiotic use, HBO2 therapy may be used as an adjunctive and safe treatment to improve clinical outcomes in patients with sternal infection and osteomyelitis after sternotomy and cardiothoracic surgery.

Background One aspect of brain death is cardiovascular deregulation because asystole invariably occurs shortly after its diagnosis. A suitable neural substrate for mechanistic delineation of this aspect of brain death resides in the rostral ventrolateral medulla (RVLM). RVLM is the origin of a life-and-death signal that our laboratory detected from blood pressure of comatose patients that disappears before brain death ensues. At the same time, transcriptional upregulation of heme oxygenase-1 in RVLM by hypoxia-inducible factor-1α (HIF-1α) plays a pro-life role in experimental brain death, and HIF-1α is subject to sumoylation activated by transient cerebral ischemia. It follows that sumoylation of HIF-1α in RVLM in response to hypoxia may play a modulatory role on brain stem cardiovascular regulation during experimental brain death. Methodology/Principal Findings A clinically relevant animal model that employed mevinphos as the experimental insult in Sprague-Dawley rat was used. Biochemical changes in RVLM during distinct phenotypes in systemic arterial pressure spectrum that reflect maintained or defunct brain stem cardiovascular regulation were studied. Western blot analysis, EMSA, ELISA, confocal microscopy and immunoprecipitation demonstrated that drastic tissue hypoxia, elevated levels of proteins conjugated by small ubiquitin-related modifier-1 (SUMO-1), Ubc9 (the only known conjugating enzyme for the sumoylation pathway) or HIF-1α, augmented sumoylation of HIF-1α, nucleus-bound translocation and enhanced transcriptional activity of HIF-1α in RVLM neurons took place preferentially during the pro-life phase of experimental brain death. Furthermore, loss-of-function manipulations by immunoneutralization of SUMO-1, Ubc9 or HIF-1α in RVLM blunted the upregulated nitric oxide synthase I/protein kinase G signaling cascade, which sustains the brain stem cardiovascular regulatory machinery during the pro-life phase. Conclusions/Significance We conclude that sumoylation of HIF-1α in RVLM ameliorates brain stem cardiovascular regulatory failure during experimental brain death via upregulation of nitric oxide synthase I/protein kinase G signaling. This information should offer new therapeutic initiatives against this fatal eventuality.

Background Elevated baseline C-reactive protein (CRP) levels are associated with increased risk for developing cardiovascular disease. Several CRP gene variants have been associated with altered baseline CRP levels in ambulatory populations. However, the influence of CRP gene variants on CRP levels during inflammatory states, such as surgery, is largely unexplored. We describe the association between candidate CRP gene variants and postoperative plasma CRP levels in patients undergoing primary, elective coronary artery bypass graft (CABG) surgery with cardiopulmonary bypass (CPB). Methods Using a multicenter candidate gene association study design, we examined the association between seventeen candidate CRP single nucleotide polymorphisms (SNPs) and inferred haplotypes, and altered postoperative CRP levels in 604 patients undergoing CABG surgery with CPB. Perioperative CRP levels were measured immediately prior to surgery, post-CPB and on postoperative days (POD) 1–4. Results CRP levels were significantly elevated at all postoperative time points when compared with preoperative levels (P < 0.0001). After adjusting for clinical covariates, the minor allele of the synonymous coding SNP, rs1800947 was associated with lower peak postoperative CRP levels (P = 2.4 × 10-4) and lower CRP levels across all postoperative time points (P = 4.8 × 10-5). rs1800947 remained highly significant after Bonferroni adjustment for multiple comparisons. Conclusion We identified a CRP gene SNP associated with lower postoperative CRP levels in patients undergoing CABG surgery with CPB. Further investigation is needed to clarify the significance of this association between CRP gene variants and the acute-phase rise in postoperative CRP levels with regard to the risk of adverse postoperative outcomes.

BACKGROUND: Induction of the heat shock response (HSR) and increased expression of the heat shock proteins (HSPs) provide mechanisms to ensure proper protein folding, trafficking, and disposition. The importance of HSPs is underscored by the understanding that protein mis-folding and aggregation contribute centrally to the pathogenesis of neurodegenerative diseases. METHODOLOGY/PRINCIPAL FINDINGS: We used a cell-based hsp70-luciferease reporter gene assay system to identify agents that modulate the HSR and show here that clinically relevant concentrations of the FDA-approved ALS drug riluzole significantly increased the heat shock induction of hsp70-luciferse reporter gene. Immuno-Western and -cytochemical analysis of HSF1 show that riluzole increased the amount of cytosolic HSF1 to afford a greater activation of HSF1 upon heat shock. The increased HSF1 contributed centrally to the cytoprotective activity of riluzole as hsf1 gene knockout negated the synergistic activity of riluzole and conditioning heat shock to confer cell survival under oxidative stress. Evidence of a post-transcriptional mechanism for the increase in HSF1 include: quantitation of mRNA(hsf1) by RT-PCR showed no effect of either heat shock or riluzole treatment; riluzole also increased the expression of HSF1 from a CMV-promoter; analysis of the turnover of HSF1 by pulse chase and immunoprecipitation show that riluzole slowed the decay of [(35)S]labeled-HSF1. The effect of riluzole on HSF1 was qualitatively different from that of MG132 and chloroquine, inhibitors of the proteasome and lysosome, respectively, and appeared to involve the chaperone-mediated autophagy pathway as RNAi-mediated knockdown of CMA negated its effect. CONCLUSION/SIGNIFICANCE: We show that riluzole increased the amount of HSF1 to amplify the HSR for cytoprotection. Our study provides novel insight into the mechanism that regulates HSF1 turnover, and identifies the degradation of HSF1 as a target for therapeutics intervention.

Intensive care unit (ICU)-acquired bacteremia (IAB) is associated with high medical expenditure and mortality. Mechanically ventilated patients represent one third of all patients admitted to ICU, but the clinical features and outcomes in mechanically ventilated patients who develop IAB remain unknown. We conducted a 3-year retrospective observational cohort study, and 1,453 patients who received mechanical ventilation on ICU admission were enrolled. Among patients enrolled, 126 patients who had developed IAB ≧48 hours after ICU admission were identified. The study patients were divided into IAB and no IAB groups, and clinical characteristics of IAB based on specific bacterial species were further analyzed. The multivariate Cox regression analysis showed that ventilator support for chronic obstructive pulmonary disease and congestive heart failure, and patients admitted from nursing home were the independent risk factors for developing IAB. Patients with IAB were significantly associated with longer length of ICU stay, prolonged ventilator use, lower rate of successful weaning, and higher rate of ventilator dependence and ICU mortality as compared to those without IAB. IAB was the independent risk factor for ICU mortality (HR, 1.510, 95% CI 1.054-1.123; p = 0.010). The clinical characteristics of IAB related to specific bacterial species included IAB due to Pseudomonas aeruginosa being likely polymicrobial, lung source and prior antibiotic use; Escherichia coli developing earlier and from urinary tract source; methicillin-resistant Staphylococcus aureus related to central venous catheter and multiple sets of positive hemoculture; and Elizabethkingia meningoseptica significantly associated with delayed/inappropriate antibiotic treatment. In summary, IAB was significantly associated with poor patient outcomes in mechanically ventilated ICU patients. The clinical features related to IAB and clinical characteristics of IAB based on specific bacterial species identified in our study may be utilized to refine the management of IAB.

Osmoregulation, the cellular response to environmental changes of osmolarity and ionic strength, is important for the survival of living organisms. We have demonstrated previously that an exposure of mammalian cells to hypo-osmotic stress, either in growth medium (30% growth medium and 70% water) or in binary solution containing sorbitol and water, prominently induced the DNA-binding activity of the heat-shock transcription factor (HSF1) [Huang, Caruccio, Liu and Chen (1995) Biochem. J. 307, 347-352]. Since hyperosmotic and hypo-osmotic stress usually elicit opposite biological responses, we wondered what would be the effect of hyperosmotic stress on HSF activation. In this study we have examined the HSF DNA-binding activity in HeLa cells maintained in the sorbitol/water binary solution over a wide concentration range (0.1-0.9 M) and in Dulbecco's medium supplemented with sorbitol or NaCl. We found that HSF-binding activity could be induced prominently under both hypo-osmotic (0.1-0.25 M) and hyperosmotic conditions (0.50-0.90 M). In both cases, HSF activation was observed within 5 min after changing the osmotic pressure. The activation was accompanied by both HSF trimerization and nuclear translocation, and appeared to be independent of protein synthesis. The effects of hypo- or hyper-osmotic stress on HSF activation could be reversed once the cells were returned to iso-osmotic conditions (0.30 M) with a half-life () of 25 min or less. This rapid turnover of the osmotic-stress-induced HSF-binding activity was inhibited by cycloheximide, a potent inhibitor of protein synthesis. Unlike heat shock, activation of HSF by either hypo- or hyper-osmotic stress did not lead to an accumulation of heat-shock protein 70 (HSP70) mRNA in HeLa cells. We propose that HSF activation during osmotic stress may serve physiological functions independent of the synthesis of heat-shock proteins.

Background Postoperative ventricular dysfunction (VnD) occurs in 9–20% of coronary artery bypass graft (CABG) surgical patients and is associated with increased postoperative morbidity and mortality. Understanding genetic causes of postoperative VnD should enhance patient risk stratification and improve treatment and prevention strategies. We aimed to determine if genetic variants associate with occurrence of in-hospital VnD after CABG surgery. Methods A genome-wide association study identified single nucleotide polymorphisms (SNPs) associated with postoperative VnD in male subjects of European ancestry undergoing isolated primary CABG surgery with cardiopulmonary bypass. VnD was defined as the need for ≥2 inotropes or mechanical ventricular support after CABG surgery. Validated SNPs were assessed further in two replication CABG cohorts and meta-analysis was performed. Results Over 100 SNPs were associated with VnD (P2.1) of developing in-hospital VnD after CABG surgery. However, three genetic loci identified by meta-analysis were more modestly associated with development of postoperative VnD. Studies of larger cohorts to assess these loci as well as to define other genetic mechanisms and related biology that link genetic variants to postoperative ventricular dysfunction are warranted.

Background Human peripheral blood monocytes (Mo) consist of subsets distinguished by expression of CD16 (FCγRIII) and chemokine receptors. Classical CD16- Mo express CCR2 and migrate in response to CCL2, while a minor CD16+ Mo subset expresses CD16 and CX3CR1 and migrates into tissues expressing CX3CL1. CD16+ Mo produce pro-inflammatory cytokines and are expanded in certain inflammatory conditions including sepsis and HIV infection. Results To gain insight into the developmental relationship and functions of CD16+ and CD16- Mo, we examined transcriptional profiles of these Mo subsets in peripheral blood from healthy individuals. Of 16,328 expressed genes, 2,759 genes were differentially expressed and 228 and 250 were >2-fold upregulated and downregulated, respectively, in CD16+ compared to CD16- Mo. CD16+ Mo were distinguished by upregulation of transcripts for dendritic cell (DC) (SIGLEC10, CD43, RARA) and macrophage (MΦ) (CSF1R/CD115, MafB, CD97, C3aR) markers together with transcripts relevant for DC-T cell interaction (CXCL16, ICAM-2, LFA-1), cell activation (LTB, TNFRSF8, LST1, IFITM1-3, HMOX1, SOD-1, WARS, MGLL), and negative regulation of the cell cycle (CDKN1C, MTSS1), whereas CD16- Mo were distinguished by upregulation of transcripts for myeloid (CD14, MNDA, TREM1, CD1d, C1qR/CD93) and granulocyte markers (FPR1, GCSFR/CD114, S100A8-9/12). Differential expression of CSF1R, CSF3R, C1QR1, C3AR1, CD1d, CD43, CXCL16, and CX3CR1 was confirmed by flow cytometry. Furthermore, increased expression of RARA and KLF2 transcripts in CD16+ Mo coincided with absence of cell surface cutaneous lymphocyte associated antigen (CLA) expression, indicating potential imprinting for non-skin homing. Conclusion These results suggest that CD16+ and CD16- Mo originate from a common myeloid precursor, with CD16+ Mo having a more MΦ – and DC-like transcription program suggesting a more advanced stage of differentiation. Distinct transcriptional programs, together with their recruitment into tissues via different mechanisms, also suggest that CD16+ and CD16- Mo give rise to functionally distinct DC and MΦ in vivo.