The complement-inhibitory activity of CD59 resides in its capacity to block incorporation of C9 into membrane C5b-9.
A human E membrane protein that inhibits lysis by the purified human C5b-9 proteins was isolated and characterized. After final purification, the protein migrated as an 18- to 20-kDa band by SDS-PAGE. Elution from gel slices and functional assay after SDS-PAGE (nonreduced) confirmed that all C5b-9 inhibitory activity of the purified protein resided in the 18- to 20-kDa band. Phosphatidylinositol-specific phospholipase C digestion of the purified protein abolished 50% of its C5b-9 inhibitory activity, and removed approximately 15% of the protein from human E. Western blots of normal and paroxysmal nocturnal hemoglobinuria E revealed an absence of the 18- to 20-kDa protein in the paroxysmal nocturnal hemoglobinuria E cells. The identity of this E protein with leukocyte Ag CD59 (P18, HRF20) was confirmed immunochemically and by N-terminal amino acid sequence analysis. A blocking antibody raised against the purified protein reacted with a single 18- to 20-kDa band on Western blots of human erythrocyte membranes. Prior incubation of human E with the F(ab) of this antibody increased subsequent lysis by the purified human C5b-9 proteins. Potentiation of C5b-9-mediated lysis was observed when erythrocytes were preincubated with this blocking antibody before C5b-9 assembly was initiated, or, when this antibody was added after 30 min, 0 degrees C incubation of C5b-8-treated E with C9. Chicken E incubated with purified CD59 were used to further characterize the mechanism of its C-inhibitory activity. Preincorporation of CD59 into these cells inhibited lysis by C5b-9, regardless of whether CD59 was added before or after assembly of the C5b-8 complex. When incorporated into the membrane, CD59 inhibited binding of 125I-C9 to membrane C5b-8 and reduced the extent of formation of SDS-resistant C9 polymer. The inhibitory effect of CD59 on 125I-C9 incorporation was most pronounced at near-saturating input of C9 (to C5b-8). By contrast, CD59 did not inhibit either C5b67 deposition onto the cell surface, or, binding of 125I-C8 to preassembled membrane C5b67. Taken together, these data suggest that CD59 exerts its C-inhibitory activity by limiting incorporation of multiple C9 into the membrane C5b-9 complex.


Cirrhotic patients at increased risk of peptic ulcer bleeding: a nationwide population-based cohort study.
Few large population-based studies have compared the occurrence of peptic ulcer bleeding (PUB) in cirrhotic and noncirrhotic patients. To investigate if cirrhotic patients have higher risk of PUB than the general population and to identify possible risk factors of PUB in cirrhotic patients. Using the National Health Insurance Research Database, a nationwide population-based dataset in Taiwan and matching age, gender, comorbidities and ulcerogenic medication by propensity score, 4013 cirrhotic patients, 8013 chronic hepatitis patients and 7793 normal controls were compared. The log-rank test was used to analyse differences in accumulated PUB-free survival rates between the groups. Cox proportional hazard regressions were performed to evaluate independent risk factors for PUB in all patients and identified risk factors of PUB in cirrhotic patients. During the 7-year follow-up, cirrhotic patients had significantly higher incidences of PUB than chronic hepatitis patients and controls, respectively (P < 0.001 by log-rank test). By Cox proportional hazard regression analysis, cirrhosis was independently associated with increased risk of PUB (hazard ratio: 4.22; 95% CI 3.37-5.29, P < 0.001) after adjusting for age, gender, economic status, underlying comorbidities and ulcerogenic medication. Age, male, diabetes, chronic renal disease, history of gastro-oesophageal variceal bleeding and use of nonsteroidal anti-inflammatory drugs were risk factors for PUB in cirrhotic patients. Cirrhotic patients have a significantly higher risk of peptic ulcer bleeding after adjustments for possible confounding factors like age, gender, economic status, underlying comorbidities and ulcerogenic medication.


Validity of biopsy-based drug effects in a diet-induced obese mouse model of biopsy-confirmed NASH.
Compounds in clinical development for nonalcoholic steatohepatitis (NASH) improve liver histopathology in diet-induced obese mouse models of biopsy-confirmed NASH. Since the biopsy section used for histopathological evaluation represents only < 1% of the whole mouse liver, we evaluated how well biopsy-based quantitative image analyses correlate to stereology-based whole-liver quantitative changes upon drug treatment. Male leptin-deficient Lepob/Lepob mice were fed the Amylin liver NASH (AMLN) diet for 16 weeks before stratification into treatment groups using a biopsy-based evaluation of type I collagen αI (col1a1) levels. Mice were treated for 8 weeks with either vehicle (PO, QD), liraglutide (0.4 mg/kg, SC, QD), elafibranor (30 mg/kg, PO, QD) or INT-767 (10 mg/kg, PO, QD). Terminal quantitative histological assessment of liver lipid (hematoxylin-eosin staining), inflammation (galectin-3 immunohistochemistry (IHC); gal-3), and fibrosis (col1a1 IHC) was performed on terminal liver biopsies and compared with stereologically sampled serial sections spanning the medial, left and right lateral lobe of the liver. The distribution of liver lipid and fibrosis was markedly consistent across lobes, whereas inflammation showed some variability. While INT-767 and liraglutide significantly reduced total liver weight by 20 and 48%, respectively, elafibranor tended to exacerbate hepatomegaly in Lepob/Lepob-NASH mice. All three compounds markedly reduced biopsy-based relative liver lipid content. Elafibranor and INT-767 significantly reduced biopsy-based relative gal-3 levels (P < 0.001), whereas INT-767 and liraglutide tended to reduce relative col1a1 levels. When changes in liver weight was accounted for, both INT-767 and liraglutide significantly reduced biopsy-based total col1a1 content. Although minor differences in absolute and relative liver lipid, inflammation and fibrosis levels were observed across lobes, the interpretation of drug-induced effects were consistent with biopsy-based conclusions. Notably, the incorporation of changes in total liver mass revealed that liraglutide's efficacy reached statistical significances for all analyzed parameters. In conclusion, in-depth analyses of liver homogeneity demonstrated that drug-induced improvement in liver biopsy-assessed histopathology is representative for overall liver effects assessed using stereology. Importantly, these findings reveal how changes in whole-liver mass should be considered to provide a deeper understanding of apparent drug treatment efficacy in preclinical NASH studies.


A coupled diffusion-kinetics model for analysis of contact-area FRAP experiment.
Kinetic rates and binding affinity of receptor-ligand interactions are important determinants of cell adhesion. Measurements of these parameters in fluid phase using soluble molecules (i.e., three-dimensionial parameters) do not necessarily correlate with their counterparts measured when both binding partners are respectively anchored to two apposing surfaces (i.e., two-dimensional (2D) parameters). Moreover, 2D affinities measured by different methods can differ by orders of magnitude. Here we describe a coupled diffusion-reaction model for the fluorescence recovery after photobleaching experiment previously used to demonstrate the dynamics of adhesive bonds in the contact area. Applying the mathematical model to the contact area fluorescence recovery after photobleaching experiment enables in situ measurements of 2D kinetic rates of the adhesion molecules and their retarded diffusion in a stable contact area. The mathematical properties of the model are characterized in this article and its experimental validation will be presented in the companion article.


Adverse Drug Event-based Stratification of Tumor Mutations: A Case Study of Breast Cancer Patients Receiving Aromatase Inhibitors.
Adverse drug events (ADEs) are a critical factor for selecting cancer therapy options. The underlying molecular mechanisms of ADEs associated with cancer therapy drugs may overlap with their antineoplastic mechanisms; an aspect of toxicity. In the present study, we develop a novel knowledge-driven approach that provides an ADE-based stratification (ADEStrata) of tumor mutations. We demonstrate clinical utility of the ADEStrata approach through performing a case study of breast invasive carcinoma (BRCA) patients receiving aromatase inhibitors (AI) from The Cancer Genome Atlas (TCGA) (n=212), focusing on the musculoskeletal adverse events (MS-AEs). We prioritized somatic variants in a manner that is guided by MS-AEs codified as 6 Human Phenotype Ontology (HPO) terms. Pathway enrichment and hierarchical clustering of prioritized variants reveals clusters associated with overall survival. We demonstrated that the prediction of per-patient ADE propensity simultaneously identifies high-risk patients experiencing poor outcomes. In conclusion, the ADEStrata approach could produce clinically and biologically meaningful tumor subtypes that are potentially predictive of the drug response to the cancer therapy drugs.


Modeling of subglacial hydrological development following rapid supraglacial lake drainage.
The rapid drainage of supraglacial lakes injects substantial volumes of water to the bed of the Greenland ice sheet over short timescales. The effect of these water pulses on the development of basal hydrological systems is largely unknown. To address this, we develop a lake drainage model incorporating both (1) a subglacial radial flux element driven by elastic hydraulic jacking and (2) downstream drainage through a linked channelized and distributed system. Here we present the model and examine whether substantial, efficient subglacial channels can form during or following lake drainage events and their effect on the water pressure in the surrounding distributed system. We force the model with field data from a lake drainage site, 70 km from the terminus of Russell Glacier in West Greenland. The model outputs suggest that efficient subglacial channels do not readily form in the vicinity of the lake during rapid drainage and instead water is evacuated primarily by a transient turbulent sheet and the distributed system. Following lake drainage, channels grow but are not large enough to reduce the water pressure in the surrounding distributed system, unless preexisting channels are present throughout the domain. Our results have implications for the analysis of subglacial hydrological systems in regions where rapid lake drainage provides the primary mechanism for surface-to-bed connections. Model for subglacial hydrological analysis of rapid lake drainage eventsLimited subglacial channel growth during and following rapid lake drainagePersistence of distributed drainage in inland areas where channel growth is limited.


Corrections and clarifications.
Figure 3 on page 253 of the report "Block of Ca(2+) wave and Ca(2+) oscillation by antibody to the inositol 1,4,5-trisphosphate receptor in fertilized hamster eggs" by Si. Miyazaki et al. (10 July, p. 251) was printed so that the sperm drawn on the computer display were not visible. Also, the scale bar for (F), which was 50 microm, was not clear. The correct figure is reproduced below. [See figure in the PDF file].


A physeal-sparing fibular collateral ligament and proximal tibiofibular joint reconstruction in a skeletally immature athlete.
The purpose of this report was to describe the surgical technique for and outcomes after a modified physeal-sparing posterolateral corner reconstruction in a 12-year-old skeletally immature male with a mid-substance fibular collateral ligament tear, a proximal posterior tibiofibular ligament tear, and an anterior cruciate ligament avulsion fracture of the medial tibial eminence. A modified physeal-sparing posterolateral corner reconstruction was used to provide a near-anatomic reconstruction of the fibular collateral ligament and proximal posterior tibiofibular ligament. An anterior cruciate ligament repair was also performed. Varus stress radiographs obtained at 6 months postoperatively demonstrated resolution of lateral knee stability. Physical examination results demonstrated stability to anterior tibial translation and a stable proximal tibiofibular joint. Computed tomography showed that the surgical technique successfully avoided breeching the patient's physes.


Anatomy of Lamina in the Subaxial Cervical Spine With the Special Reference to Translaminar Screws: CT and Cadaveric Analysis With Screw Trajectory Simulation.
A cadaveric study. Translaminar screws were initially developed for C2 fixation. Since then, their usage has expanded to include the subaxial cervical spine, and thoracic and lumbar spine. To the best of our knowledge, special anatomy for inserting translaminar screws in the subaxial cervical spine has not been studied. To report the special anatomy for inserting translaminar screws in the subaxial cervical spine. A total of 18 cadaveric spines were harvested from C3 to C7 and 1 mm computed tomography (CT) scans and 3D reconstructions were obtained. Bilateral translaminar screw entry points and trajectories were simulated at each level from C3 to C7 utilizing Kodak Carestream/Pacs Ver 10.2. Constructs were selected to achieve maximal bony purchase with 1 screw, designated the "primary screw." The contralateral screw, designated the "secondary screw," was selected to achieve the optimal allowable diameter possible while avoiding a simulated cortical breach, which was not always necessarily the "best purchase" diameter. Initial screw diameters selected were 3.5 mm; however, in the event that a narrower portion was encountered, then a 3.0 mm diameter screw was utilized instead. The crossing area of both screws were calculated geometrically. Maximal thickness of the lamina was considered in determining the diameter of screws. Whenever possible, 3.5 mm screws were selected in both lamina (3.5/3.5 mm); however, if a 3.5 mm screw was utilized as the primary screw, but the permissible range (P) for the secondary screw was <3.5 mm, then a hybrid construct was utilized (3.5/3.0 mm). In cases where P was <3 mm, then both screws were studied at 3 mm (3.0/3.0 mm). Screw diameters that optimized trajectory and bony purchase, while remaining within the permissible range, were analyzed, tabulated, and recorded. On CT, along the trajectory of the screws, the image was cut and measured in terms of screw length, the narrowest portion of the lamina, vertical angle, and horizontal angle in both primary and secondary screws. On the individually separated cervical spine segments in cadavers (11 of 18), we performed caliper measurements on the same portions that were measured on CT. It could not be exactly the same portions, however, due to the 3-dimensional characteristics of the specimens. For C3, only 1 specimen allowed 2 screws (3/3 mm), while the remaining specimens permitted a unilateral primary screw (3.5 or 3 mm) only. For C4, 37% of specimens allowed 2 screws (3.5/3 mm or 3/3 mm), but the rest allowed only a unilateral primary screw (3.5 or 3 mm). For C5, 58% allowed 2 screws (3.5/3.5, 3.5/3, or 3/3 mm). For C6, 89% of specimen allowed 2 screws (3.5/3.5, 3.5/3, or 3/3 mm). For C7, all levels allowed 2 screws (3.5/3.5, 3.5/3, 4/4, 4/3, 4.5/3, 4.5/3.5, or 4/3.5 mm). On CT, the average lengths of the 1- and 2-degree screws were 26.14 and 24.01 mm, respectively. The average vertical and horizontal angles were 22.26 and 40.66 degrees for the 1-degree screw, and 3.45 and 45.59 degrees for the 2-degree screw. On cadavers, the average lengths of the 1- and the 2-degree screws were 22.58 and 23.44 mm, respectively. The average vertical and horizontal angles were 23.67 and 54.44 degrees for the 1-degree screw, and 2.28 and 54.89 degrees for the 2-degree screw. This is a report of the anatomy of the lamina in the subaxial cervical spine with the special reference to translaminar screws. It was analyzed with CT and cadaveric spines along with simulated screw trajectories. For the 1-degree translaminar screw, the entry point is the distance of the diameter of desired screw superior to the inferior margin of lamina-spinous process junction. The trajectory should be targeted toward the most superomedial corner of lateral mass. For the 2-degree translaminar screw, the entry point is the distance of the diameter of desired screw below the superior margin of lamina-spinous process junction, and the target is the most superolateral corner of lateral mass, which is typically horizontal. Further studies are needed to assess the feasibility of translaminar screw insertion in the actual subaxial cervical spine.


Prospective multicenter study of the impact of oncotype DX colon cancer assay results on treatment recommendations in stage II colon cancer patients.
The Oncotype DX colon cancer assay is a clinically validated predictor of recurrence risk in stage II colon cancer patients. This prospective study evaluated the impact of recurrence score (RS) results on physician recommendations regarding adjuvant chemotherapy in T3, mismatch repair-proficient (MMR-P) stage II colon cancer patients. Patients and Methods. Stage IIA colon cancer patients were enrolled in 17 centers. Patient tumor specimens were assessed by the RS test (quantitative reverse transcription-polymerase chain reaction) and mismatch repair (immunohistochemistry). For each patient, the physician's recommended postoperative treatment plan of observation, fluoropyrimidine monotherapy, or combination therapy with oxaliplatin was recorded before and after the RS and mismatch repair results were provided. Results. Of 221 enrolled patients, 141 patients had T3 MMR-P tumors and were eligible for the primary analysis. Treatment recommendations changed for 63 (45%; 95% confidence interval: 36%-53%) of these 141 T3 MMR-P patients, with intensity decreasing for 47 (33%) and increasing for 16 (11%). Recommendations for chemotherapy decreased from 73 patients (52%) to 42 (30%), following review of RS results by physician and patient. Increased treatment intensity was more often observed at higher RS values, and decreased intensity was observed at lower values (p = .011). Conclusion. Compared with traditional clinicopathological assessment, incorporation of the RS result into clinical decision making was associated with treatment recommendation changes for 45% of T3 MMR-P stage II colon cancer patients in this prospective multicenter study. Use of the RS assay may lead to overall reduction in adjuvant chemotherapy use in this subgroup of stage II colon cancer patients.


Differential volatile signatures from skin, naevi and melanoma: a novel approach to detect a pathological process.
Early detection of melanoma is of great importance to reduce mortality. Discovering new melanoma biomarkers would improve early detection and diagnosis. Here, we present a novel approach to detect volatile compounds from skin. We used Head Space Solid Phase Micro-Extraction (HS-SPME) and gas chromatography/mass spectrometry (GC/MS) to identify volatile signatures from melanoma, naevi and skin samples. We hypothesized that the metabolic state of tissue alters the profile of volatile compounds. Volatiles released from fresh biopsy tissue of melanoma and benign naevus were compared based on their difference in frequency distribution and their expression level. We also analyzed volatile profiles from frozen tissue, including skin and melanoma. Three volatiles, 4-methyl decane, dodecane and undecane were preferentially expressed in both fresh and frozen melanoma, indicating that they are candidate biomarkers. Twelve candidate biomarkers evaluated by fuzzy logic analysis of frozen samples distinguished melanoma from skin with 89% sensitivity and 90% specificity. Our results demonstrate proof-of-principle that there is differential expression of volatiles in melanoma. Our volatile metabolomic approach will lead to a better understanding of melanoma and can enable development of new diagnostic and treatment strategies based on altered metabolism.


Epigenomic analysis reveals a dynamic and context-specific macrophage enhancer landscape associated with innate immune activation and tolerance.
Chromatin states and enhancers associate gene expression, cell identity and disease. Here, we systematically delineate the acute innate immune response to endotoxin in terms of human macrophage enhancer activity and contrast with endotoxin tolerance, profiling the coding and non-coding transcriptome, chromatin accessibility and epigenetic modifications. We describe the spectrum of enhancers under acute and tolerance conditions and the regulatory networks between these enhancers and biological processes including gene expression, splicing regulation, transcription factor binding and enhancer RNA signatures. We demonstrate that the vast majority of differentially regulated enhancers on acute stimulation are subject to tolerance and that expression quantitative trait loci, disease-risk variants and eRNAs are enriched in these regulatory regions and related to context-specific gene expression. We find enrichment for context-specific eQTL involving endotoxin response and specific infections and delineate specific differential regions informative for GWAS variants in inflammatory bowel disease and multiple sclerosis, together with a context-specific enhancer involving a bacterial infection eQTL for KLF4. We show enrichment in differential enhancers for tolerance involving transcription factors NFκB-p65, STATs and IRFs and prioritize putative causal genes directly linking genetic variants and disease risk enhancers. We further delineate similarities and differences in epigenetic landscape between stem cell-derived macrophages and primary cells and characterize the context-specific enhancer activities for key innate immune response genes KLF4, SLAMF1 and IL2RA. Our study demonstrates the importance of context-specific macrophage enhancers in gene regulation and utility for interpreting disease associations, providing a roadmap to link genetic variants with molecular and cellular functions.


Micro-RNA-195 and -451 regulate the LKB1/AMPK signaling axis by targeting MO25.
Recently, MicroRNAs (miR) and AMP-kinase (AMPK) have emerged as prominent players in the development of cardiac hypertrophy and heart failure. We hypothesized that components of the adenosine monophosphate-activated kinase (AMPK) pathway are targeted by miRs and alter AMPK signaling during pathological cardiac stress. Using a mouse model of hypertrophic cardiomyopathy (HCM), we demonstrated early elevation of miR-195 and miR-451 in HCM hearts, which targets MO25, a central component of the MO25/STRAD/LKB1 complex that acts as an upstream kinase for AMPK. We show functional targeting of MO25 by miR-195 and -451. Further in vitro interrogation of MO25 as a functional target validated this hypothesis where over-expression of miR-195 in C2C12 cells knocked down MO25 expression levels and downstream AMPK signaling (phosphorylation of Acetyl CoA carboxylase [ACC] and AMPK activity assay), similar to MO25 knockdown in C2C12 cells by siRNA. Parallel changes were measured in 60 day R403Q HCM male hearts that were rescued by short-term administration of AICAR, an AMPK agonist. Elevated miR-195 targets the LKB1/AMPK signaling axis in HCM progression and implicates a functional role in HCM disease progression. MiR-195 may serve as potential therapeutics or therapeutic targets for heart disease.


Intraocular pressure efficacy of glaucoma medications versus placebo in phase II compared to later phase trials.
This review aimed to compare the predictive value between the untreated reduction in intraocular pressure (IOP) from baseline or placebo measured in early phase clinical trials to phase III and IV results for glaucoma medicines. Published, placebo-controlled, randomised, parallel, single-masked or double-masked clinical trials with at least one phase II, III and IV study available were reviewed. This study included 50 articles evaluating 9 medicines from 59 active arms and 18 placebo arms. For all studies the phase II IOP reduction from placebo showed less decrease compared to the decrease from baseline (p<0.04). For all medicines, reductions from morning baseline in phase II did not predict better than the decrease from placebo for phase III (p=0.15) or IV (p=0.08) reductions in IOP. In contrast, diurnal IOP reduction from baseline in phase II predicted decreases better than placebo in phase III (p=0.007) and IV (p=0.02). Generally, for prostaglandins, β blockers and carbonic anhydrase inhibitors for the morning trough and diurnal curve there was no difference in pressure reduction from baseline for phase II compared to phase III or IV (p≥0.23). In contrast, where comparisons were available for the decrease in pressure from placebo there were differences for phase II compared to phase III and phase IV (p≤0.02). This study suggests that in early phase glaucoma trials, using the reduction from untreated baseline in general better approximates the results of later regulatory and post-commercialisation trials than the decrease from placebo.


Bariatric surgery and renal function: a precarious balance between benefit and harm.
Medical treatment of obesity and lifestyle modification have limited effectiveness in treating it in morbidly obese individuals. Importantly, bariatric surgery is regarded as the only therapy that is effective in maintaining significant weight loss in morbidly obese individuals. Despite the fact that bariatric surgery-induced weight loss is associated with a significant decrease in morbidity and mortality and improvement in renal function, bariatric surgery has recently been shown to be associated with a significant risk of nephrolithiasis. The main risk factor for nephrolithiasis is increased excretion of urinary oxalate. In this review, we discuss the association between bariatric surgery, an increased risk of renal stone formation and oxalate nephropathy.


Neurotransmitter substance P mediates pancreatic cancer perineural invasion via NK-1R in cancer cells.
Pancreatic cancer significantly affects the quality of life due to the severe abdominal pain. However, the underlying mechanism is not clear. This study aimed to determine the relationship between Substance P (SP) and pancreatic cancer perineural invasion (PNI) as well as the mechanism of SP mediating pancreatic cancer PNI, which causes pain in patients with pancreatic cancer. Human pancreatic cancer cells and newborn dorsal root ganglions (DRG) were used to determine the expression of SP or NK-1R in pancreatic cancer cells and DRGs cells by QT-PCR and Western blotting. The effects of SP on pancreatic cancer cell proliferation and invasion were analyzed using MTT assay and Transwell Matrigel invasion assay, respectively. Alterations in the neurotropism of pancreatic cancer cells were assessed by coculture system, which mimics the interaction of tumor/neuron in vivo. SP is not only widely distributed in the neurite outgrowth from newborn DRGs but also expressed in MIA PaCa-2 and BxPC-3 cells. NK-1R is found to be overexpressed in the pancreatic cancer cell lines examined. SP induces cancer cell proliferation and invasion as well as the expression of matrix metalloproteinase (MMP)-2 in pancreatic cancer cells, and NK-1R antagonists inhibit these effects. Furthermore, SP promotes neurite outgrowth and the migration of pancreatic cancer cell cluster to the DRGs, which is blocked by NK-1R antagonists in the coculture model. Our results suggest that SP plays an important role in the development of pancreatic cancer metastasis and PNI, and blocking the SP/NK-1R signaling system is a novel strategy for the treatment of pancreatic cancer.


How do some plants tolerate low levels of soil zinc? Mechanisms of zinc efficiency in crop plants.
Researchers are beginning to understand how some plant genotypes can maintain reasonable growth and yields under low soil Zn, a trait termed zinc efficiency (ZE). Several studies have shown no correlation between ZE and root Zn uptake, Zn translocation to shoot, or shoot Zn accumulation. Furthermore, it has not been possible to conclusively link differences in leaf subcellular Zn compartmentation with ZE. However, biochemical Zn utilization, including the ability to maintain the activity of Zn requiring enzymes in response to Zn deficiency may be a key component of ZE. The next logical step in investigations of this trait is research on the genetic and molecular basis for ZE, in order to better understand the relationship between Zn utilization and ZE, and to identify the gene(s) controlling ZE. Progress in this research area could provide the knowledge to facilitate the engineering of Zn-efficient plant varieties, which could help both crop production on marginal soils as well as possibly improve the micronutrient density of food crops to help address significant human nutrition problems related to micronutrient deficiency.


Conversion of a single-facility pediatric antimicrobial stewardship program to multi-facility application with computerized provider order entry and clinical decision support.
Antimicrobial stewardship programs (ASPs) help meet quality and safety goals with regard to antimicrobial use. Prior to CPOE implementation, the ASP at our pediatric tertiary hospital developed a paper-based order set containing recommendations for optimization of dosing. In adapting our ASP for CPOE, we aimed to preserve consistency in our ASP recommendations and expand ASP expertise to other hospitals in our health system. Nine hospitals in our health system adopted pediatric CPOE and share a common domain (Cerner Millenium). ASP clinicians developed sixty individual electronic order sets (vendor reference PowerPlans) to be used independently or as part of larger electronic order sets. Analysis of incidents reported during CPOE implementation and medication variances reports was used to determine the effectiveness of the ASP adaptation. 769 unique PowerPlans were used 15,889 times in the first 30 days after CPOE implementation. Of these, 43 were PowerPlans included in the ASP design and were used a total of 1149 times (7.2% of all orders). During CPOE implementation, 437 incidents were documented, 1.1% of which were associated with ASP content or workflow. Additionally, analysis of medication variance following CPOE implementation showed that ASP errors accounted for 2.9% of total medication variances. ASP content and workflow accounted for proportionally fewer incidents than expected as compared to equally complex and frequently used CPOE content. Well-defined ASP recommendations and modular design strengthened successful CPOE implementation, as well as the adoption of specialized pediatric ASP expertise with other facilities.


Benzodiazepine binding site occupancy by the novel GABAA receptor subtype-selective drug 7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine (TPA023) in rats, primates, and humans.
The GABA(A) receptor alpha2/alpha3 subtype-selective compound 7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine (TPA023; also known as MK-0777) is a triazolopyridazine that has similar, subnanomolar affinity for the benzodiazepine binding site of alpha1-, alpha2-, alpha3-, and alpha5-containing GABA(A) receptors and has partial agonist efficacy at the alpha2 and alpha3 but not the alpha1 or alpha5 subtypes. The purpose of the present study was to define the relationship between plasma TPA023 concentrations and benzodiazepine binding site occupancy across species measured using various methods. Thus, occupancy was measured using either in vivo [(3)H]flumazenil binding or [(11)C]flumazenil small-animal positron emission tomography (microPET) in rats, [(123)I]iomazenil gamma-scintigraphy in rhesus monkeys, and [(11)C]flumazenil PET in baboons and humans. For each study, plasma-occupancy curves were derived, and the plasma concentration of TPA023 required to produce 50% occupancy (EC(50)) was calculated. The EC(50) values for rats, rhesus monkeys, and baboons were all similar and ranged from 19 to 30 ng/ml, although in humans, the EC(50) was slightly lower at 9 ng/ml. In humans, a single 2-mg dose of TPA023 produced in the region of 50 to 60% occupancy in the absence of overt sedative-like effects. Considering that nonselective full agonists are associated with sedation at occupancies of less than 30%, these data emphasize the relatively nonsedating nature of TPA023.


Bleeding complications in laparoscopic cholecystectomy: Incidence, mechanisms, prevention and management.
Laparoscopic cholecystectomy (LC) has established itself firmly as the 'gold standard' for the treatment of gallstone disease, but it can, at times, be associated with significant morbidity and mortality. Existing literature has focused almost exclusively on the biliary complications of this procedure, but other complications such as significant haemorrhage can also be encountered, with an immediate mortality if not recognized and treated in a timely manner. Publications in English language literature that have reported the complication of bleeding during or after the performance of LC were identified and accessed. The results thus obtained were tabulated and analyzed to get a true picture of this complication, its mechanism and preventive measures. Bleeding has been reported to occur with an incidence of up to nearly 10% in various series, and can occur at any time during LC (during trocar insertion, dissection technique or slippage of clips/ ligatures) or in the postoperative period. It can range from minor haematomas to life-threatening injuries to major intra-abdominal vessels (such as aorta, vena cava and iliacs). Good surgical technique, awareness and early recognition and management of such cases are keys to success when dealing with this problem.


The complete chloroplast genome sequence of tung tree (Vernicia fordii): Organization and phylogenetic relationships with other angiosperms.
Tung tree (Vernicia fordii) is an economically important tree widely cultivated for industrial oil production in China. To better understand the molecular basis of tung tree chloroplasts, we sequenced and characterized its genome using PacBio RS II sequencing platforms. The chloroplast genome was sequenced with 161,528 bp in length, composed with one pair of inverted repeats (IRs) of 26,819 bp, which were separated by one small single copy (SSC; 18,758 bp) and one large single copy (LSC; 89,132 bp). The genome contains 114 genes, coding for 81 protein, four ribosomal RNAs and 29 transfer RNAs. An expansion with integration of an additional rps19 gene in the IR regions was identified. Compared to the chloroplast genome of Jatropha curcas, a species from the same family, the tung tree chloroplast genome is distinct with 85 single nucleotide polymorphisms (SNPs) and 82 indels. Phylogenetic analysis suggests that V. fordii is a sister species with J. curcas within the Eurosids I. The nucleotide sequence provides vital molecular information for understanding the biology of this important oil tree.


Are obese women more likely to participate in a mobile mammography program?
Mobile mammography services are typically offered as a means to increase access and adherence to mammography screenings. As mobile mammography becomes a viable strategy to increase screening, a 3 year study of such a state-wide program in WV found surprisingly high rates of obesity within the study population. Thus, the objectives were to: (1) describe the demographic characteristics and comorbidities of women who utilized the WV program, and (2) determine the association between body mass index (BMI) and personal health and screening history, preventive care and wellness behaviors, nutrition and exercise behaviors, and demographics. Data collected from 1,099 women, age 40 and above, were analyzed using descriptive statistics, bivariate analyses, and a multivariate regression model. The majority (60.4 %) were married, had an income <$25,000 (59.2 %), and had health insurance (53.5 %). Major comorbidities were hypertension (49 %) and high cholesterol (43.9 %). Based on BMI scores, 884 participants were either overweight (26.6 %), mildly obese (27.7 %), moderately obese (15.1 %), or severely obese (11.1 %). Bivariate analyses indicated that increasing BMI was significantly associated with factors such as having hypertension or diabetes, limited daily activities, perceived health, and not smoking or drinking. The regression model was significant (p < 0.001; R2 = 0.425) indicating that women who engaged in preventive care behaviors were less likely to be obese than those who did not. The WV mobile mammography program appeared to attract women who were disproportionately obese and had multiple comorbidities, thus providing a great opportunity for targeted interventions related to improving preventive care and screening behaviors.


Increased intrinsic pumping of intestinal lymphatics following hemorrhage in anesthetized sheep.
The return of fluid and protein to the bloodstream by the lymphatic circulation may play an important role in reconstituting intravascular volume following hemorrhage. In this study, we have defined the lymph-flow changes that occur in cannulated mesenteric lymphatics following a 25% blood loss in anesthetized sheep and investigated the effects of hemorrhage on the intrinsic contractile activity (lymph pumping) of these vessels in vivo using a new model system. The removal of 25% of the calculated blood volume resulted in increases in lymph flow over a 6-hour period, with peak changes to 3.5 times the prebleed levels. Systemic arterial pressures dropped to roughly 50% of control values immediately following the bleed and returned to control in 3 hours. To directly assess the effects of hemorrhage on lymphatic pumping, a segment of intestinal lymphatic was isolated from all lymph input and supplied with fluid from a reservoir. While there was no net pressure driving fluid through the duct, a transmural distending pressure was applied to the vessel, which stimulated resting lymphatic contractions and fluid pumping. A 25% blood loss resulted in increased activity of the lymph pump; up to 6 times more fluid was propelled through this "isolated" vessel in vivo than in similar preparations in sheep that were not bled (p less than 0.01). Measurements of fluid pulse pressures in this preparation indicated increased pumping frequency and/or force after hemorrhage compared with prebleed levels. We conclude that lymphatic contractile activity is stimulated after a blood loss independent of changes in lymph formation and speculate that this mechanism may play an important role in the reexpansion of the vascular space.


Description and Comparative Genomics of Macrococcus caseolyticus subsp. hominis subsp. nov., Macrococcus goetzii sp. nov., Macrococcus epidermidis sp. nov., and Macrococcus bohemicus sp. nov., Novel Macrococci From Human Clinical Material With Virulence Potential and Suspected Uptake of Foreign DNA by Natural Transformation.
The genus Macrococcus is a close relative of the genus Staphylococcus. Whilst staphylococci are widespread as human pathogens, macrococci have not yet been reported from human clinical specimens. Here we investigated Gram-positive and catalase-positive cocci recovered from human clinical material and identified as Macrococcus sp. by a polyphasic taxonomic approach and by comparative genomics. Relevant phenotypic, genotypic and chemotaxonomic methods divided the analyzed strains into two separate clusters within the genus Macrococcus. Comparative genomics of four representative strains revealed enormous genome structural plasticity among the studied isolates. We hypothesize that high genomic variability is due to the presence of a com operon, which plays a key role in the natural transformation of bacilli and streptococci. The possible uptake of exogenous DNA by macrococci can contribute to a different mechanism of evolution from staphylococci, where phage-mediated horizontal gene transfer predominates. The described macrococcal genomes harbor novel plasmids, genomic islands and islets, as well as prophages. Capsule gene clusters, intracellular protease, and a fibronectin-binding protein enabling opportunistic pathogenesis were found in all four strains. Furthermore, the presence of a CRISPR-Cas system with 90 spacers in one of the sequenced genomes corresponds with the need to limit the burden of foreign DNA. The highly dynamic genomes could serve as a platform for the exchange of virulence and resistance factors, as was described for the methicillin resistance gene, which was found on the novel composite SCCmec-like element containing a unique mec gene complex that is considered to be one of the missing links in SCC evolution. The phenotypic, genotypic, chemotaxonomic and genomic results demonstrated that the analyzed strains represent one novel subspecies and three novel species of the genus Macrococcus, for which the names Macrococcus caseolyticus subsp. hominis subsp. nov. (type strain CCM 7927T = DSM 103682T), Macrococcus goetzii sp. nov. (type strain CCM 4927T = DSM 103683T), Macrococcus epidermidis sp. nov. (type strain CCM 7099T = DSM 103681T), and Macrococcus bohemicus sp. nov. (type strain CCM 7100T = DSM 103680T) are proposed. Moreover, a formal description of Macrococcus caseolyticus subsp. caseolyticus subsp. nov. and an emended description of the genus Macrococcus are provided.


Application of an aqueous two-phase systems high-throughput screening method to evaluate mAb HCP separation.
Aqueous two-phase systems (ATPSs) as separation technique have regained substantial interest from the biotech industry. Biopharmaceutical companies faced with increasing product titers and stiffening economic competition reconsider ATPS as an alternative to chromatography. As the implementation of an ATPS is material, time, and labor intensive, a miniaturized and automated screening process would be beneficial. In this article such a method, its statistical evaluation, and its application to a biopharmaceutical separation task are shown. To speed up early stage ATPS profiling an automated application of the cloud-point method for binodal determination was developed. PEG4000-PO(4) binodals were measured automatically and manually and were found to be identical within the experimental error. The ATPS screening procedure was applied to a model system and an industrial separation task. PEG4000-PO(4) systems at a protein concentration of 0.75 mg/mL were used. The influence of pH, NaCl addition, and tie line length was investigated. Lysozyme as model protein, two monoclonal antibodies, and a host cell protein pool were used. The method was found to yield partition coefficients identical to manually determined values for lysozyme. The monoclonal antibodies were shifted from the bottom into the upper phase by addition of NaCl. This shift occurred at lower NaCl concentration when the pH of the system was closer to the pI of the distributed protein. Addition of NaCl, increase in PEG4000 concentration and pH led to significant loss of the mAb due to precipitation. Capacity limitations of these systems were thus demonstrated. The chosen model systems allowed a reduction of up to 50% HCP with a recovery of greater than 95% of the target proteins. As these values might not be industrially relevant when compared to current chromatographic procedures, the developed screening procedure allows a fast evaluation of more suitable and optimized ATPS system for a given task.


HBx relieves chromatin-mediated transcriptional repression of hepatitis B viral cccDNA involving SETDB1 histone methyltransferase.
Maintenance of the covalently closed circular HBV DNA (cccDNA) that serves as a template for HBV transcription is responsible for the failure of antiviral therapies. While studies in chronic hepatitis patients have shown that high viremia correlates with hyperacetylation of cccDNA-associated histones, the molecular mechanisms controlling cccDNA stability and transcriptional regulation are still poorly understood. This study aimed to decipher the role of chromatin and chromatin modifier proteins on HBV transcription. We analyzed the chromatin structure of actively transcribed or silenced cccDNA by infecting primary human hepatocytes and differentiated HepaRG cells with wild-type virus or virus deficient (HBVX-) for the expression of hepatitis B virus X protein (HBx), that is required for HBV expression. In the absence of HBx, HBV cccDNA was transcriptionally silenced with the concomitant decrease of histone 3 (H3) acetylation and H3K4me3, increase of H3 di- and tri-methylation (H3K9me) and the recruitment of heterochromatin protein 1 factors (HP1) that correlate with condensed chromatin. SETDB1 was found to be the main histone methyltransferase responsible for the deposition of H3K9me3 and HBV repression. Finally, full transcriptional reactivation of HBVX- upon HBx re-expression correlated with an increase of histone acetylation and H3K4me3, and a concomitant decrease of HP1 binding and of H3K9me3 on the cccDNA. Upon HBV infection, cellular mechanisms involving SETDB1-mediated H3K9me3 and HP1 induce silencing of HBV cccDNA transcription through modulation of chromatin structure. HBx is able to relieve this repression and allow the establishment of active chromatin.


Architecture of the RNA polymerase II-TFIIF complex revealed by cross-linking and mass spectrometry.
Higher-order multi-protein complexes such as RNA polymerase II (Pol II) complexes with transcription initiation factors are often not amenable to X-ray structure determination. Here, we show that protein cross-linking coupled to mass spectrometry (MS) has now sufficiently advanced as a tool to extend the Pol II structure to a 15-subunit, 670 kDa complex of Pol II with the initiation factor TFIIF at peptide resolution. The N-terminal regions of TFIIF subunits Tfg1 and Tfg2 form a dimerization domain that binds the Pol II lobe on the Rpb2 side of the active centre cleft near downstream DNA. The C-terminal winged helix (WH) domains of Tfg1 and Tfg2 are mobile, but the Tfg2 WH domain can reside at the Pol II protrusion near the predicted path of upstream DNA in the initiation complex. The linkers between the dimerization domain and the WH domains in Tfg1 and Tfg2 are located to the jaws and protrusion, respectively. The results suggest how TFIIF suppresses non-specific DNA binding and how it helps to recruit promoter DNA and to set the transcription start site. This work establishes cross-linking/MS as an integrated structure analysis tool for large multi-protein complexes.


Serosurvey of selected zoonotic agents in polar bears (Ursus maritimus).
Between 1982 and 1999 blood samples were collected from 500 polar bears (Ursus maritimus) captured in the Beaufort and Chukchi seas, to determine the seroprevalence of Brucella species, Toxoplasma gondii, and Trichinella species infections. The bears were classified into four age groups, cubs, yearlings, subadults and adults. Brucella and Toxoplasma antibodies were detected by agglutination (a buffered acidified card antigen and rapid automated presumptive test for brucellosis and a commercial latex agglutination test for toxoplasmosis); an ELISA was used to detect Trichinella antibodies. The overall seroprevalence of Brucella species was 5 per cent, and subadults and yearlings were 2-62 times (95 per cent confidence interval 1.02 to 6.82) more likely to be seropositive for Brucella species than adults and their cubs. The antibody prevalence for Toxoplasma gondii was 6 per cent, and for Trichinella species 55.6 per cent. The prevalence of antibodies to Trichinella species increased with age (P<0.001).


Influence of magnesium on biochemical parameters of iron and oxidative stress in patients with type 2 diabetes.
Studies have shown that oxidative stress, found in patients with type 2 diabetes, may be due to changes in the metabolism of minerals, such as magnesium and iron. Data related to compartmentalization of these minerals in diabetes are scarce and controversial. This study assessed the influence of magnesium on biochemical parameters of iron and oxidative stress in patients with type 2 diabetes. A case-control study in male and female subjects aged 27-59 years, divided into two groups: type 2 diabetes (n=40) and control (n=48). Intake of magnesium and iron was assessed by three-day food record. Plasma, erythrocyte and urinary levels of magnesium, serum iron, ferritin, total iron binding capacity, fasting glucose, glycated hemoglobin, insulin, creatinine clearance and plasma thiobarbituric acid reactive substances (TBARS) were analyzed. Magnesium intake and plasma magnesium were lower in diabetic subjects. There was low urinary magnesium excretion, with no difference between groups. Although normal, the diabetic group had lower serum iron and ferritin concentrations compared to control subjects. Plasma TBARS in diabetic patients was higher than control while creatinine clearance was lower. An inverse correlation between erythrocyte magnesium and serum iron and ferritin was observed in the diabetes group. Diabetes induced hypomagnesemia and this, associated with chronic hyperglycemia, may have enhanced oxidative stress. Erythrocyte magnesium may have contributed to prevent iron overload and worsening of oxidative stress and hyperglycemic status.


Ocean acidification increases the toxicity of contaminated sediments.
Ocean acidification (OA) may alter the behaviour of sediment-bound metals, modifying their bioavailability and thus toxicity. We provide the first experimental test of this hypothesis with the amphipod Corophium volutator. Amphipods were exposed to two test sediments, one with relatively high metals concentrations (Σmetals 239 mg kg(-1) ) and a reference sediment with lower contamination (Σmetals 82 mg kg(-1) ) under conditions that mimic current and projected conditions of OA (390-1140 μatm pCO2 ). Survival and DNA damage was measured in the amphipods, whereas the flux of labile metals was measured in the sediment and water column (WC) using Diffusive Gradients in Thin-films. The contaminated sediments became more acutely toxic to C. volutator under elevated pCO2 (1140 μatm). There was also a 2.7-fold increase in DNA damage in amphipods exposed to the contaminated sediment at 750 μatm pCO2 , as well as increased DNA damage in organisms exposed to the reference sediment, but only at 1140 μatm pCO2 . The projected pCO2 concentrations increased the flux of nickel and zinc to labile states in the WC and pore water. However, the increase in metal flux at elevated pCO2 was equal between the reference and contaminated sediments or, occasionally, greater from reference sediments. Hence, the toxicological interaction between OA and contaminants could not be explained by e ffects of pH on metal speciation. We propose that the additive physiological effects of OA and contaminants will be more important than changes in metal speciation in determining the responses of benthos to contaminated sediments under OA. Our data demonstrate clear potential for near-future OA to increase the susceptibility of benthic ecosystems to contaminants. Environmental policy should consider contaminants within the context of changing environmental conditions. Specifically, sediment metals guidelines may need to be reevaluated to afford appropriate environmental protection under future conditions of OA.


Phytophthora sojae avirulence effector Avr3b is a secreted NADH and ADP-ribose pyrophosphorylase that modulates plant immunity.
Plants have evolved pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) to protect themselves from infection by diverse pathogens. Avirulence (Avr) effectors that trigger plant ETI as a result of recognition by plant resistance (R) gene products have been identified in many plant pathogenic oomycetes and fungi. However, the virulence functions of oomycete and fungal Avr effectors remain largely unknown. Here, we combined bioinformatics and genetics to identify Avr3b, a new Avr gene from Phytophthora sojae, an oomycete pathogen that causes soybean root rot. Avr3b encodes a secreted protein with the RXLR host-targeting motif and C-terminal W and Nudix hydrolase motifs. Some isolates of P. sojae evade perception by the soybean R gene Rps3b through sequence mutation in Avr3b and lowered transcript accumulation. Transient expression of Avr3b in Nicotiana benthamiana increased susceptibility to P. capsici and P. parasitica, with significantly reduced accumulation of reactive oxygen species (ROS) around invasion sites. Biochemical assays confirmed that Avr3b is an ADP-ribose/NADH pyrophosphorylase, as predicted from the Nudix motif. Deletion of the Nudix motif of Avr3b abolished enzyme activity. Mutation of key residues in Nudix motif significantly impaired Avr3b virulence function but not the avirulence activity. Some Nudix hydrolases act as negative regulators of plant immunity, and thus Avr3b might be delivered into host cells as a Nudix hydrolase to impair host immunity. Avr3b homologues are present in several sequenced Phytophthora genomes, suggesting that Phytophthora pathogens might share similar strategies to suppress plant immunity.


Spotlight on the Underdogs-An Analysis of Underrepresented Alternaria Mycotoxins Formed Depending on Varying Substrate, Time and Temperature Conditions.
Alternaria (A.) is a genus of widespread fungi capable of producing numerous, possibly health-endangering Alternaria toxins (ATs), which are usually not the focus of attention. The formation of ATs depends on the species and complex interactions of various environmental factors and is not fully understood. In this study the influence of temperature (7 °C, 25 °C), substrate (rice, wheat kernels) and incubation time (4, 7, and 14 days) on the production of thirteen ATs and three sulfoconjugated ATs by three different Alternaria isolates from the species groups A. tenuissima and A. infectoria was determined. High-performance liquid chromatography coupled with tandem mass spectrometry was used for quantification. Under nearly all conditions, tenuazonic acid was the most extensively produced toxin. At 25 °C and with increasing incubation time all toxins were formed in high amounts by the two A. tenuissima strains on both substrates with comparable mycotoxin profiles. However, for some of the toxins, stagnation or a decrease in production was observed from day 7 to 14. As opposed to the A. tenuissima strains, the A. infectoria strain only produced low amounts of ATs, but high concentrations of stemphyltoxin III. The results provide an essential insight into the quantitative in vitro AT formation under different environmental conditions, potentially transferable to different field and storage conditions.


Inhalation versus total intravenous anesthesia for lumbar disc herniation: comparison of hemodynamic effects, recovery characteristics, and cost.
The clinical effects, recovery characteristics, and costs of total intravenous anesthesia (TIVA), sevoflurane, and isoflurane anesthesia have been measured in various out-patient operations, but have not been evaluated in patients undergoing laminectomy or discectomy. In the current study, the authors assessed the hemodynamic characteristics, recovery, and cost analyzes after laminectomy and discectomy operations, comparing TIVA, sevoflurane, and isoflurane anesthesia. Sixty American Society of Anesthesiologists I and II patients were randomly divided into three groups, each consisting of 20 patients. Group I received propofol-alfentanil, Group 2 received sevoflurane-N2O, and Group 3 received isoflurane-N2O. At the end of surgery, the anesthetics were discontinued, and recovery from anesthesia was assessed by measuring the time until spontaneous eye opening and the time until response to verbal commands. The drug and delivery costs were calculated in United States dollars. No significant differences were found in the demographic data. Heart rate and mean arterial pressure decreased significantly after induction of anesthesia in the TIVA group, compared to the two other groups ( P < .05 for both comparisons). The fastest recovery was seen in the TIVA group. Incidences of postoperative nausea, vomiting, and pain were significantly reduced after TIVA ( P < .05 for both comparisons). Thus, TIVA patients required fewer additional drugs and showed the lowest additional costs in the post-anesthesia care unit. However, the total cost was significantly higher in the TIVA group than in the sevoflurane and isoflurane groups (52.73 dollars, 29.99 dollars, and 24.14 dollars, respectively) ( P < .05). Total intravenous anesthesia was associated with the highest intraoperative cost but provided the most rapid recovery from anesthesia, and the least frequent postoperative side effects.


Identification of metabotropic glutamate receptor antagonists using an automated high-throughput screening system.
Antagonists to the human metabotropic glutamate receptor subtype 5a(mGluR(5a)) have been implicated as potential therapeutics for the treatment of a variety of nervous system disorders, including pain, anxiety, and Parkinson's disease. To discover novel antagonists to the mGluR(5a), a functional assay measuring agonist-induced intracellular calcium release was developed. The assay was used for the high-throughput screening of a large collection of compounds in single wells using a fully automated robotic platform. Primary high-throughput screening hits were subjected to a combination of data analysis and counterscreening assays to identify several compounds with both efficacy and selectivity for the metabotropic glutamate receptor target.


Specific aspects of consanguinity: some examples from the Tunisian population.
Located at the cross-road between Europe and Africa, Tunisia is a North African country of 11 million inhabitants. Throughout its history, it has been invaded by different ethnic groups. These historical events, and consanguinity, have impacted on the spectrum and frequency of genetic diseases in Tunisia. Investigations of Tunisian families have significantly contributed to elucidation of the genetic bases of rare disorders, providing an invaluable resource of cases due to particular familial structures (large family size, consanguinity and share of common ancestors). In the present study, we report on and review different aspects of consanguinity in the Tunisian population as a case study, representing several features common to neighboring or historically related countries in North Africa and the Middle East. Despite the educational, demographic and behavioral changes that have taken place during the last four decades, familial and geographical endogamy still exist at high frequencies, especially in rural areas. The health implications of consanguinity in Tunisian families include an increased risk of the expression of autosomal recessive diseases and particular phenotypic expressions. With new sequencing technologies, the investigation of consanguineous populations provides a unique opportunity to better evaluate the impact of consanguinity on the genome dynamic and on health, in addition to a better understanding of the genetic bases of diseases.


Effects of colonization asymmetries on metapopulation persistence.
Ocean currents, prevailing winds, and the hierarchical structures of river networks are known to create asymmetries in re-colonization between habitat patches. The impacts of such asymmetries on metapopulation persistence are seldom considered, especially rarely in theoretical studies. Considering three classical models (the island, the stepping stone and the distance-dependent model), we explore how metapopulation persistence is affected by (i) asymmetry in dispersal strength, in which the colonization rate between two patches differs in direction, and (ii) asymmetry in connectivity, in which the overall colonization pattern displays asymmetry (circulating or dendritic networks). Viability can be drastically reduced when directional bias in dispersal strength is higher than 25%. Re-colonization patterns that allow for strong local connectivity provide the highest persistence compared to systems that allow circulation. Finally, asymmetry has relatively weak effects when metapopulations maintain strong general connectivity.


Bacterial Symbionts in Lepidoptera: Their Diversity, Transmission, and Impact on the Host.
The insect's microbiota is well acknowledged as a "hidden" player influencing essential insect traits. The gut microbiome of butterflies and moths (Lepidoptera) has been shown to be highly variable between and within species, resulting in a controversy on the functional relevance of gut microbes in this insect order. Here, we aim to (i) review current knowledge on the composition of gut microbial communities across Lepidoptera and (ii) elucidate the drivers of the variability in the lepidopteran gut microbiome and provide an overview on (iii) routes of transfer and (iv) the putative functions of microbes in Lepidoptera. To find out whether Lepidopterans possess a core gut microbiome, we compared studies of the microbiome from 30 lepidopteran species. Gut bacteria of the Enterobacteriaceae, Bacillaceae, and Pseudomonadaceae families were the most widespread across species, with Pseudomonas, Bacillus, Staphylococcus, Enterobacter, and Enterococcus being the most common genera. Several studies indicate that habitat, food plant, and age of the host insect can greatly impact the gut microbiome, which contributes to digestion, detoxification, or defense against natural enemies. We mainly focus on the gut microbiome, but we also include some examples of intracellular endosymbionts. These symbionts are present across a broad range of insect taxa and are known to exert different effects on their host, mostly including nutrition and reproductive manipulation. Only two intracellular bacteria genera (Wolbachia and Spiroplasma) have been reported to colonize reproductive tissues of Lepidoptera, affecting their host's reproduction. We explore routes of transmission of both gut microbiota and intracellular symbionts and have found that these microbes may be horizontally transmitted through the host plant, but also vertically via the egg stage. More detailed knowledge about the functions and plasticity of the microbiome in Lepidoptera may provide novel leads for the control of lepidopteran pest species.


Mybl2, downregulated during colon epithelial cell maturation, is suppressed by miR-365.
Altered profiles of gene expression reflect the reprogramming of intestinal epithelial cells during their maturation along the crypt-luminal axis. To focus on genes important in this process, and how they in turn are regulated, we identified 14 transcripts commonly downregulated in expression during lineage-specific maturation of the immortalized cell lines Caco-2 (absorptive), HT29Cl16E (goblet), and HT29Cl19A (secretory) induced by contact inhibition of growth or the short-chain fatty acid butyrate. One such gene, Mybl2 (Myb-related protein B), has been linked to the stem cell phenotype, and we report is also markedly suppressed in maturing cells along the crypt-luminal axis in vivo. Mybl2 is not significantly downregulated transcriptionally during colon cell maturation, but we identified a potential micro-RNA (miRNA)-binding sequence in the Mybl2 3'-untranslated region that mediates reporter gene suppression in differentiating colon cells. Accordingly, miRNAs predicted to bind this functional target are upregulated in differentiating colon epithelial cells in vitro and in vivo; expression of one of these, hsa-miR-365 (but not hsa-324-5p), suppresses Mybl2 protein expression in proliferating Caco-2 cells. These data demonstrate that miRNA silencing plays an important role in regulating gene expression in maturing colon epithelial cells, and that utilizing a target-centered approach, rather than profiling global miRNA expression, can identify physiologically relevant, functional miRNAs.


A laser unequal path interferometer for the optical shop.
The application of laser technology has been extended to optical shop testing by incorporating a cw, helium-neon gas laser in a package that houses a modified Twyman-Green interferometer. This modification provides for optical testing over large path differences with an auxiliary set of lenses used in the long path and a small reference flat used in the short path of the interferometer. With this technique, f/0.7 spherical mirrors have been tested (at the center of curvature) to an accuracy of 1/10 wavelength at the surface, and various other optical systems have been tested in both double pass and single pass. Two of the advantages of this testing method are (1) the capability of testing spherical concave surfaces without physically contacting the surface and (2) the ability to use small reference surfaces for large optical components or systems. The device known as a laser unequal path interferometer can be used with a set of null lenses to qualify aspheric surfaces. The unit is portable and capable of testing in any orientation under various environmental conditions. Several applications of this device are presented to illustrate its versatility.


Mitochondrial dynamics regulates migration and invasion of breast cancer cells.
Mitochondria are highly dynamic and undergo constant fusion and fission that are essential for maintaining physiological functions of cells. Although dysfunction of mitochondria has been implicated in tumorigenesis, little is known about the roles of mitochondrial dynamics in metastasis, the major cause of cancer death. In the present study, we found a marked upregulation of mitochondrial fission protein dynamin-related protein 1 (Drp1) expression in human invasive breast carcinoma and metastases to lymph nodes. Compared with non-metastatic breast cancer cells, mitochondria also were more fragmented in metastatic breast cancer cells that express higher levels of total and active Drp1 and less mitochondrial fusion protein 1 (Mfn1). Silencing Drp1 or overexpression of Mfn1 resulted in mitochondria elongation or clusters, respectively, and significantly suppressed metastatic abilities of breast cancer cells. In contrast, silencing Mfn proteins led to mitochondrial fragmentation and enhanced metastatic abilities of breast cancer cells. Interestingly, these manipulations of mitochondrial dynamics altered the subcellular distribution of mitochondria in breast cancer cells. For example, silencing Drp1 or overexpression of Mfn1 inhibited lamellipodia formation, a key step for cancer metastasis, and suppressed chemoattractant-induced recruitment of mitochondria to lamellipodial regions. Conversely, silencing Mfn proteins resulted in more cell spreading and lamellipodia formation, causing accumulation of more mitochondria in lamellipodia regions. More importantly, treatment with a mitochondrial uncoupling agent or adenosine triphosphate synthesis inhibitor reduced lamellipodia formation and decreased breast cancer cell migration and invasion, suggesting a functional importance of mitochondria in breast cancer metastasis. Together, our findings show a new role and mechanism for regulation of cancer cell migration and invasion by mitochondrial dynamics. Thus targeting dysregulated Drp1-dependent mitochondrial fission may provide a novel strategy for suppressing breast cancer metastasis.


Changes in cytokine production and T cell subpopulations in experimentally induced zinc-deficient humans.
We have utilized an experimental model of human zinc deficiency for study of cytokines production by TH1 and TH2 cells. Additionally, we determined ratios of CD4+ to CD8+ and CD4+ CD45RA+ to CD4+CD45RO+ cells and percentages of CD73+ T cytolytic cells in the CD8+ subset. The data were collected during baseline, at the end of the zinc-restricted period, and following zinc repletion. Our results showed that functions of TH1 cells, as evidenced by production of interferon-gamma, interleukin-2 (IL-2), and tumor necrosis factor-alpha, were decreased, whereas functions of TH2 cells (production of IL-4, IL-6, and IL-10) were unaffected by zinc deficiency. Thus an imbalance between TH1 and TH2 cells resulted because of zinc deficiency in humans. Our studies also showed that zinc may be required for regeneration of new CD4+ T lymphocytes and maintenance of T cytolytic cells. We conclude that an imbalance between TH1 and TH2 cells, decreased recruitment of T naive cells, and decreased percentage of T cytolytic cells may account for decreased cell-mediated immune functions in zinc-deficient subjects.


Underreporting of maternal mortality in Taiwan: A data linkage study.
This study examined the extent to which maternal mortality in Taiwan is underreported in officially published mortality statistics. We used National Health Insurance claims data collected from two million samples, which were linked with the officially published mortality data, to identify women aged 15-49 years, who were admitted to a hospital with pregnancy-related diagnoses during 2000-2009 and died during the pregnancy or within 42 days after the termination of pregnancy. Based on these linked data, we identified 26 maternal deaths, only nine of which were reported in the original officially published mortality data; thus, the rate of underreporting was 65% [(26 - 9)/26]. The revised maternal mortality ratio was 14.1 deaths per 100,000 live births (95% confidence interval: 8.7-19.5), which was approximately three times higher than the official reported ratio of 4.9 (95% confidence interval: 1.7-8.1). The most common cause of maternal deaths was amniotic fluid embolism (n = 10), followed by eclampsia and preeclampsia (n = 4). Approximately two-thirds of the maternal deaths in Taiwan were unreported in the officially published mortality data. Hence, routine nationwide data linkage is essential to monitor maternal mortality in Taiwan accurately.


Kv1.3 Controls Mitochondrial Dynamics during Cell Cycle Progression.
The voltage-gated potassium channel Kv1.3 is a potential therapeutic target for obesity and diabetes. The genetic ablation and pharmacological inhibition of Kv1.3 lead to a lean phenotype in rodents. The mechanism of regulation of body weight and energy homeostasis involves Kv1.3 expression in different organs, including white and brown adipose tissues. Here, we show that Kv1.3 promotes the proliferation of preadipocytes through the control of mitochondrial dynamics. Kv1.3 is expressed in mitochondria exhibiting high affinity for the perinuclear population. The mitochondrial network is highly dynamic during the cell cycle, showing continuous fusion-fission events. The formation of a hyperfused mitochondrial network at the G1/S phase of the cell cycle is dependent on Kv1.3 expression. Our results demonstrate that Kv1.3 promotes preadipocyte proliferation and differentiation by controlling mitochondrial membrane potential and mitochondrial dynamics at the G1 phase of the cell cycle.


An animal model for human melanoma.
Experimental animal models that are directly relevant to human melanoma are lacking. We propose the Angora goat as a potentially useful field model with experimental potential and to this end have examined the prevalence and site distribution of all skin cancers in 28 Angora goat herds in Queensland, Australia. The prevalence of benign melanocytic lesions (lentigines) and their experimental induction by sunlight were also investigated. Among 1731 goats over 2 years of age, 139 malignant skin tumors were excised from 95 affected animals. The prevalence of squamous cell carcinoma (SCC) was 3.8% and of melanoma, 2.2%. Main site of occurrence of melanoma (83%) was the dorsal surface of the ear; in contrast SCC occurred mostly (84%) on the perineum. Lentigines were darker and more prevalent on the exposed compared with the unexposed surface of the ear in Angoras, analogous to the higher prevalence of nevi on the exposed compared with the less exposed inner surface of the arm in humans. Lentigines, which were also found on the perineum though lighter in color than on the dorsal ear, were absent in young animals under 3 months but were numerous in 1-3 year olds. Furthermore in an experimental substudy eight goats, having one flank repeatedly shorn and the contralateral flank left unshorn, revealed consistently more solar lentigines on the shorn flank (P < 0.05) when both sides were examined after 9 months. Histopathological examination of paired skin biopsies from five of these goats also showed more abundant pigmentation in skin from the exposed, as compared with the unexposed flank. These findings indicate that sunlight induces tumors and lentigines in goats in a highly site-specific manner. The Angora goat model may suggest paradigms for explaining the site differences observed for human melanoma and may also be useful in the future clarification of molecular changes following carcinogenic levels of sun exposure.


Cervical cancer screening among immigrants and ethnic minorities: a systematic review using the Health Belief Model.
To systematically review all studies examining sociocultural factors influencing cervical cancer screening among immigrant and ethnic minorities in the United States along the theoretical framework of the Health Belief Model. MEDLINE/PubMed, Cumulative Index to Nursing and Allied Health Literature, EMBASE, and Cochrane database searches were conducted searching for English language, US-based studies to examine minority and immigrant populations within the theoretical framework of the Health Belief Model. Fifty-five of more than 3,381 potentially relevant articles were included in the final analysis. Commonly held beliefs across several cultural groups emerged including the following: fatalistic attitudes, a lack of knowledge about cervical cancer, fear of Pap smears threatening one's virginity, as well as beliefs that a Pap smear is unnecessary unless one is ill. Beliefs unique to specific cultural groups included: body-focused notions among Hispanics, as childbirth, menses, sex, and stress were considered to play a role in one's susceptibility to cancer. African Americans identified administrative processes in establishing health care as barriers to screening, whereas Asian immigrants held a variety of misconceptions concerning one's susceptibility to cancer as well as stigmatization imposed by their own community and providers. Health care providers and policy makers must be cognizant of the various sociocultural factors influencing health-related beliefs and health care utilization among immigrant and ethnic minorities in the United States. Culturally relevant screening strategies and programs that address these sociocultural factors must be developed to address the growing disparity in cervical cancer burden among underserved, resource-poor populations in the United States.


Evidence for IFN-gamma as a mediator of the lethality of endotoxin and tumor necrosis factor-alpha.
Current evidence indicates that endogenously produced peptide cytokines, most notably TNF-alpha and IL-1, mediate the lethality of experimental endotoxemia. Because circulating serum levels of IFN-gamma can be detected soon after TNF-alpha and IL-1 in response to endotoxin, we investigated the role of IFN-gamma in endotoxin and TNF-alpha lethality. Specific neutralizing antibodies to murine TNF-alpha (anti-TNF-alpha Ab) or murine IFN gamma (anti-IFN-gamma Ab) produced in our laboratory protected mice against the lethality of Escherichia coli endotoxin (LPS) administered 6 h later. Serum IFN-gamma levels 2 h after i.v. LPS were lower in mice treated with anti-TNF-alpha Ab compared to mice that received nonimmune IgG (median less than 2.5 vs 3.0 U/ml, P2 less than 0.05). In contrast, serum TNF-alpha levels 1 h after i.v. LPS peaked more than fourfold higher in mice treated with anti-IFN-gamma Ab compared to controls (median greater than 6400 vs 1405 pg/ml, p2 less than 0.05). Doses of TNF-alpha (300 micrograms/kg) and IFN-gamma (50,000 U) which were well tolerated when given individually were synergistically lethal in combination (0% lethality vs 100% lethality, P2 less than 0.001), and were associated with higher serum levels of IL-6 than with either cytokine alone. Anti-IFN-gamma Ab provided complete protection against exogenous human rTNF-alpha at the LD100 dose (1400 micrograms/kg, p2 less than 0.001), and in fact prevented lethality at doses four- to fivefold greater than the LD100 human rTNF-alpha (up to 6000 micrograms/kg). We conclude that IFN-gamma is synergistic with TNF-alpha, is essential for the lethality of LPS and TNF-alpha, and may have modulating effects on the negative control of serum levels of TNF-alpha after LPS in mice.


Vaping in the Workplace: Awareness and Support for E-cigarette Workplace Policies.
Objectives: Clean indoor air policies have been an important tool protecting the health of working adults. The purpose of this study was to examine awareness of and support for e-cigarette-free workplace policies among working adults in the United States. Methods: Employees of companies with at least 150 employees (N = 1607, ages 18-65 years) were recruited from an opt-in national panel for an online survey. Results: Nearly half of respondents (48.4%) reported that their employer had a written policy addressing e-cigarette use, 30.2% reported their employer did not have such a policy, and 21.4% reported that they did not know. Most respondents (73.5%) supported e-cigarette-free workplaces, including the majority of current e-cigarette users (53.5%). Multiple regression modeling found that odds of support for e-cigarette workplace policies was significantly higher among never (OR = 1.77, 95% CI: 1.19-2.64) and former e-cigarette users (OR = 1.71, 95% CI: 1.15-2.54) relative to current users. Policy support also varied by perceived harm and other perceptions of workplace vaping, and demographic and workplace characteristics. Conclusions: E-cigarette-free workplace policies have high levels of support among employees, including both current and former e-cigarette users. Policies restricting workplace vaping can improve the health of all employees.


Ultrasound-based quantification of vitreous floaters correlates with contrast sensitivity and quality of life.
Clinical evaluation of floaters lacks quantitative assessment of vitreous structure. This study used quantitative ultrasound (QUS) to measure vitreous opacities. Since floaters reduce contrast sensitivity (CS) and quality of life (Visual Function Questionnaire [VFQ]), it is hypothesized that QUS will correlate with CS and VFQ in patients with floaters. Twenty-two eyes (22 subjects; age = 57 ± 19 years) with floaters were evaluated with Freiburg acuity contrast testing (FrACT; %Weber) and VFQ. Ultrasonography used a customized probe (15-MHz center frequency, 20-mm focal length, 7-mm aperture) with longitudinal and transverse scans taken in primary gaze and a horizontal longitudinal scan through premacular vitreous in temporal gaze. Each scan set had 100 frames of log-compressed envelope data. Within each frame, two regions of interest (ROIs) were analyzed (whole-central and posterior vitreous) to yield three parameters (energy, E; mean amplitude, M; and percentage of vitreous filled by echodensities, P50) averaged over the entire 100-frame dataset. Statistical analyses evaluated E, M, and P50 correlations with CS and VFQ. Contrast sensitivity ranged from 1.19%W (normal) to 5.59%W. All QUS parameters in two scan positions within the whole-central ROI correlated with CS (R > 0.67, P < 0.001). P50 in the nasal longitudinal position had R = 0.867 (P < 0.001). Correlations with VFQ ranged from R = 0.52 (P < 0.013) to R = 0.65 (P < 0.001). Quantitative ultrasound provides quantitative measures of vitreous echodensity that correlate with CS and VFQ, providing objective assessment of vitreous structure underlying the functional disturbances induced by floaters, useful to quantify vitreous disease severity and the response to therapy.


Peripheral δ-opioid receptors attenuate the exercise pressor reflex.
In rats with ligated femoral arteries, the exercise pressor reflex is exaggerated, an effect that is attenuated by stimulation of peripheral μ-opioid receptors on group IV metabosensitive afferents. In contrast, δ-opioid receptors are expressed mostly on group III mechanosensitive afferents, a finding that prompted us to determine whether stimulation of these opioid receptors could also attenuate the exaggerated exercise pressor reflex in "ligated" rats. We found femoral arterial injection of [D-Pen2,D-Pen5]enkephalin (DPDPE; 1.0 μg), a δ-opioid agonist, significantly attenuated the pressor and cardioaccelerator components of the exercise pressor reflex evoked by hindlimb muscle contraction in both rats with ligated and patent femoral arteries. DPDPE significantly decreased the pressor responses to muscle mechanoreflex activation, evoked by tendon stretch, in ligated rats only. DPDPE (1.0 μg) had no effect in either group on the pressor and cardioaccelerator responses to capsaicin (0.2 μg), which primarily stimulates group IV afferents. DPDPE (1.0 μg) had no effect on the pressor and cardioaccelerator responses to lactic acid (24 mM), which stimulates group III and IV afferents, in rats with patent femoral arteries but significantly decreased the pressor response in ligated rats. Western blots revealed the amount of protein comprising the δ-opioid receptor was greater in dorsal root ganglia innervating hindlimbs with ligated femoral arteries than in dorsal root ganglia innervating hindlimbs with patent femoral arteries. Our findings support the hypothesis that stimulation of δ-opioid receptors on group III afferents attenuated the exercise pressor reflex.


Maternal administration of cannabidiol promotes an anti-inflammatory effect on the intestinal wall in a gastroschisis rat model.
Gastroschisis (GS) is an abdominal wall defect that results in histological and morphological changes leading to intestinal motility perturbation and impaired absorption of nutrients. Due to its anti-inflammatory, antioxidant, and neuroprotective effects, cannabidiol (CBD) has been used as a therapeutic agent in many diseases. Our aim was to test the effect of maternal CBD in the intestine of an experimental model of GS. Pregnant rats were treated over 3 days with CBD (30 mg/kg) after the surgical induction of GS (day 18.5 of gestation) and compared to controls. Fetuses were divided into 4 groups: 1) control (C); 2) C+CBD (CCBD); 3) gastroschisis (G), and 4) G+CBD (GCBD). On day 21.5 of gestation, the fetuses were harvested and evaluated for: a) body weight (BW), intestinal weight (IW), and IW/BW ratio; b) histometric analysis of the intestinal wall; c) immunohistochemically analysis of inflammation (iNOS) and nitrite/nitrate level. BW: GCBD was lower than CCBD (P<0.005), IW and IW/BW ratio: GCBD was smaller than G (P<0.005), GCBD presented lower thickness in all parameters compared to G (P<0.005), iNOS and nitrite/nitrate were lower concentration in GCBD than to G (P<0.005). Maternal use of CBD had a beneficial effect on the intestinal loops of GS with decreased nitrite/nitrate and iNOS expression.


Emerging role of selective autophagy in human diseases.
Autophagy was originally described as a highly conserved system for the degradation of cytosol through a lysosome-dependent pathway. In response to starvation, autophagy degrades organelles and proteins to provide metabolites and energy for its pro-survival effects. Autophagy is recognized as playing a role in the pathogenesis of disease either directly or indirectly, through the regulation of vital processes such as programmed cell death, inflammation, and adaptive immune mechanisms. Recent studies have demonstrated that autophagy is not only a simple metabolite recycling system, but also has the ability to degrade specific cellular targets, such as mitochondria, cilia, and invading bacteria. In addition, selective autophagy has also been implicated in vesicle trafficking pathways, with potential roles in secretion and other intracellular transport processes. Selective autophagy has drawn the attention of researchers because of its potential importance in clinical diseases. Therapeutic strategies to target selective autophagy rather than general autophagy may maximize clinical benefit by enhancing selectivity. In this review, we outline the principle components of selective autophagy processes and their emerging importance in human disease, with an emphasis on pulmonary diseases.


Factors influencing constipation in advanced cancer patients: a prospective study of opioid dose, dantron dose and physical functioning.
The relationship between opioid dose, dantron dose, bowel function and physical functioning (measured with the modified Barthel Index) was determined in a sample of 50 inpatients with advanced cancer. Data were collected prospectively from chart review and patient interviews one week after admission to allow for protocol-driven management of constipation to be established. Bowel scores were significantly reduced in 35 patients treated with opioids compared with 15 patients not treated with opioids. Within the opioid group, however, there was no relationship between opioid dose, bowel score, dantron dose or Barthel Index. Higher doses of dantron were associated with better physical functioning (but not opioid dose) suggesting that for any given dose of opioid, fitter patients were treated with larger doses of laxatives. Factors other than opioid dose and physical functioning may be more important in contributing to constipation in this group of patients. Less potent opioid drugs, such as codeine, are just as likely to cause constipation as more potent opioids.


Emerging Technologies to Image Tissue Metabolism.
Due to the implication of altered metabolism in a large spectrum of tissue function and disease, assessment of metabolic processes becomes essential in managing health. In this regard, imaging can play a critical role in allowing observation of biochemical and physiological processes. Nuclear imaging methods, in particular positron emission tomography, have been widely employed for imaging metabolism but are mainly limited by the use of ionizing radiation and the sensing of only one parameter at each scanning session. Observations in healthy individuals or longitudinal studies of disease could markedly benefit from non-ionizing, multi-parameter imaging methods. We therefore focus this review on progress with the non-ionizing radiation methods of MRI, hyperpolarized magnetic resonance and magnetic resonance spectroscopy, chemical exchange saturation transfer, and emerging optoacoustic (photoacoustic) imaging. We also briefly discuss the role of nuclear and optical imaging methods for research and clinical protocols.


Increased infiltration of CCR4-positive regulatory T cells in prostate cancer tissue is associated with a poor prognosis.
Regulatory T cells (Tregs) play important roles in the suppression of immune responses, including antitumor immune responses. C-C chemokine receptor 4 (CCR4) is highly expressed on effector Tregs, and anti-CCR4 antibody is attracting attention as a novel immunotherapeutic agent for solid tumors. This study aimed to evaluate the expression of CCR4-positive Tregs (CCR4+Tregs) in prostate cancer and estimate the clinical potential of CCR4-targeting therapy for prostate cancer. A total of 15 radical prostatectomy (RP) specimens and 60 biopsy specimens from individuals diagnosed with prostate cancer were analyzed to evaluate the infiltration of CCR4+Tregs in prostate cancer. The relationships between the number of CCR4+Tregs and clinical parameters were investigated in RP and biopsy specimens. Moreover, the total number of Tregs, CCR4+Tregs, and T cells and the ratio of CCR4+Tregs to Tregs and T cells in biopsy specimens were compared between patients with poor prognosis who progressed to castration-resistant prostate cancer (CRPC) within 12 months (n = 13) and those with good prognosis who were stable with hormone-sensitive prostate cancer over 12 months (n = 47). Furthermore, biopsy specimens were divided into two groups: low and high CCR4+Treg expression groups and the prognosis was compared between them. There was a higher expression of CCR4+Tregs in RP specimens with a higher (≥8) Gleason score than in those with a lower (<8) Gleason score (P = .041). In biopsy specimens, 65.9% Tregs were positive for CCR4. The number of CCR4+Tregs positively correlated with clinical stage (P < .001) and Gleason score (P = .006). The total number of Tregs and CCR4+Tregs significantly increased in the poor prognosis group compared with that in the good prognosis group (P = .024 and .01, respectively). Furthermore, patients with lower CCR4+Treg expression levels showed a significantly longer time to progression to CRPC (not reached vs 27.3 months; P < .001) and median survival time (not reached vs 69.0 months; P = .014) than those with higher expression levels. CCR4+Tregs are highly infiltrated in the prostate tissue of patients with poor prognosis with potential to progress to CRPC. Furthermore, the degree of infiltration of CCR4+Tregs is related to the prognosis of prostate cancer.


Adherence to self-managed exercises for patients with persistent subacromial pain: the Ad-Shoulder feasibility study.
Exercise is recommended for patients with subacromial pain. It has been suggested that good exercise adherence improves clinical outcomes. Despite this, little attention has been paid to the need for behavioural frameworks to enhance adherence to home exercise programmes for patients with subacromial pain. A feasibility study with pre-post design was used. Participants aged > 18 years, with subacromial pain, who had received conservative treatment during the past 6 months, were recruited. The Ad-Shoulder intervention consisted of 1-5 individual sessions provided over 3 months and was based on 5 self-management skills, which aimed to enhance the patients' self-efficacy and adherence to self-managed exercises. The primary objectives were assessed according to predefined progression criteria: (1) the recruitment rate (10 patients enrolled within 12 weeks), (2) follow-up rate (≥ 80% on all self-reported measures), (3) objective physical activity measures (≥ 80% of participants would contribute valid data at each time point), (4) adherence with the self-managed exercises (≥ 80% of the participants would adhere to ≥ 80% of the assigned home exercise programme), (5) fidelity of the delivery of the intervention (the therapists delivered the intervention according to the protocol) and (6) adverse events (< 30% would report adverse events (including mild)). The results were reported using descriptive statistics. Eleven patients were recruited during 16 weeks. Ten patients completed the self-reported measures at baseline and week 12. Objective physical activity measures were successfully obtained for 100% (11/11) at baseline, 64% (7/11) at week six and 82% at week 12. Fifty-five percent (6/11) of the participants satisfactorily completed at least 80% of their home exercise programme. All sessions were delivered according to the protocol. None of the patients reported any adverse events. Objective physical activity data measures at baseline and week 12, follow-up, the physiotherapists' fidelity to the intervention and adverse events met our pre-specified progression criteria. Recruitment and adherence to the self-managed exercise programme were both below the anticipated level. Further intervention development is necessary to understand whether adherence to the self-managed exercises could be enhanced and additional methods of recruitment would need to be considered, including additional recruitment sites, in any planning for a future main trial. ClinicalTrials.gov, NCT04190836 , Registered December 9, 2019-retrospectively registered.


Leaky gut and autoimmune diseases.
Autoimmune diseases are characterized by tissue damage and loss of function due to an immune response that is directed against specific organs. This review is focused on the role of impaired intestinal barrier function on autoimmune pathogenesis. Together with the gut-associated lymphoid tissue and the neuroendocrine network, the intestinal epithelial barrier, with its intercellular tight junctions, controls the equilibrium between tolerance and immunity to non-self antigens. Zonulin is the only physiologic modulator of intercellular tight junctions described so far that is involved in trafficking of macromolecules and, therefore, in tolerance/immune response balance. When the zonulin pathway is deregulated in genetically susceptible individuals, autoimmune disorders can occur. This new paradigm subverts traditional theories underlying the development of these diseases and suggests that these processes can be arrested if the interplay between genes and environmental triggers is prevented by re-establishing the zonulin-dependent intestinal barrier function. Both animal models and recent clinical evidence support this new paradigm and provide the rationale for innovative approaches to prevent and treat autoimmune diseases.


Traditional plant functional groups explain variation in economic but not size-related traits across the tundra biome.
Plant functional groups are widely used in community ecology and earth system modelling to describe trait variation within and across plant communities. However, this approach rests on the assumption that functional groups explain a large proportion of trait variation among species. We test whether four commonly used plant functional groups represent variation in six ecologically important plant traits. Tundra biome. Data collected between 1964 and 2016. 295 tundra vascular plant species. We compiled a database of six plant traits (plant height, leaf area, specific leaf area, leaf dry matter content, leaf nitrogen, seed mass) for tundra species. We examined the variation in species-level trait expression explained by four traditional functional groups (evergreen shrubs, deciduous shrubs, graminoids, forbs), and whether variation explained was dependent upon the traits included in analysis. We further compared the explanatory power and species composition of functional groups to alternative classifications generated using post hoc clustering of species-level traits. Traditional functional groups explained significant differences in trait expression, particularly amongst traits associated with resource economics, which were consistent across sites and at the biome scale. However, functional groups explained 19% of overall trait variation and poorly represented differences in traits associated with plant size. Post hoc classification of species did not correspond well with traditional functional groups, and explained twice as much variation in species-level trait expression. Traditional functional groups only coarsely represent variation in well-measured traits within tundra plant communities, and better explain resource economic traits than size-related traits. We recommend caution when using functional group approaches to predict tundra vegetation change, or ecosystem functions relating to plant size, such as albedo or carbon storage. We argue that alternative classifications or direct use of specific plant traits could provide new insights for ecological prediction and modelling.


Structure of ZnCl2 Melt. Part I: Raman Spectroscopy Analysis Driven by Ab Initio Methods.
The structure of molten ZnCl2 is investigated using a combination of computer simulation and experimental methods. Ab initio molecular dynamics (AIMD) is used to model the structure of ZnCl2 at 600 K. The structure factors and pair distribution functions derived from AIMD show a good match with those previously measured by neutron diffraction (ND). In addition, Raman spectroscopy is used to investigate the structure of liquid ZnCl2 and identify the relative fractions of constituent structural units. To ascertain the assignment of each Raman mode, a series of ZnCl2 crystalline prototypes are modeled and the corresponding Raman modes are derived by first-principles calculations. Curve fitting of experimental Raman spectra using these mode assignments shows excellent agreement with both AIMD and ND. These results confirm the presence of significant fractions of edge-sharing tetrahedra in liquid ZnCl2. The presence of these structural motifs has significant impact on the fragility of this tetrahedral glass-forming liquid. The assignment of Raman bands present in molten ZnCl2 is revised and discussed in view of these results.


Intramuscular botulinum toxin in complex regional pain syndrome: case series and literature review.
Pain associated with Complex Regional Pain Syndrome (CRPS) is frequently excruciating and intractable. The use of botulinum toxin for relief of CRPS-associated pain has not been well described. To assess whether intramuscular botulinum toxin injections cause relief of pain caused by CRPS, and to assess the risks of this treatment. Retrospective chart review. Outpatient clinic. 37 patients with spasm/dystonia in the neck and/or upper limb girdle muscles. EMG-guided injection of Botulinum Toxin - A (BtxA), 10-20 units per muscle. Total dose used was 100 units in each patient. Local pain score was measured on an 11-point Likert scale, 4 weeks after BtxA injections. Mean pain score decreased by 43% (8.2 ± 0.8 to 4.5 ± 1.1, P < 0.001). 97% patients had significant pain relief. One patient had transient neck drop after the injections. This is a retrospective study, it lacks a control group and hence the placebo effect cannot be eliminated. This study does not provide information on the efficacy of this treatment after 4 weeks. Intramuscular injection of botulinum toxin in the upper limb girdle muscles was beneficial for short term relief of pain caused by CRPS. The incidence of complications was low (2.7%).