The population structure of Acinetobacter baumannii: expanding multiresistant clones from an ancestral susceptible genetic pool.
Outbreaks of hospital infections caused by multidrug resistant Acinetobacter baumannii strains are of increasing concern worldwide. Although it has been reported that particular outbreak strains are geographically widespread, little is known about the diversity and phylogenetic relatedness of A. baumannii clonal groups. Sequencing of internal portions of seven housekeeping genes (total 2,976 nt) was performed in 154 A. baumannii strains covering the breadth of known diversity and including representatives of previously recognized international clones, and in 19 representatives of other Acinetobacter species. Restricted amounts of diversity and a star-like phylogeny reveal that A. baumannii is a genetically compact species that suffered a severe bottleneck in the recent past, possibly linked to a restricted ecological niche. A. baumannii is neatly demarcated from its closest relative (genomic species 13TU) and other Acinetobacter species. Multilocus sequence typing analysis demonstrated that the previously recognized international clones I to III correspond to three clonal complexes, each made of a central, predominant genotype and few single locus variants, a hallmark of recent clonal expansion. Whereas antimicrobial resistance was almost universal among isolates of these and a novel international clone (ST15), isolates of the other genotypes were mostly susceptible. This dichotomy indicates that antimicrobial resistance is a major selective advantage that drives the ongoing rapid clonal expansion of these highly problematic agents of nosocomial infections.


Formation and structure of scales in the Australian lungfish, Neoceratodus forsteri (Osteichthyes: Dipnoi).
The large elasmoid scales of the Australian lungfish, Neoceratodus forsteri, are formed within the dermis by unpigmented scleroblasts, growing within a collagenous dermal pocket below a thick glandular epidermis. The first row of scales, on the trunk of the juvenile lungfish, appears below the lateral line of the trunk, single in this species, at around stage 53. The scales, initially circular in outline, develop anteriorly and posteriorly from the point of initiation in the mid-trunk region, and rows are added alternately below the line, and above the line, until they reach the dorsal or ventral midline, or the margins of the fins. Scales develop later on the ventral surface of the head, from a separate centre of initiation. Scales consist of three layers, all produced by scleroblasts of dermal origin. The outermost layer of interlocking plates, or squamulae, consists of a mineralised matrix of fine collagen fibrils, covered by unmineralised collagen and a single layer of cells. Squamulae of the anterior and lateral surfaces are ornamented with short spines, and the mineralised tissue of the posterior surface is linked to the pouch by collagen fibrils. The innermost layer, known as elasmodin, consists of bundles of thick collagen fibrils and cells arranged in layers. An intermediate layer, made up of collagen fibrils, links the outer and inner layers. The elasmoid scales of N. forsteri can be compared with scale types among other osteichthyan groups, although the cellsand canaliculi in the mineralised squamulae bear little resemblance to typical bone.


Novel immunologic approaches to the management of malignant melanoma.
In the past decade, the discovery of tumor antigens recognized by T cells has revolutionized the tumor vaccine field. The appreciation that peptides are bound to and restricted by major histocompatibility class I and II molecules for immune recognition has encouraged a number of early-phase clinical trials of peptide vaccines. I summarize herein the rationale for and the results of a number of clinical trials of peptide vaccines for melanoma, suggesting that immune and clinical responses can be seen in those with metastatic and resected disease using a variety of surrogate assays. The challenge for the future is to correlate the results of immunologic assays with clinical benefit in patients with advanced cancer.


CDKL5 knockout leads to altered inhibitory transmission in the cerebellum of adult mice.
Mutations in the X-linked cyclin-dependent kinase-like 5 gene (CDKL5) are associated to severe neurodevelopmental alterations including motor symptoms. In order to elucidate the neurobiological substrate of motor symptoms in CDKL5 syndrome, we investigated the motor function, GABA and glutamate pathways in the cerebellum of CDKL5 knockout female mice. Behavioural data indicate that CDKL5-KO mice displayed impaired motor coordination on the Rotarod test, and altered steps, as measured by the gait analysis using the CatWalk test. A higher reduction in spontaneous GABA efflux, than that in glutamate, was observed in CDKL5-KO mouse cerebellar synaptosomes, leading to a significant increase of spontaneous glutamate/GABA efflux ratio in these animals. On the contrary, there were no differences between groups in K(+) -evoked GABA and glutamate efflux. The anatomical analysis of cerebellar excitatory and inhibitory pathways showed a selective defect of the GABA-related marker GAD67 in the molecular layer in CDKL5-KO mice, while the glutamatergic marker VGLUT1 was unchanged in the same area. Fine cerebellar structural abnormalities such as a reduction of the inhibitory basket 'net' estimated volume and an increase of the pinceau estimated volume were also observed in CDKL5-KO mice. Finally, the BDNF mRNA expression level in the cerebellum, but not in the hippocampus, was reduced compared with WT animals. These data suggest that CDKL5 deletion during development more markedly impairs the establishment of a correct GABAergic cerebellar network than that of glutamatergic one, leading to the behavioural symptoms associated with CDKL5 mutation.


A realistic brain tissue phantom for intraparenchymal infusion studies.
The goal of this study was to validate a simple, inexpensive, and robust model system to be used as an in vitro surrogate for in vivo brain tissues in preclinical and exploratory studies of infusion-based intraparenchymal drug and cell delivery. Agarose gels of varying concentrations and porcine brain were tested to determine the infusion characteristics of several different catheters at flow rates of 0.5 and 1 microl per minute by using bromophenol blue (BPB) dye (molecular weight [MW] approximately 690) and gadodiamide (MW approximately 573). Magnetic resonance (MR) imaging and videomicroscopy were used to measure the distribution of these infusates, with a simultaneous measurement of infusion pressures. In addition, the forces of catheter penetration and movement through gel and brain were measured. Agarose gel at a 0.6% concentration closely resembles in vivo brain with respect to several critical physical characteristics. The ratio of distribution volume to infusion volume of agarose was 10 compared with 7.1 for brain. The infusion pressure of the gel demonstrated profiles similar in configuration and magnitude to those of the brain (plateau pressures 10-20 mm Hg). Gadodiamide infusion in agarose closely resembled that in the brain, as documented using T1-weighted MR imaging. Gadodiamide distribution in agarose gel was virtually identical to that of BPB dye, as documented by MR imaging and videomicroscopy. The force profile for insertion of a silastic catheter into agarose gel was similar in magnitude and configuration to the force profile for insertion into the brain. Careful insertion of the cannula using a stereotactic guide is critical to minimize irregularity and backflow of infusate distribution. Agarose gel (0.6%) is a useful surrogate for in vivo brain in exploratory studies of convection-enhanced delivery.


Galleria mellonella as an infection model to investigate virulence of Vibrio parahaemolyticus.
Non-toxigenic V. parahaemolyticus isolates (tdh-/trh-/T3SS2-) have recently been isolated from patients with gastroenteritis. In this study we report that the larvae of the wax moth (Galleria mellonella) are susceptible to infection by toxigenic or non-toxigenic clinical isolates of V. parahaemolyticus. In comparison larvae inoculated with environmental isolates of V. parahaemolyticus did not succumb to disease. Whole genome sequencing of clinical non-toxigenic isolates revealed the presence of a gene encoding a nudix hydrolase, identified as mutT. A V. parahaemolyticus mutT mutant was unable to kill G. mellonella at 24 h post inoculation, indicating a role of this gene in virulence. Our findings show that G. mellonella is a valuable model for investigating screening of possible virulence genes of V. parahaemolyticus and can provide new insights into mechanisms of virulence of atypical non-toxigenic V. parahaemolyticus. These findings will allow improved genetic tests for the identification of pathogenic V. parahaemolyticus to be developed and will have a significant impact for the scientific community.


GC-MS and GC-IRD studies on the six ring regioisomeric dimethoxybenzoylpiperazines (DMBzPs).
The dimethoxybenzoylpiperazines show mass spectra characteristic for this set of six regioisomeric substances and the position of ring substitution for the dimethoxy groups can be determined by vapor phase infrared methods. The dimethoxybenzoylpiperazines are characterized by several fragment ions unique to the mass spectra for this set of regioisomeric compounds. Ions at m/z 165 and 182 indicate the presence of the dimethoxybenzoyl and dimethoxybenzamide groups while low mass ions at m/z 56, 69 and 85 are characteristic of the piperazine ring in these isomeric compounds. The mass spectra for these regioisomeric dimethoxybenzoylpiperazines are almost identical and this method alone does not provide for the confirmation of identity of any one of the isomers to the exclusion of the other compounds. The perfluoroacyl derivatives of the six regioisomers were resolved by gas chromatography and their mass spectra showed some differences in relative abundance of fragment ions without the appearance of any unique fragments for specific confirmation of structure. Gas chromatography with infrared detection (GC-IRD) provides direct confirmatory data for the differentiation between the regioisomeric underivatized dimethoxybenzoylpiperazines.


Single-Cell Transcriptome Analysis of Mouse Liver Cell-Specific Tropism and Transcriptional Dysregulation Following Intravenous Administration of AAVrh.10 Vectors.
Capitalizing on liver tropism of adeno-associated viral (AAV) vectors, intravenous vector administration is commonly used to genetically modify hepatocytes, a strategy currently in clinical trials for a number of liver-based hereditary disorders. Although hepatocytes are known to exhibit extensive phenotypic heterogeneity influenced by liver zonation and dietary cycle, there is little data available for the tropism capacity, as well as the potential transcriptional dysregulation, of AAV vectors for specific liver cell types. To assess these issues, we employed single-cell RNA sequencing of the mouse liver after intravenous administration of the liver tropic AAVrh.10 vector to characterize cell-specific AAV-mediated transgene expression and transcriptome dysregulation. Wild-type 8-week-old male C57Bl/6 mice under normal feed cycle were randomly divided into three groups and intravenously administered phosphate-buffered saline (PBS), AAVrh.10Null (no transgene), or AAVrh.10mCherry (marker gene). Overall, a total of 46,500 liver cells were sequenced. The single-cell transcriptomic profiles were grouped into three separate clusters of hepatocytes (Ttr-enriched "Hep1," Tat-enriched "Hep2," and Alb-enriched "Hep3") and multiple other cell types. The hepatocyte diversity was driven by glucose and lipid homeostasis signaling. Assessment of the transgene expression demonstrated that AAVrh.10 is primarily Hep1-tropic, with a 10-gene signature positively correlated with AAVrh.10-mediated transgene expression. The transgene expression was less in Hep2 and Hep3 cells with a high receptor tyrosine kinase phenotype. Importantly, AAVrh.10 vector interactions with the liver markedly altered the transcriptional patterns of all cell types, with modified genes enriched in pathways of complement and coagulation cascade, cytochrome P450, peroxisome, antigen processing and presentation, and endoplasmic reticulum protein processing. These observations provide insights into the liver cell-specific consequences of AAV-mediated liver gene transfer, far beyond the well-known organ-specific expression of the vector-delivered transgene.


Lung transplantation in patients with cystic fibrosis: the Israeli experience.
Lung transplantation is a well-established therapeutic option for end-stage lung disease in cystic fibrosis. Although it confers a clear survival advantage, outcome differs among centers according to local experience, patient selection, transplantation procedure, and postoperative care. To evaluate the national Israeli experience with lung transplantation in patients with CF. We reviewed the medical charts of all CF patients who underwent lung transplantation between January 1996 and June 2005 at the two Israeli centers that perform this procedure. Eighteen transplantations were performed in 17 patients. Mean patient age at transplantation was 25.3 +/- 9.1 years, and mean duration of follow-up in survivors (n=14) was 37.2 months (range 1-113 months). The actuarial survival rate was 88% at 1 year and 74% at 5 years. Pulmonary function, expressed as percent of predicted normal forced expiratory volume in 1 sec, improved from 22.4 +/- 8.1% to 76 +/- 16.8% at one year after transplantation. Bronchiolitis obliterans syndrome was diagnosed in 5 patients (29%), of whom 2 died and 2 are currently candidates for retransplantation. Median time to onset of BOS was 34.2 months (range 17-64 months). In Israel, the early and intermediate-term results of lung transplantation for cystic fibrosis are encouraging. BOS remains a major complication that threatens long-term outcome.


[Personality disorders and "psychopathy" in sex offenders imprisoned in forensic-psychiatric hospitals--SKID-II- and PCL-R-results in patients with impulse control disorder and paraphilia].
In order to clarify differential-diagnostic questions 47 mentally ill sex offenders (with impulse control disorder and paraphilia) were assessed with regard to comorbidity of personality disorders and "psychopathy". For this examination the SKID-II for personality disorders and the Psychopathy Checklist (PCL-R) were used. 72 % of the sex offenders showed at least one personality disorder. The highest prevalence was found for cluster-B disorders, first of all the antisocial personality disorder. Using a PCL-R cut off score of 25 (for Europe) 10 of the 47 subjects (21 %) were diagnosed as "psychopaths", whereas being a "psychopath" is associated with a number of different personality disorders. The importance of structured diagnostic with regard to comorbidity of personality disorders in sex offenders imprisoned in maximum security psychiatric hospitals is stressed, and furthermore there is evidence that the PCL-R is a valid instrument for prognosis, but not a psychological-psychiatric instrument for the assessment of personality disorders.


Chromogranin A, B and C: immunohistochemical localization in ovine pituitary and the relationship with hormone-containing cells.
Chromogranins A, B and C, three distinct groups of proteins found in bovine chromaffin granules, were also found to be present in the pituitary using immunoblotting techniques. Their distribution was therefore studied in the normal ram pituitary using an immunoperoxidase technique applied to semithin serial sections and compared with that of some of the hormones of the anterior pituitary. Chromogranin-immunoreactivity was found in gonadotrophs (all three), thyrotrophs (A with some positive for C) and corticotrophs (a fraction with A and fewer with B and C). The mammotrophs and somatotrophs were negative. Chromogranin C was the only one of the three to be located in the pars nervosa, whilst chromogranin B was rarely found in the pars intermedia. The results suggest that chromogranins A, B and C are not always stored together, some hormone-containing cells do not contain immunohistologically detectable levels of the chromogranins.


Systemic collateral supply in patients with chronic thromboembolic and primary pulmonary hypertension: assessment with multi-detector row helical CT angiography.
To compare retrospectively the frequency of systemic collateral supply in patients who have chronic thromboembolic pulmonary hypertension with the frequency of systemic collateral supply in patients who have primary pulmonary hypertension by using multi-detector row helical computed tomographic (CT) angiography. For this review, neither institutional board approval nor informed consent was required. Thirty-six consecutive patients, including 22 patients (four men, 18 women; mean age, 46.0 years) with chronic thromboembolic pulmonary hypertension (group 1) and 14 patients (five men, nine women; mean age, 63.0 years) with primary pulmonary hypertension (group 2), underwent multisection spiral CT angiography of the pulmonary and systemic circulations with a four- (n = 17) or 16- (n = 19) detector row scanner. CT angiograms were assessed for the presence of abnormal bronchial and/or nonbronchial systemic arteries, CT features of pulmonary hypertension, and right ventricular dysfunction. Vascular and parenchymal signs of chronic pulmonary embolism were specifically analyzed on CT angiograms of group 1 patients. Comparative analyses were performed by using the chi(2) or the Fisher exact test for categoric data. An unpaired bilateral Wilcoxon rank sum test was used for continuous data. A chi(2) goodness-of-fit test was used to compare observed proportions with equal proportions. The degree of pulmonary hypertension was comparable in groups 1 and 2. Abnormally enlarged systemic arteries were identified in 16 (73%) of 22 patients from group 1 and in two (14%) of 14 patients from group 2 (P = .002). The systemic collateral supply in group 1 comprised enlargement of both bronchial and nonbronchial systemic arteries in nine (56%) of the 16 patients; the remaining seven patients had an exclusive enlargement of bronchial systemic arteries (n = 6, 38%) or nonbronchial (n = 1, 6%) systemic arteries. A total of 31 enlarged nonbronchial systemic arteries were depicted, including 13 inferior phrenic arteries, 10 intercostal arteries, seven internal mammary arteries, and one lateral thoracic artery. The mean +/- standard deviation of abnormal nonbronchial systemic arteries per patient was 1.4 +/- 1.9. No relationship was found between the mean number of abnormally enlarged nonbronchial systemic arteries and the CT angiographic features of chronic pulmonary embolism. These results demonstrate the higher frequency of abnormally enlarged bronchial and nonbronchial systemic arteries in patients who have chronic thromboembolic pulmonary hypertension compared with patients who have primary pulmonary hypertension; this finding could help distinguish these two entities on CT angiograms.


[A novel coronavirus, MERS-CoV].
A novel human coronavirus was identified in Saudi Arabia and Qatar as the causative agent of severe acute respiratory diseases in 2012. The virus was termed Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and is taken notice of important coronavirus caused severe diseases to human after the outbreak of severe acute respiratory syndrome (SARS) coronavirus. There is a lot of unknown characterization regarding MERS-CoV because of less than one year after finding the first case. MERS-CoV was related to the 2C betacoronavirus clade and is closely related to Tylonycteris bat coronavirus HKU4 and Pipistrellus bat coronavirus HKU5. Thus, bats are thought to be natural hosts of this virus. Recently, there were reports supposed to be cases of human to human infection. There are growing concerns about spread of infection.


Osteopontin small interfering RNA protects mice from fulminant hepatitis.
Osteopontin (OPN) has been implicated in various helper T cell type 1 immunity-mediated diseases including rheumatoid arthritis (RA), multiple sclerosis (MS), Crohn's disease, and fulminant hepatitis. Increased expression of OPN has been detected in pathological foci of these diseases. RA and fulminant hepatitis have been successfully treated by administration of neutralizing anti-OPN antibody in mice. Antibody treatment may elicit side effects including allergic reactions against heterologous antibody proteins, thus necessitating humanization of antibody. To provide alternative means to neutralize OPN function, in this study we explored the possibility of using OPN small interfering RNA (siRNA) to silence OPN gene expression. In vitro, OPN siRNA efficiently silenced the expression of both exogenous and endogenous OPN gene. After hydrodynamic intravenous injection of OPN siRNA, OPN siRNA was efficiently delivered to the liver, which resulted in the efficient silencing of OPN gene expression in liver. In a murine model of concanavalin A (ConA)-induced fulminant hepatitis, OPN expression was elevated in liver and severe hepatic necrosis was induced. Importantly, after OPN siRNA treatment, the OPN expression level in liver was significantly reduced and liver tissue injury was ameliorated, as reflected by the significant reduction of serum alanine aminotransferase levels and almost normal liver histology. Thus, this study indicates that OPN siRNA delivery has therapeutic potential in various inflammatory diseases in which OPN play a critical role by silencing OPN gene expression in vivo.


Measurement of Cervical Sagittal Alignment Parameters on X-Ray Films of Adults without Severe Spinal Deformity Whose Shoulder Hides the Lower Cervical Column.
The Cobb angle between the lower endplate of C2 and C7 (C2L-C7L angle) is a traditional parameter used for the assessment of the cervical alignment. However, when the lower cervical column is masked by the shoulder, measurements are difficult. In the present study, we inspected 191 X-ray films, measured the Cobb angle between C2L and the endplates at the several levels of the lower cervical column, and assessed their usefulness of such measurements for the determination of cervical sagittal alignment. We obtained X-ray films on 191 patients ranging in age from 20 to 93 years. The Cobb angle between C2L and the C7 upper (C7U), the C6 lower (C6L), the C6 upper (C6U), and the C5 lower endplate (C5L) was measured and compared with the C2L-C7L angle. C7L was identified in 116 of 191 patients (60.7%). Except for C2L-C7U angle (P = 0.55), the difference in the mean between C2L-C7L angle and the angle between C2L and the other endplates was statistically significant (P < 0.05). There was a very strong correlation between C2L-C7L angle and C2L-C7U angle (r = 0.99), C2L-C6L angle (r = 0.96), C2L-C6U angle (r = 0.94), and C2L-C5L angle (r = 0.86). To measure the C2L-C7L angle on unclear X-ray films, C7U can be substituted for C7L. Our measurement data for the C6 and C5 endplates were statistically different; however, the correlation between the C2L-C7L angle and C2L-C6U angle, C2L-C6L angle or C2L-C5L angle was very strong. In patients with unclear lower vertebral bodies, cervical sagittal alignment can be predicted by using adjacent endplates.


The Simbios National Center: Systems Biology in Motion.
Physics-based simulation is needed to understand the function of biological structures and can be applied across a wide range of scales, from molecules to organisms. Simbios (the National Center for Physics-Based Simulation of Biological Structures, http://www.simbios.stanford.edu/) is one of seven NIH-supported National Centers for Biomedical Computation. This article provides an overview of the mission and achievements of Simbios, and describes its place within systems biology. Understanding the interactions between various parts of a biological system and integrating this information to understand how biological systems function is the goal of systems biology. Many important biological systems comprise complex structural systems whose components interact through the exchange of physical forces, and whose movement and function is dictated by those forces. In particular, systems that are made of multiple identifiable components that move relative to one another in a constrained manner are multibody systems. Simbios' focus is creating methods for their simulation. Simbios is also investigating the biomechanical forces that govern fluid flow through deformable vessels, a central problem in cardiovascular dynamics. In this application, the system is governed by the interplay of classical forces, but the motion is distributed smoothly through the materials and fluids, requiring the use of continuum methods. In addition to the research aims, Simbios is working to disseminate information, software and other resources relevant to biological systems in motion.


Oxidative stress responses of juvenile tambaqui Colossoma macropomum after short-term anesthesia with benzocaine and MS-222.
The present study aimed to evaluate the effects of benzocaine and tricaine methanesulfonate on oxidative stress parameters of juvenile tambaqui tissues. Fish (n=80) were anesthetized with benzocaine (100 mg L-1) or tricaine (240 mg L-1) and two control groups were used (non-anesthetized fish and fish exposed to ethanol-only). After anesthetic induction 10 fish/anesthetic were euthanized after 3, 12 and 24 hours post-anesthesia and tissue samplings (gills, liver and brain) were performed. Samples were submitted to analyses of enzyme activity glutathione-S-transferase (GST), cellular lipid peroxidation (TBARS) and total antioxidant capacity (ACAP). ACAP increased in gills of benzocaine treatment after 12 hours. The liver showed a reduction in ACAP of tricaine treatment after 12 hours. Both anesthetic treatments showed an increase of ACAP at 24 hours compared to control group. The activity of the GST enzyme increased in the gills for treatments benzocaine and tricaine after 3 and 12 hours. Liver showed increased GST activity (benzocaine after 24 hours and tricaine after 3 and 24 hours). Lipid damage decreased in gills (both anesthetics) and brain (tricaine) after 24 hours. The results demonstrate that benzocaine and tricaine did not cause oxidative damage in juvenile tambaqui under the experimental conditions herein established.


Delivery Room Nurses' Experiences of Caring for Stillborn Babies and Their Parents.
The purpose of the study was to have an in-depth understanding of delivery room nurses'experiences of caring for stillborn babies and their parents. After obtaining approval from the Institutional Review Board, data were collected from March 1 to August 30, 2016. A total of six nurses participated in the study. Semi-structured in-depth interviews were conducted and the data subsequently analyzed. Using thematic analysis, six themes were defined and refined: Emotional distress as a result of encountering stillborn babies; Caring for the deceased baby; Concerns for the mother's pain; Consideration for the father; Conflicts between accepting and dismissing parents' requests; and Strong mindset for the stillborn babies and their parents. The results showed that the nurses' experiences of caring for stillborn babies and their parents might be a difficult and lonely process. However, it could also be a process of attaining maturity by finding meaning and value in one's work. It would be helpful for the delivery room nurses to share examples of effective communication, train through simulation, and learn useful strategies that each nurse can use.


Survival and causes of death in cirrhotic and in noncirrhotic patients with primary hemochromatosis.
We analyzed survival and causes of death among 163 patients with primary hemochromatosis diagnosed between 1959 and 1983. The mean follow-up period was 10.5 +/- 5.6 years (+/- S.D.). Cumulative survival was 92 per cent at 5 years, 76 per cent at 10 years, 59 per cent at 15 years, and 49 per cent at 20 years. Life expectancy was reduced in patients with cirrhosis of the liver as compared with those without cirrhosis (P less than or equal to 0.05), in patients with diabetes mellitus as compared with those without diabetes (P less than or equal to 0.002), and in patients who could not be depleted of iron during the first 18 months of venesection therapy as compared with those who could be depleted (P less than or equal to 0.001). Prognosis was not influenced by sex (P less than or equal to 0.5). Patients without cirrhosis had a life expectancy that was not different from that expected in an age- and sex-matched normal population. Analysis of the causes of death in 53 patients, as compared with the normal population, showed that liver cancer was 219 times more frequent among the patients (16 patients), cardiomyopathy was 306 times more frequent (3 patients), liver cirrhosis was 13 times more frequent (10 patients), and diabetes mellitus was 7 times more frequent (3 patients). Death rates for other causes, including extrahepatic carcinomas (seven patients), were not different from the rates expected. We conclude that patients with hemochromatosis diagnosed in the precirrhotic stage and treated by venesection have a normal life expectancy, whereas cirrhotic patients have a shortened life expectancy and a high risk of liver cancer even when complete iron depletion has been achieved.


Imaging diagnosis of dural and direct cavernous carotid fistulae.
Arteriovenous fistulae of the cavernous sinus are rare and difficult to diagnose. They are classified into dural cavernous sinus fistulae or direct carotid-cavernous fistulae. Despite the similarity of symptoms between both types, a precise diagnosis is essential since the treatment is specific for each type of fistula. Imaging findings are remarkably similar in both dural cavernous sinus fistulae and carotid-cavernous fistulae, but it is possible to differentiate one type from the other. Amongst the available imaging methods (Doppler ultrasonography, computed tomography, magnetic resonance imaging and digital subtraction angiography), angiography is considered the gold standard for the diagnosis and classification of cavernous sinus arteriovenous fistulae. The present essay is aimed at didactically presenting the classification and imaging findings of cavernous sinus arteriovenous fistulae.


The Campylobacter jejuni Dps homologue is important for in vitro biofilm formation and cecal colonization of poultry and may serve as a protective antigen for vaccination.
In this work, we investigated the Campylobacter jejuni dps (DNA binding protein from starved cells) gene for a role in biofilm formation and cecal colonization in poultry. In vitro biofilm formation assays were conducted with stationary-phase cells in cell culture plates under microaerophilic conditions. These studies demonstrated a significant (>50%) reduction in biofilm formation by the C. jejuni dps mutant compared to that by the wild-type strain. Studies in poultry also demonstrated the importance of the dps gene in host colonization by C. jejuni. Real-time PCR analysis of mRNA extracted from the cecal contents of poultry infected with wild-type C. jejuni indicated that the dps gene is upregulated 20-fold during poultry colonization. Cecal colonization was greater than 5 log CFU lower in chicks infected with the dps mutant than chicks infected with the wild-type C. jejuni strain. Moreover, the dps mutant failed to colonize 75% of the chicks following challenge with 10(5) CFU. Preliminary studies were conducted in chicks by parenteral vaccination with a recombinant Dps protein or through oral vaccination with a recombinant attenuated Salmonella enterica strain synthesizing the C. jejuni Dps protein. No reduction in C. jejuni was noted in chicks vaccinated with the parenteral recombinant protein, whereas, a 2.5-log-unit reduction of C. jejuni was achieved in chicks vaccinated with the attenuated Salmonella vector after homologous challenge. Taken together, this work demonstrated the importance of Dps for biofilm formation and poultry colonization, and the study also provides a basis for continued work using the Dps protein as a vaccine antigen when delivered through a Salmonella vaccine vector.


Conservation of natural enemies in cotton: comparative selectivity of acetamiprid in the management of Bemisia tabaci.
The integrated control concept emphasizes the importance of both chemical and biological control for pest suppression in agricultural systems. A two-year field study was conducted to evaluate the selectivity of acetamiprid for the control of Bemisia tabaci (Gennadius) in cotton compared with a proven selective regime based on the insect growth regulators (IGRs) pyriproxyfen and buprofezin. Acetamiprid was highly effective in controlling all stages of B tabaci compared with an untreated control, and generally produced lower pest densities than the IGR regime. Univariate analyses indicated that nine of 17 taxa of arthropod predators were significantly depressed with the use of acetamiprid compared with an untreated control, including common species such as Geocoris punctipes (Say), Orius tristicolor (White), Chrysoperla carnea Stephens sensu lato, Collops vittatus (Say), Hippodamia convergens Guérin-Méneville, and Drapetis nr divergens. Compared with results from independent, concurrent studies using mixtures of broad-spectrum insecticides at the same research site, acetamiprid depressed populations of fewer predator taxa; but, for eight predator taxa significantly affected by both regimes, the average population reduction was roughly equal. In contrast, only four taxa were significantly reduced in the IGR regime compared with the untreated control and three of these were omnivores that function primarily as plant pests. Principal response curves analyses (a time-dependent, multivariate ordination method) confirmed these patterns of population change for the entire predator community. Predator:prey ratios generally increased with the use of both IGRs and acetamiprid compared with an untreated control, but ratios were consistently higher with IGRs. Parasitism by aphelinid parasitoids was unaffected or depressed slightly in all insecticide regimes compared with the control. Because of its high efficacy, acetamiprid may play an important role in later stages of B tabaci control where less emphasis is placed on selectivity. However, our results suggest that acetamiprid would be a poor substitute for the currently used IGRs in the initial stage of control where insecticide selectivity is crucial to a functional integrated control program for B tabaci in cotton.


Effect of chocolate and mate tea on the lipid profile of individuals with HIV/AIDS on antiretroviral therapy: A clinical trial.
HIV/AIDS is generally associated with dyslipidemia and oxidative imbalance, which are caused by the infection itself and by antiretroviral therapy (ART). The flavonoids, found in cocoa and yerba mate, have antioxidant and hypolipidemic properties. The aim of this study was to evaluate the effects of the consumption of dark chocolate and mate tea on the lipid profiles of individuals with HIV/AIDS who are undergoing ART. A randomized, double-blind, placebo-controlled crossover clinical trial was conducted with 92 patients receiving ART for ≥6 mo and with viral suppression. The participants were randomized to receive either 65 g of chocolate (with 2148 mg polyphenols) or placebo chocolate (without polyphenols) or 3 g of mate tea (with 107 mg total phenols and 84.24 mg chlorogenic acid) or placebo mate (without polyphenols) for 15 d each, separated by a washout period of 15 d. The lipid profile, including determination of electronegative low-density lipoprotein, was determined after each intervention. The data were analyzed by analysis of variance using the pkcross procedure of the Stata 11.0 software. Analysis of variance revealed a significant overall difference in mean high-density lipoprotein cholesterol (HDL-C) between all supplements (P = 0.047). Using the paired t test, the effect was attributed to the consumption of dark chocolate (P = 0.046). The other parameters investigated were not improved. The consumption of dark chocolate for 15 d improved HDL-C concentrations of individuals with HIV/AIDS undergoing ART, possibly due to the presence of fatty acids (stearic acid), polyphenols, and theobromine. This fact is important for the cardiovascular protection of these individuals.


Use of peak systolic strain as an index of regional left ventricular function: comparison with tissue Doppler velocity during dobutamine stress and myocardial ischemia.
The goals of this study were to examine peak systolic strain as an index of regional function in an animal model of inotropic stress and ischemia, and to compare these results with peak systolic myocardial tissue Doppler velocity (MDV). Myocardial tissue Doppler velocity is an objective measure of regional left ventricular responses to inotropic stimulation and ischemia, but it is affected by tethering from adjacent segments and translational movement. Myocardial Doppler strain (epsilon, relative change in length) is a more local measure of contractility, which can now be derived noninvasively from MDV. Eight dogs underwent graded dobutamine infusion followed by coronary occlusion. Epicardial 2-dimensional echocardiography and color MDV of the left ventricle were obtained and digitized from the short-axis view at baseline and with dobutamine doses of 2, 4, and 8 microg/kg per minute. These were repeated 0, 10, 20, 45, and 90 seconds after occlusion of the left anterior descending artery (LAD) (n = 3) or circumflex coronary artery (n = 5). Dobutamine was continued at 8 microg/kg per minute during coronary occlusion. The peak systolic radial MDV (cm/s) and systolic strain (epsilon(s), percent thickening) in the anterior and posterior walls were measured off-line at each stage. Dobutamine caused an increase in MDV (P =.0001) and epsilon(s) (P =.09) above baseline values. Coronary occlusion caused a reduction in wall motion; after 45 seconds, all nonperfused segments were hypokinetic. There was a corresponding decrease in MDV and epsilon(s), but this occurred earlier for epsilon(s), and the difference between ischemic and nonischemic segments was greater for epsilon(s) than for MDV (P <. 03). Nonischemic regions trended to an increase in epsilon(s) (compensatory hyperkinesis), whereas MDV trended downward, probably reflecting the global decrease in left ventricular function. Both MDV and epsilon(s) increase with dobutamine and decrease during ischemia. epsilon(s) appears to respond to local ischemia earlier than MDV, perhaps because it is a more local measure. Thus epsilon(s) may prove to be an accurate parameter for the clinical recognition of regional ischemia.


Causal frequency-specific contributions of frontal spatiotemporal patterns induced by non-invasive neurostimulation to human visual performance.
Neural oscillatory activity is known to play a crucial role in brain function. In the particular domain of visual perception, specific frequency bands in different brain regions and networks, from sensory areas to large-scale frontoparietal systems, have been associated with distinct aspects of visual behavior. Nonetheless, their contributions to human visual cognition remain to be causally demonstrated. We hereby used non-uniform (and thus non-frequency-specific) and uniform (frequency-specific) high-beta and gamma patterns of noninvasive neurostimulation over the right frontal eye field (FEF) to isolate the behavioral effects of oscillation frequency and provide causal evidence that distinct visual behavioral outcomes could be modulated by frequency-specific activity emerging from a single cortical region. In a visual detection task using near-threshold targets, high-beta frequency enhanced perceptual sensitivity (d') without changing response criterion (beta), whereas gamma frequency shifted response criterion but showed no effects on perceptual sensitivity. The lack of behavioral modulations by non-frequency-specific patterns demonstrates that these behavioral effects were specifically driven by burst frequency. We hypothesize that such frequency-coded behavioral impact of oscillatory activity may reflect a general brain mechanism to multiplex functions within the same neural substrate. Furthermore, pathological conditions involving impaired cerebral oscillations could potentially benefit in the near future from the use of neurostimulation to restore the characteristic oscillatory patterns of healthy systems.


Increased risk of papillary thyroid cancer related to hormonal factors in women.
Strikingly higher rates of papillary thyroid cancer in women compared with men suggest that hormonal factors may be involved in the development of this cancer. A number of independent studies have investigated the association between hormonal factors and papillary thyroid cancer risk in women but yielded conflicting and inconclusive findings. We performed a meta-analysis of all currently published studies to provide better estimates for the risk of papillary thyroid cancer related to menstrual, reproductive, and other hormonal factors in women. Six cohort studies and three case-control ones were included into our study after a comprehensive literature search. The pooled relative risk (RR) with 95 % confidence interval (95 % CI) implicated that late age at menopause was associated with an increased risk of papillary thyroid cancer (RR = 1.39, 95 % CI 1.03-1.89, P = 0.032). No significant association was demonstrated between papillary thyroid cancer risk and other hormone-related factors, including oral contraceptive, hormone replacement therapy, age at menarche, parity, age at first birth, menopausal status, and breast feeding. Subgroup analysis by study design confirmed those associations. Sensitivity analysis did not materially alter the pooled results. The meta-analysis firstly suggests that late age at menopause is a risk factor for papillary thyroid cancer.


Multiscale modeling of a rectifying bipolar nanopore: Comparing Poisson-Nernst-Planck to Monte Carlo.
In the framework of a multiscale modeling approach, we present a systematic study of a bipolar rectifying nanopore using a continuum and a particle simulation method. The common ground in the two methods is the application of the Nernst-Planck (NP) equation to compute ion transport in the framework of the implicit-water electrolytemodel. The difference is that the Poisson-Boltzmann theory is used in the Poisson-Nernst-Planck (PNP) approach, while the Local Equilibrium Monte Carlo (LEMC) method is used in the particle simulation approach (NP+LEMC) to relate the concentration profile to the electrochemical potential profile. Since we consider a bipolar pore which is short and narrow, we perform simulations using two-dimensional PNP. In addition, results of a non-linear version of PNP that takes crowding of ions into account are shown. We observe that the mean field approximation applied in PNP is appropriate to reproduce the basic behavior of the bipolar nanopore (e.g., rectification) for varying parameters of the system (voltage, surface charge,electrolyte concentration, and pore radius). We present current data that characterize the nanopore's behavior as a device, as well as concentration, electrical potential, and electrochemical potential profiles.


[New approaches for excellent operative field during tracheobronchial anastomoses in sleeve pneumonectomy: cardiopulmonary bypass and diseased lung ventilation].
Cardiopulmonary bypass (CPB) and diseased lung ventilation were presented as new methods of oxygenation during carinal reconstruction. Two cases of left sleeve pneumonectomy (SP) were performed for bronchial gland carcinoma through clamshell incisions. Adequate oxygenation and excellent operative fields were provided by CPB in both patients. The postoperative courses of these patients were uneventful, although they developed temporary pulmonary edema that needed mechanical ventilation and appropriate diuretics for several days. One case of patient with squamous cell carcinoma after chemotherapy was successfully treated by right SP through midline sternotomy. Adequate oxygenation and excellent operative fields was obtained by diseased right lung ventilation in this patient. The postoperative course of the patient was uneventful without mechanical ventilation support. These procedures of oxygenation in this paper are considered to be safe and effective methods for carinal reconstruction.


Effects of cationic antimicrobial peptides on liquid-preserved boar spermatozoa.
Antibiotics are mandatory additives in semen extenders to control bacterial contamination. The worldwide increase in resistance to conventional antibiotics requires the search for alternatives not only for animal artificial insemination industries, but also for veterinary and human medicine. Cationic antimicrobial peptides are of interest as a novel class of antimicrobial additives for boar semen preservation. The present study investigated effects of two synthetic cyclic hexapeptides (c-WFW, c-WWW) and a synthetic helical magainin II amide derivative (MK5E) on boar sperm during semen storage at 16 °C for 4 days. The standard extender, Beltsville Thawing Solution (BTS) containing 250 µg/mL gentamicin (standard), was compared to combinations of BTS with each of the peptides in a split-sample procedure. Examination revealed peptide- and concentration-dependent effects on sperm integrity and motility. Negative effects were more pronounced for MK5E than in hexapeptide-supplemented samples. The cyclic hexapeptides were partly able to stimulate a linear progressive sperm movement. When using low concentrations of cyclic hexapeptides (4 µM c-WFW, 2 µM c-WWW) sperm quality was comparable to the standard extender over the course of preservation. C-WFW-supplemented boar semen resulted in normal fertility rates after AI. In order to investigate the interaction of peptides with the membrane, electron spin resonance spectroscopic measurements were performed using spin-labeled lipids. C-WWW and c-WFW reversibly immobilized an analog of phosphatidylcholine (PC), whereas MK5E caused an irreversible increase of PC mobility. These results suggest testing the antimicrobial efficiency of non-toxic concentrations of selected cyclic hexapeptides as potential candidates to supplement/replace common antibiotics in semen preservation.


Extrapolation of the evidence on teratogenicity of chemicals between humans and experimental animals: chemicals other than drugs.
Epidemiologic literature regarding the possible association between malformations and 23 exposures or occupations other than pharmaceutical products, was analysed. The qualitative level of scientific evidence was classified into four categories: high (ethanol, methylmercury, PCBs, laboratory work), limited (anesthetic gases, carbon monoxide), low (hexachlorophene, LSD, nitrous oxide, smelter work, tobacco), and inadequate (all other exposures). Human data for exposures belonging to categories "high" and "limited" were quantitatively compared to results of animal teratogenicity tests of the relevant chemicals. Ethanol, methylmercury, and PCBs have caused malformations in experimental animals, and the effective doses have ranged from 0.2 to 8.0 times the effective human doses. Ethanol and PCBs caused similar types of lesions in some animal species as have been observed in humans.


Future growth and development of hysteroscopy.
In less than two decades, hysteroscopy has evolved into a practical technique for the evaluation of the uterine cavity, with well-established indications such as evaluation of abnormal uterine bleeding and abnormal hysterograms as well as the treatment of intrauterine adhesions or the septate uterus, the removal of misplaced or embedded intrauterine devices, and the removal of submucous leiomyomas. As the use of hysteroscopy increases and more practitioners utilize it, new instrumentation and techniques are beginning to evolve, simplifying and facilitating not only a diagnostic examination, but also the more difficult and complex surgical interventions. Flexible endoscopes are being tested for possible use in the uterine cavity, and operative hysteroscopes with practical inflow and outflow accessory channels have been introduced. The accessory operative instrumentation has also been expanded and refined, and portable units for office examinations have been developed. The trend in the development of instrumentation is toward simplicity, effectiveness, and refinement in lenses with wider fields of view and accessory channels without compromising the total outer diameter of the endoscopes. As clinical applications expand as a result of increased use and proficiency in hysteroscopy, new applications undoubtedly will follow, such as closer study of the endometrium with and without additional magnification. This latter may permit a better understanding of the receptivity of the endometrium to the embryo, which may help in predicting successful nidations. Portable laser units with the capability to use the CO2 fiber undoubtedly will increase laser use through hysteroscopy, and in the near future, photodynamic therapy may become the best approach to the selective treatment of intrauterine lesions. The uterotubal junction will remain an attractive area for endoscopists to approach the fallopian tubes transcervically and eventually to accomplish tubal closure safely and effectively with the possibility of future reversibility. The hysteroscope will play an important role in new reproductive technologies, particularly those related to the gamete intrafallopian transfer via the uterine side and perhaps also for direct embryo transfers under visual control. The future of hysteroscopy thus is promising. The present diagnostic and therapeutic applications will not only become a standard of treatment but will expand as gynecologists will gain proficiency and confidence in this endoscopic method.


A simple and sensitive assay for distinguishing the expression of ricin and Ricinus communis agglutinin genes in developing castor seed (R. communis L.).
Castor oil is the only commercial source of ricinoleic acid and has numerous industrial applications. Among the factors limiting domestic production of castor oil is the presence of the toxin ricin and its less toxic homologue Ricinus communis agglutinin (RCA) in seeds. Although the sequences of ricin and RCA genes are known, their transcriptional expression patterns have not been distinguished due to their high degree of sequence similarity. As the information is critical for assessing success in developing a ricin-free castor crop using genetic silencing, we have designed a gene specific reverse transcription-polymerase chain reaction (RT-PCR) assay to examine the expression of the ricin and RCA genes in developing seeds. The results show that the ricin and RCA mRNA are highly abundant in seeds during the development of endosperm, and the expression pattern is similar to that observed in the Northern analysis. The RT-PCR results can be confirmed by a simple RT-PCR-based restriction fragment analysis.


Influence of maxillary antrolith on the clinical outcome of implants placed simultaneously with osteotome sinus floor elevation: A retrospective radiographic study.
Large antroliths and those located adjacent to the sinus floor can affect clinical interventions and increase the difficulty of implant placement performed simultaneously with osteotome sinus floor elevation surgery. This retrospective study investigated the clinical outcomes of implants placed simultaneously with osteotome sinus floor elevation subjacent to maxillary antroliths. Twenty implants inserted subjacent to or intruding into the antrolith after sinus floor elevation were evaluated in 18 patients. Cone-beam computed tomography (CBCT) was used to measure antrolith size and membrane thickness at sites of osteotome sinus floor elevation. Periapical radiographs were used to assess the height of grafted bone. Generalized estimating equation (GEE) analysis was performed to correlate the occurrence of antroliths with patient background characteristics and dental outcomes, based on a sample population of 239, among whom 33 presented antroliths. The 20 implants remained clinically stable over a mean follow-up period of 42.4 months. The mean thickness of the sinus membrane at osteotome sites was 5.4 ± 3.3 mm. None of the cases presented sinus membrane perforation or sinus symptoms following osteotome intervention. The mean gain in the height of grafted sinus bone was 4.0 ± 1.4 mm at the last follow-up. The occurrence of antroliths was higher among females and the elderly (>49 years old). The multivariable GEE analysis showed that the adjusted odds ratio for the occurrence of antroliths with root canal fillings was significantly lower than those without root canal fillings (odds ratio = 0.33; 95% confidence interval = 0.11-0.96). Our findings indicate that osteotome sinus floor elevation is a surgical procedure with a risk <17%. Thorough planning based on CBCT and careful management during surgery can eliminate the negative effects of antroliths on implant performance.


Exposure of trees to drought-induced die-off is defined by a common climatic threshold across different vegetation types.
Increases in drought and temperature stress in forest and woodland ecosystems are thought to be responsible for the rise in episodic mortality events observed globally. However, key climatic drivers common to mortality events and the impacts of future extreme droughts on tree survival have not been evaluated. Here, we characterize climatic drivers associated with documented tree die-off events across Australia using standardized climatic indices to represent the key dimensions of drought stress for a range of vegetation types. We identify a common probabilistic threshold associated with an increased risk of die-off across all the sites that we examined. We show that observed die-off events occur when water deficits and maximum temperatures are high and exist outside 98% of the observed range in drought intensity; this threshold was evident at all sites regardless of vegetation type and climate. The observed die-off events also coincided with at least one heat wave (three consecutive days above the 90th percentile for maximum temperature), emphasizing a pivotal role of heat stress in amplifying tree die-off and mortality processes. The joint drought intensity and maximum temperature distributions were modeled for each site to describe the co-occurrence of both hot and dry conditions and evaluate future shifts in climatic thresholds associated with the die-off events. Under a relatively dry and moderate warming scenario, the frequency of droughts capable of inducing significant tree die-off across Australia could increase from 1 in 24 years to 1 in 15 years by 2050, accompanied by a doubling in the occurrence of associated heat waves. By defining commonalities in drought conditions capable of inducing tree die-off, we show a strong interactive effect of water and high temperature stress and provide a consistent approach for assessing changes in the exposure of ecosystems to extreme drought events.


The role of translation elongation factor eEF1A in intracellular alkalinization-induced tumor cell growth.
The formation of a pH gradient, which is characterized by intracellular alkalinization and extracellular acidification, plays a key role in the growth and metastasis of tumor cells. However, the underlying mechanisms of alkalinization-induced cell growth are not known. In this study, we investigated the roles of eukaryotic translation elongation factor 1 alpha (eEF1A) in alkalinization-induced cell growth. In all cell lines tested (NIH3T3, HEK293, and HeLa), cell growth was affected by the modulation of intracellular pH. In general, weak intracellular alkalinization produced increased cell growth, whereas intracellular acidification resulted in decreased cell growth. It is interesting to note that portions of actin-bound eEF1A proteins were gradually reduced from acidic to alkaline conditions, suggesting an increase in levels of functionally active, free-form eEF1A. Over-expression of eEF1A caused increased cell growth in HeLa cells. It should be noted that dissociation of eEF1A from actin by transfection with the actin-binding domain deleted eEF1A construct further increased cell growth under acidic conditions, whereas most of the intact eEF1A was bound to actin. Conversely, knockdown of eEF1A by treatment with eEF1A1 and eEF1A2 siRNAs nullified the effects of alkalinization-induced cell growth. The above findings suggest that an increase in free-form eEF1A under alkaline conditions plays a critical role in alkalinization-induced cell growth.


Polymorphisms in the ABCB1 gene in phenobarbital responsive and resistant idiopathic epileptic Border Collies.
Variation in the ABCB1 gene is believed to play a role in drug resistance in epilepsy. Variation in the ABCB1 gene encoding the permeability-glycoprotein could have an influence on phenobarbital (PB) resistance, which occurs with high frequency in idiopathic epileptic Border Collies (BCs). Two hundred and thirty-six client-owned BCs from Switzerland and Germany including 25 with idiopathic epilepsy, of which 13 were resistant to PB treatment. Prospective and retrospective case-control study. Data were collected retrospectively regarding disease status, antiepileptic drug (AED) therapy, and drug responsiveness. The frequency of a known mutation in the ABCB1 gene (4 base-pair deletion in the ABCB1 gene [c.296_299del]) was determined in all BCs. Additionally, the ABCB1 coding exons and flanking sequences were completely sequenced to search for additional variation in 41 BCs. Association analyses were performed in 2 case-control studies: idiopathic epileptic and control BCs and PB-responsive and resistant idiopathic epileptic BCs. One of 236 BCs (0.4%) was heterozygous for the mutation in the ABCB1 gene (c.296_299del). A total of 23 variations were identified in the ABCB1 gene: 4 in exons and 19 in introns. The G-allele of the c.-6-180T > G variation in intron 1 was significantly more frequent in epileptic BCs resistant to PB treatment than in epileptic BCs responsive to PB treatment (P(raw) = .0025). A variation in intron 1 of the ABCB1 gene is associated with drug responsiveness in BCs. This might indicate that regulatory mutations affecting the expression level of ABCB1 could exist, which may influence the reaction of a dog to AEDs.


Cellular and extracellular remodeling with the development and recovery from tachycardia-induced cardiomyopathy: changes in fibrillar collagen, myocyte adhesion capacity and proteoglycans.
The myocardial extracellular matrix (ECM) is composed of three important constituents: (1) fibrillar collagen, (2) a basement membrane, and (3) proteoglycans. Structural or compositional changes in these ECM components may affect left ventricular (LV) function as well as influence overall LV geometry. Accordingly, this study examined the relationship between changes in these ECM components to changes in LV function and geometry which develop with the progression and regression from supraventricular tachycardia-induced cardiomyopathy (SVT). LV function and specific components of the ECM were studied in pigs with SVT cardiomyopathy (SVT:atrially paced 240 bpm, 3 weeks; n = 7), or after a 4-week recovery from SVT cardiomyopathy (post-SVT; n = 6), and in controls (n = 7). LV fractional shortening fell by 60% and end-diastolic dimension increased by 47% with SVT compared to controls. While LV fractional shortening normalized with post-SVT, end-diastolic dimension remained 40% higher than controls. Collagen concentration fell by 22% and salt extractable collagen, which reflects collagen cross-linking, increased by 41% with SVT compared to controls. Collagen concentration increased by 20%, collagen extraction normalized, and levels of collagen type III mRNA increased by 42% with post-SVT. Isolated myocyte adhesion capacity to basement membrane substrates laminin, fibronectin, and collagen type IV were examined. SVT resulted in over a 50% reduction in myocyte adhesion for all of the basement membrane components compared to controls. A normalization in isolated myocyte adhesion capacity was observed in post-SVT. The relative content and distribution of the ECM proteoglycan chondroitin sulfate was examined using immunohistochemistry. With SVT, the density of this proteoglycan increased around individual myocytes. With post-SVT, the relative distribution of chondroitin sulfate returned to control levels. Thus, SVT cardiomyopathy was associated with reduced collagen concentration and cross-linking, diminished myocyte basement membrane adhesion capacity, and increased proteoglycans. Recovery from SVT cardiomyopathy resulted in increased collagen concentration, and a normalization of myocyte adhesion capacity and proteoglycan distribution. These results suggest that changes within the ECM are a dynamic process and accompany the LV systolic and diastolic function as well as ventricular and myocyte remodeling during the progression and regression from cardiomyopathic disease.


miR-4270 suppresses hepatocellular carcinoma progression by inhibiting DNMT3A-mediated methylation of HGFAC promoter.
miR-4270 is a regulatory factor has been linked with the progression of various cancers, such as nasopharyngeal carcinoma, hepatocellular carcinoma (HCC), and gastric cancer. However, the underlying mechanisms through which miR-4270 modulates HCC development are not fully understood. miR-4270 expression levels were analyzed in various HCC cell lines and tissue samples. An online bioinformatics tool was then utilized to predict the miR-4270 target gene. The binding relationship between miR-4270 and its target gene DNMT3A was verified using dual-luciferase reporter and Ago2-RIP assays. Then, co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP) assays were conducted to investigate the association between DNMT3A and the hepatocyte growth factor activator (HGFAC) promoter region. To assess the methylation level of the HGFAC promoter, methylation-specific PCR (MSP) was employed. Furthermore, rescue analyses were carried out to evaluate the functional relevance of miR-4270 and HGFAC in the modulation of the malignant properties of HCC cells. Finally, HepG2 cells overexpressing miR-4270 were subcutaneously injected into nude mice to estimate the impact of miR-4270 on the xenograft tumor growth of HCC. A substantial miR-4270 downregulation was revealed in HCC patient samples and cell lines. miR-4270 upregulation suppressed both cell proliferation and invasion while promoting apoptosis. At the molecular level, miR-4270 was found to bind to the 3'untranslated region (3'UTR) of DNMT3A, thereby inhibiting DNMT3A-mediated methylation of the HGFAC promoter. Functional assays indicated that inhibition of miR-4270 stimulated HCC cell growth, an effect counteracted by overexpression of HGFAC. In vivo assays further verified that miR-4270 effectively suppressed the progression of HCC xenograft tumors. miR-4270 was found to mitigate the malignant characteristics of HCC by inhibiting DNMT3A-mediated methylation of the HGFAC promoter, suggesting a potential therapeutic avenue for the management of HCC.


Rbm20ΔRRM Mice, Expressing a Titin Isoform with Lower Stiffness, Are Protected from Mechanical Ventilation-Induced Diaphragm Weakness.
Diaphragm weakness frequently develops in mechanically ventilated critically ill patients and is associated with increased morbidity, including ventilator weaning failure, mortality, and health care costs. The mechanisms underlying diaphragm weakness are incompletely understood but may include the elastic properties of titin, a giant protein whose layout in the muscle's sarcomeres makes it an ideal candidate to sense ventilation-induced diaphragm unloading, resulting in downstream signaling through titin-binding proteins. In the current study, we investigated whether modulating titin stiffness affects the development of diaphragm weakness during mechanical ventilation. To this end, we ventilated genetically engineered mice with reduced titin stiffness (Rbm20ΔRRM), and robust (TtnΔIAjxn) or severely (TtnΔ112-158) increased titin stiffness for 8 h, and assessed diaphragm contractility and protein expression of titin-binding proteins. Mechanical ventilation reduced the maximum active tension of the diaphragm in WT, TtnΔIAjxn and TtnΔ112-158 mice. However, in Rbm20ΔRRM mice maximum active tension was preserved after ventilation. Analyses of titin binding proteins suggest that muscle ankyrin repeat proteins (MARPs) 1 and 2 may play a role in the adaptation of the diaphragm to mechanical ventilation, and the preservation of diaphragm contractility in Rbm20ΔRRM mice. Thus, Rbm20ΔRRM mice, expressing titin isoforms with lower stiffness, are protected from mechanical ventilation-induced diaphragm weakness, suggesting that titin elasticity may modulate the diaphragm's response to unloading during mechanical ventilation.


The 5/95 Gap on the dissemination of mental health research: The World Psychiatric Association (WPA) task force report on project with editors of low and middle income (LAMI) countries.
The World Psychiatric Association (WPA) Task Force and a small group previously convened by the WPA publications committee initiated three activities between 2006-2008 that aimed to respond to the need for greater support for psychiatry journals in LAMI countries. In a joint venture with participants from the Global Mental Health Movement the Task Force editors from LAMI countries in Africa, Asia, Eastern Europe and Latin America were contacted to identify potential journals to target for indexation (Medline and ISI). The committee analyzed the editors' applications on the following criteria: a) geographical representativeness; b) affiliation to a professional mental health society; c) regular publication of at least 4 issues per year over the past few years; d) comprehensive national and international editorial boards; e) publication of original articles, or at least abstracts, in English; f) some level of current indexation; g) evidence of a good balance between original and review articles in publications; and h) a friendly access website. The committee received 26 applications (11 from Latin America, 7 from Central Europe, 4 from Asia and 4 from Africa), and selected 8 journals, 2 from each geographical area, on the basis of the overall scores obtained for the items mentioned, to participate in an editors meeting held in Prague in September 2008. The aims of the committee are twofold: a) to concentrate support for those selected journals; and b) to assist all LAMI mental health editors in improving the quality of their journals and fulfilling the requirements for full indexation. This report summarizes the procedures conducted by the committee, the assessment of the current non-indexed journals, and offers suggestions for further action.


Reward and motivation in pain and pain relief.
Pain is fundamentally unpleasant, a feature that protects the organism by promoting motivation and learning. Relief of aversive states, including pain, is rewarding. The aversiveness of pain, as well as the reward from relief of pain, is encoded by brain reward/motivational mesocorticolimbic circuitry. In this Review, we describe current knowledge of the impact of acute and chronic pain on reward/motivation circuits gained from preclinical models and from human neuroimaging. We highlight emerging clinical evidence suggesting that anatomical and functional changes in these circuits contribute to the transition from acute to chronic pain. We propose that assessing activity in these conserved circuits can offer new outcome measures for preclinical evaluation of analgesic efficacy to improve translation and speed drug discovery. We further suggest that targeting reward/motivation circuits may provide a path for normalizing the consequences of chronic pain to the brain, surpassing symptomatic management to promote recovery from chronic pain.


Mediterranean diet, antioxidants and cancer: the need for randomized trials.
In nutritional epidemiology the traditional approach has been to assess single nutrients or food items. Now, a growing interest exists in dietary patterns. The study of dietary patterns with a whole-diet approach represents a needed and complementary methodology. Among a priori defined patterns, the highly palatable traditional Mediterranean diet has many options to be the first choice in the dietary prevention of cancer. However, sound epidemiologic evidence about its ability to prevent the most frequent cancers is scarce. In conjunction with large and well-designed cohort studies, randomized trials using a whole-diet approach and not a simple antioxidant supplement are needed in Mediterranean countries.


Berberine Protects Against Palmitate-Induced Apoptosis in Tubular Epithelial Cells by Promoting Fatty Acid Oxidation.
BACKGROUND Increased lipid accumulation in renal tubular epithelial cells (TECs) contributes to their injury and dysfunction and progression of tubulointerstitial fibrosis. Berberine (BBR), a natural plant alkaloid isolated from traditional medicine herbs, is effective in lowing serum lipid, and has a protective effect on chronic kidney disease (CKD) with dyslipidemia, including diabetic nephropathy. The aim of this study was to investigate the effect of BBR on palmitate (PA)-induced lipid accumulation and apoptosis in TECs. MATERIAL AND METHODS Human kidney proximal tubular epithelial cell line (HK-2) cells were treated with PA, BBR, and/or palmitoyltransferase 1A (CPT1A) inhibitor Etomoxir. Intracellular lipid content was assessed by Oil Red O and Nile Red staining. Cell apoptosis rate was evaluated by flow cytometry assay. The expression of apoptosis-related protein cleaved-caspase3 and fatty acid oxidation (FAO)-regulating proteins, including CPT1A, peroxisome proliferator-activated receptor α (PPARα), and PPARγ co-activator-1α (PGC1α), was measured by Western blot analysis and immunofluorescence. RESULTS In the present study, PA treatment increased intracellular lipid deposition accompanied by elevated apoptosis in TECs compared with control group, whereas the protein expression of CPT1A, PPARα, and PGC1α, did not correspondingly increase in TECs. BBR significantly up-regulated the protein expression of CPT1A, PPARα, and PGC1α in TECs treated with or without PA, and reversed PA-induced intracellular lipid accumulation and apoptosis. Moreover, the CPT1A inhibitor Etomoxir counteracted the protective effect of BBR in TECs. CONCLUSIONS These in vitro findings suggest that PA can induce intracellular lipid accumulation and apoptosis in TECs, and the mechanism may be associated with inducing defective FAO, whereas BBR can protect TECs against PA-induced intracellular lipid accumulation and apoptosis by promoting FAO.


Evaluating Machine Learning Methods of Analyzing Multiclass Metabolomics.
Multiclass metabolomic studies have become popular for revealing the differences in multiple stages of complex diseases, various lifestyles, or the effects of specific treatments. In multiclass metabolomics, there are multiple data manipulation steps for analyzing raw data, which consist of data filtering, the imputation of missing values, data normalization, marker identification, sample separation, classification, and so on. In each step, several to dozens of machine learning methods can be chosen for the given data set, with potentially hundreds or thousands of method combinations in the whole data processing chain. Therefore, a clear understanding of these machine learning methods is helpful for selecting an appropriate method combination for obtaining stable and reliable analytical results of specific data. However, there has rarely been an overall introduction or evaluation of these methods based on multiclass metabolomic data. Herein, detailed descriptions of these machine learning methods in multiple data manipulation steps are reviewed. Moreover, an assessment of these methods was performed using a benchmark data set for multiclass metabolomics. First, 12 imputation methods for imputing missing values were evaluated based on the PSS (Procrustes statistical shape analysis) and NRMSE (normalized root-mean-square error) values. Second, 17 normalization methods for processing multiclass metabolomic data were evaluated by applying the PMAD (pooled median absolute deviation) value. Third, different methods of identifying markers of multiclass metabolomics were evaluated based on the CWrel (relative weighted consistency) value. Fourth, nine classification methods for constructing multiclass models were assessed using the AUC (area under the curve) value. Performance evaluations of machine learning methods are highly recommended to select the most appropriate method combination before performing the final analysis of the given data. Overall, detailed descriptions and evaluation of various machine learning methods are expected to improve analyses of multiclass metabolomic data.


Evolution of the Systems Pharmacokinetics-Pharmacodynamics Model for Antibody-Drug Conjugates to Characterize Tumor Heterogeneity and In Vivo Bystander Effect.
The objective of this work was to develop a systems pharmacokinetics-pharmacodynamics (PK-PD) model that can characterize in vivo bystander effect of antibody-drug conjugate (ADC) in a heterogeneous tumor. To accomplish this goal, a coculture xenograft tumor with 50% GFP-MCF7 (HER2-low) and 50% N87 (HER2-high) cells was developed. The relative composition of a heterogeneous tumor for each cell type was experimentally determined by immunohistochemistry analysis. Trastuzumab-vc-MMAE (T-vc-MMAE) was used as a tool ADC. Plasma and tumor PK of T-vc-MMAE was analyzed in N87, GFP-MCF7, and coculture tumor-bearing mice. In addition, tumor growth inhibition (TGI) studies were conducted in all three xenografts at different T-vc-MMAE dose levels. To characterize the PK of ADC in coculture tumors, our previously published tumor distribution model was evolved to account for different cell populations. The evolved tumor PK model was able to a priori predict the PK of all ADC analytes in the coculture tumors reasonably well. The tumor PK model was subsequently integrated with a PD model that used intracellular tubulin occupancy to drive ADC efficacy in each cell type. The final systems PK-PD model was able to simultaneously characterize all the TGI data reasonably well, with a common set of parameters for MMAE-induced cytotoxicity. The model was later used to simulate the effect of different dosing regimens and tumor compositions on the bystander effect of ADC. The model simulations suggested that dose-fractionation regimen may further improve overall efficacy and bystander effect of ADCs by prolonging the tubulin occupancy in each cell type. SIGNIFICANCE STATEMENT: A PK-PD analysis is presented to understand bystander effect of Trastuzumab-vc-MMAE ADC in antigen (Ag)-low, Ag-high, and coculture (i.e., Ag-high + Ag-low) xenograft mice. This study also describes a novel single cell-level systems PK-PD model to characterize in vivo bystander effect of ADCs. The proposed model can serve as a platform to mathematically characterize multiple cell populations and their interactions in tumor tissues. Our analysis also suggests that fractionated dosing regimen may help improve the bystander effect of ADCs.


The role of diet in the management of gout: a comparison of knowledge and attitudes to current evidence.
Evidence supports dietary modifications in the management of gout. Despite this, the degree of implementation of this evidence by nutrition professionals and rheumatologists and those affected by gout is unknown. The present study aimed to compare usual dietary practices of patients with gout to evidence for dietary management of gout and to investigate whether the knowledge and attitudes of nutrition professionals and rheumatologists reflects current evidence. A food frequency questionnaire was used to determine usual dietary intake of patients with gout, a separate questionnaire examined gout-related dietary modifications (n = 29). Online questionnaires to examine attitudes towards dietary management of gout were completed by nutrition professionals and rheumatologists. Proportions of participants whose reported intakes were inconsistent with current evidence for the dietary management of gout were: alcohol, n = 14 (48%); beer, n = 18 (62%); seafood, n = 29 (100%); meat, n = 7 (24%); beef/pork/lamb, n = 24 (83%); dairy products, n = 12 (41%); vitamin C supplementation, n = 29 (100%). Of the 61 rheumatologists and 231 nutrition professionals who completed the online survey, the majority considered that weight loss and decreased alcohol intake were important or very important outcomes. Proportions were lower for decreased purine intake. Thirty-four (56%) rheumatologists do not refer patients with gout to dietetic services and, of those who do, the majority refer less than half. Overall, patients with gout in the present study were not implementing evidence for dietary management of their condition and complex dietary issues were evident.


Variation in virulence for mice and rhesus monkeys among St. Louis encephalitis virus strains of different origin.
The virulence characteristics of 67 strains of St. Louis encephalitis (SLE) virus isolated from various sources in North, Middle, and South America were compared in mice and rhesus monkeys. Each virus strain was titrated in mice exactly 21 days old and virulence was expressed as the ratio of intracerebral (ic)/intraperitoneal (ip) LD50. Virus strains fell into three groups: 1) high virulence (ic/ip LD50 ratio approximately 1.0); 2) intermediate virulence (variable mortality over a wide dose range); and 3) low virulence (ic/ip LD50 less than or equal to 0.00002). Virus strains isolated during Culex pipiens and Cx. nigripalpus--borne epidemics in the eastern United States were highly virulent for mice, whereas a high proportion of the endemic virus strains isolated from Cx. tarsalis in the western United States were attenuated. Virus strains isolated from birds (the usual host for SLE virus) were highly virulent, in contrast to strains from rodents and carnivores, which were attenuated. Isolates from humans exhibited variable virulence characteristics. In experimentally-infected mice, virulence correlated with high viremia, replication in extraneural tissues, and earlier neuroinvasion. Mouse virulence correlated with clinical and histopathologic markers of pathogenicity for ic inoculated rhesus monkeys. Monkeys immunized with nonpathogenic strains by subcutaneous inoculation were partially protected against ic challenge with a virulent virus strain. The virulence classification of SLE virus strains is discussed in terms of epidemiologic correlations. This classification provides a framework for future studies on the antigenic, genetic, and biochemical bases for SLE virus strain variation.


Genetic Predisposition to Hepatocarcinogenesis in Inbred and Outbred Mouse Lines Selected for High or Low Inflammatory Response.
AIRmax and AIRmin mouse strains phenotypically selected for high and low acute inflammatory responsiveness (AIR) are, respectively, susceptible or resistant to developing hepatocellular carcinoma (HCC) induced by the chemical carcinogens urethane and diethylnitrosamine (DEN). Early production of TNF-α, IL-1β, and IL-6 in the liver after DEN treatment correlated with tumor development in AIRmax mice. Transcriptome analysis of livers from untreated AIRmax and AIRmin mice showed specific gene expression profiles in each line, which might play a role in their differential susceptibility to HCC. Linkage analysis with SNP markers in F2 (AIRmax×AIRmin) intercross mice revealed two quantitative trait loci (QTL) in chromosomes 2 and 9, which are significantly associated with the number and progression of urethane-induced liver tumors. An independent linkage analysis with an intercross population from A/J and C57BL/6J inbred mice mapped regions in chromosomes 1 and 7 associated with the progression of urethane-induced liver tumors, evidencing the heterogeneity of HCC genetic control.


Prevalence and risk factors of anal human papillomavirus infection among men with anal condyloma acuminata by HIV status in ShenZhen, Southeast China: A retrospective cohort study.
Patients with anal condyloma acuminatum (CA) are at risk of developing anal cancer which is associated with oncogenic human papillomavirus (HPV) infection. Investigation of anal HPV prevalence and risk factors can provide effective strategies for the prevention of anal cancer. A retrospective study was conducted among 549 patients with anal CA in the Third People's Hospital of Shenzhen between January 2019 and October 2021. HPV prevalence and HIV antibodies were detected by fluorescent PCR and ELISA, respectively. Logistic regression model and structural equation modeling (SEM) were conducted to analyzed the risk factors of oncogenic HPV infection. The overall prevalence of HPV was 96.72%. Both HPV6 (N = 285, 51.91%) and HPV11 (N = 300, 54.64%) were more than half infected and the most frequent Hr-HPV genotype was HPV16 (N = 138, 25.14%). HIV-positive (AOR: 5.02, 95% CI: 2.98-8.60, p < 0.0001) and history of syphilis (AOR: 4.24, 95% CI: 2.31-8.46, p < 0.0001) were independent risk factors statistically associated with oncogenic HPV infection. Ever had anal sex (AOR: 3.40, 95% CI: 1.28-11.81, p = 0.0267) and age 35 years and older (AOR: 2.79, 95% CI: 1.53-5.15, p = 0.0009) were associated with HPV16 and HPV52, respectively. SEM analyses showed that HIV-positive (b = 1.549, p < 0.001) and history of syphilis (b = 1.450, p < 0.001) had significant positive effects on oncogenic HPV infection. Ever had anal sex (b = 1.243, p = 0.025) and Age (b = 0.043, p = 0.002) positively drived HPV16 and HPV52 infection, respectively. Anal CA patients who are HIV-positive, have a history of syphilis, or at least 35 years old should be considered for Hr-HPV, cytology and other anal cancer related tests to reduce the risk of cancer development.


Review of the current status of whole-body hyperthermia administered by water circulation techniques.
Since the mid 1970's, whole-body hyperthermia (WBH) has been actively investigated for the treatment of patients with cancer in the United States. Two basic techniques are currently used; one involves transcutaneous transfer of heat by circulation of heated water, and, in the other, extracorporeally circulated blood is heated. Studies which have used the water circulation method are compared as to protocol design, toxicities encountered, and responses observed with and without concomitant chemotherapy. In vitro data indicating that faster rates of heating are associated with greater cytotoxicity due to hyperthermia alone or with several anticancer drugs are discussed in relationship to the design of future techniques for administration of WBH.


Predicting microRNA precursors with a generalized Gaussian components based density estimation algorithm.
MicroRNAs (miRNAs) are short non-coding RNA molecules, which play an important role in post-transcriptional regulation of gene expression. There have been many efforts to discover miRNA precursors (pre-miRNAs) over the years. Recently, ab initio approaches have attracted more attention because they do not depend on homology information and provide broader applications than comparative approaches. Kernel based classifiers such as support vector machine (SVM) are extensively adopted in these ab initio approaches due to the prediction performance they achieved. On the other hand, logic based classifiers such as decision tree, of which the constructed model is interpretable, have attracted less attention. This article reports the design of a predictor of pre-miRNAs with a novel kernel based classifier named the generalized Gaussian density estimator (G2DE) based classifier. The G2DE is a kernel based algorithm designed to provide interpretability by utilizing a few but representative kernels for constructing the classification model. The performance of the proposed predictor has been evaluated with 692 human pre-miRNAs and has been compared with two kernel based and two logic based classifiers. The experimental results show that the proposed predictor is capable of achieving prediction performance comparable to those delivered by the prevailing kernel based classification algorithms, while providing the user with an overall picture of the distribution of the data set. Software predictors that identify pre-miRNAs in genomic sequences have been exploited by biologists to facilitate molecular biology research in recent years. The G2DE employed in this study can deliver prediction accuracy comparable with the state-of-the-art kernel based machine learning algorithms. Furthermore, biologists can obtain valuable insights about the different characteristics of the sequences of pre-miRNAs with the models generated by the G2DE based predictor.


Amniotic membrane transplantation for ocular disease: a review of the first 233 cases from the UK user group.
Amniotic membrane transplantation (AMT), as a new tool in the armamentarium of therapies available for ocular surface problems, became widely available in the UK in 1998. This study evaluates the indications for treatment, the surgical procedures used, and the results of a subset of the first AMT cases carried out by the group using this nationally available supply. This user group model provides data which is different from that obtained from uncontrolled case series, or clinical trials, and may be more representative of the outcomes that can be expected when a procedure becomes widely available. The first 233 AMTs, performed by the UK user group, were evaluated by audit and outcomes were assessed at 3 months. Of the 233 transplants, there were 126 (54.1%) valid outcome returns: the outcome for persistent epithelial defects was a healed and stable surface in 11/35 (31.4%, 95% CI 16.9 to 49.3); for chemical/thermal injuries, a healed uninflamed eye with clear cornea in 5/18 (27.8%, 95% CI 9.7 to 53.4); for bullous keratopathy a pain-free, stable surface without bullae in 4/18 (22.2%, 95% CI 6.4 to 47.6); for ocular surface reconstruction, an epithelialised uninflamed conjunctiva without scarring in 12/23 (52.2%, 95% CI 30.6 to 73.2); and for limbal stem cell deficiency, a corneal phenotype in 4/7 (57.1%). The operative technique least associated with failure was use of a bandage contact lens at the end of the procedure (OR 0.19, 95% CI 0.06 to 0.59, p = 0.004). Previous treatment with topical steroids was significantly associated with failure (OR 5.70, 95% CI 1.77 to 18.43, p = 0.004). Although the outcome criteria used in this study were stringent, and the follow-up duration was short, the results of AMT by this user group were generally less favourable than those of previously reported case series. Controlled clinical trials would improve the quality of evidence for use of amniotic membrane in ocular disease.


A SAGE based approach to human glomerular endothelium: defining the transcriptome, finding a novel molecule and highlighting endothelial diversity.
Large scale transcript analysis of human glomerular microvascular endothelial cells (HGMEC) has never been accomplished. We designed this study to define the transcriptome of HGMEC and facilitate a better characterization of these endothelial cells with unique features. Serial analysis of gene expression (SAGE) was used for its unbiased approach to quantitative acquisition of transcripts. We generated a HGMEC SAGE library consisting of 68,987 transcript tags. Then taking advantage of large public databases and advanced bioinformatics we compared the HGMEC SAGE library with a SAGE library of non-cultured ex vivo human glomeruli (44,334 tags) which contained endothelial cells. The 823 tags common to both which would have the potential to be expressed in vivo were subsequently checked against 822,008 tags from 16 non-glomerular endothelial SAGE libraries. This resulted in 268 transcript tags differentially overexpressed in HGMEC compared to non-glomerular endothelia. These tags were filtered using a set of criteria: never before shown in kidney or any type of endothelial cell, absent in all nephron regions except the glomerulus, more highly expressed than statistically expected in HGMEC. Neurogranin, a direct target of thyroid hormone action which had been thought to be brain specific and never shown in endothelial cells before, fulfilled these criteria. Its expression in glomerular endothelium in vitro and in vivo was then verified by real-time-PCR, sequencing and immunohistochemistry. Our results represent an extensive molecular characterization of HGMEC beyond a mere database, underline the endothelial heterogeneity, and propose neurogranin as a potential link in the kidney-thyroid axis.


In-plane optical anisotropy in ReS2 flakes determined by angle-resolved polarized optical contrast spectroscopy.
Various in-plane anisotropic properties are observed for the layered semiconducting transition metal dichalcogenide (TMD), rhenium disulfide (ReS2) due to its reduced symmetry. The understanding of these unique anisotropic behaviors in ReS2 will promote its applications in optoelectronics. In this work, angle-resolved polarized optical contrast spectroscopy has proved to be an efficient, quantitative, and non-destructive method to probe the optical anisotropy in ReS2 flakes with different thicknesses. The contrast value of ReS2 displays the maximum intensity when the polarization of incident light is along the Re-Re chain direction, while the contrast shows the minimum value when the polarization is perpendicular. An empirical equation for in-plane anisotropic refractive index calculation has been proposed and the angle-resolved polarized optical contrasts of 1-3-layer ReS2 are calculated. The calculation results show good agreements with the experimental observations. This indicates that the proposed equation is indeed appropriate for the quantitative understanding of birefringence and dichroism in ReS2 flakes. Our results not only shed light on the identification of crystal axes in anisotropic materials by using angle-resolved polarized contrast spectroscopy, but also provide quantitative information about anisotropy in anisotropic materials such as ReS2.


Characterization of a type I collagen alpha 2(I) glycine-586 to valine substitution in osteogenesis imperfecta type IV. Detection of the mutation and prenatal diagnosis by a chemical cleavage method.
A chemical cleavage method for detecting mismatched bases in heteroduplexes formed between patient mRNA and control cDNA probes was employed to identify a single base mutation in a heterozygous case of osteogenesis imperfecta type IV. The parents' fibroblast mRNA did not contain the mutation. The region of the mRNA mismatch was amplified by using the polymerase chain reaction, cloned and sequenced. A point mutation of G to U at base-pair 2162 of the collagen alpha 2(I) mRNA resulted in the substitution of glycine by valine at amino acid position 586 of the helix. This substitution disrupted the critical Gly-Xaa-Yaa repeating unit of the collagen triple helix and resulted in helix destabilization, as evidenced by a decreased thermal stability. This local disturbance to helix propagation from the C-terminus to the N-terminus led to the overmodification of the collagen helix downstream towards the N-terminus. However, collagen secretion in vitro was normal, and the clinical phenotype probably resulted from the secretion into the extracellular matrix of the mutant collagen combined with a decrease in collagen production to 65% of control values. The rapid detection of the osteogenesis imperfecta mutation by using the chemical cleavage method afforded the opportunity to apply the technique to prenatal diagnosis in the next pregnancy of the mother of the osteogenesis imperfecta patient. The absence of a mismatched base in chorionic villus mRNA and control cDNA heteroduplexes indicated that the foetus did not carry the mutation, which was confirmed by the subsequent delivery of a normal baby.


Fc-rosette inhibition by hypocomplementaemic systemic lupus erythematosus sera.
Human red cells sensitized with one of the Rh antisera (Ripley) form rosettes (Fc-rosette) with human B lymphocytes and the rosettes are well inhibited by aggregated human IgG. Since sera of hypocomplementaemic patients with systemic lupus erythematosus (SLE) have frequently been reported to contain immune complexes, they were used for the inhibition of Fc-rosette formation in this study. The results of Fc-rosette inhibition rates of the sera were inversely correlated with the serum CH50 levels. When the sera were separated into top, middle, and bottom fractions by ultracentrifugation, the bottom fractions showed more effective inhititions than the others. Similarly, the strongest inhibition was found in the void volume of the serum separated by Sephadex G200 gel filtration. Reduction and alkylation of IgG resulted in the loss of reactivity with Fc receptor of B lymphocytes, and the rosette inhibiting activities of the SLE sera were markedly reduced after reduction and alkylation. Some of anti-HLA sera were inhibitory for the Fc-rosette formation, while the tested sera did not contain anti-HLA activity assessed by the microcytotoxicity test. These results indicated that circulating immune complexes contained in the sera inhibit the rosette formation, and that the Fc-rosette inhibition test is a simple and relatively sensitive method for the detection of circulating immune complexes. Antinuclear antibody activities of the sera were tested by the indirect immunofluorescent method; however, clear correlations were not obtained between Fc-rosette inhibition rates and staining patterns of antinuclear antibodies. On the other hand, the positive groups of LE-test exhibited slightly greater inhibition rates of the rosette than the negative groups.


Phosphorylation of the cAMP-dependent protein kinase (PKA) regulatory subunit modulates PKA-AKAP interaction, substrate phosphorylation, and calcium signaling in cardiac cells.
Subcellular compartmentalization of the cAMP-dependent protein kinase (PKA) by protein kinase A-anchoring proteins (AKAPs) facilitates local protein phosphorylation. However, little is known about how PKA targeting to AKAPs is regulated in the intact cell. PKA binds to an amphipathic helical region of AKAPs via an N-terminal domain of the regulatory subunit. In vitro studies showed that autophosphorylation of type II regulatory subunit (RII) can alter its affinity for AKAPs and the catalytic subunit (PKA(cat)). We now investigate whether phosphorylation of serine 96 on RII regulates PKA targeting to AKAPs, downstream substrate phosphorylation and calcium cycling in primary cultured cardiomyocytes. We demonstrated that, whereas there is basal phosphorylation of RII subunits, persistent maximal activation of PKA results in a phosphatase-dependent loss of RII phosphorylation. To investigate the functional effects of RII phosphorylation, we constructed adenoviral vectors incorporating mutants which mimic phosphorylated (RIIS96D), nonphosphorylated (RIIS96A) RII, or wild-type (WT) RII and performed adenoviral infection of neonatal rat cardiomyocytes. Coimmunoprecipitation showed that more AKAP15/18 was pulled down by the phosphomimic, RIIS96D, than RIIS96A. Phosphorylation of phospholamban and ryanodine receptor was significantly increased in cells expressing RIIS96D versus RIIS96A. Expression of recombinant RII constructs showed significant effects on cytosolic calcium transients. We propose a model illustrating a central role of RII phosphorylation in the regulation of local PKA activity. We conclude that RII phosphorylation regulates PKA-dependent substrate phosphorylation and may have significant implications for modulation of cardiac function.


Combination of small interfering RNAs mediates greater inhibition of human hepatitis B virus replication and antigen expression.
To evaluate the inhibitory effect mediated by combination of small interfering RNAs (siRNAs) targeting different sites of hepatitis B virus (HBV) transcripts on the viral replication and antigen expression in vitro. (1) Seven siRNAs targeting surface (S), polymerase (P) or precore (PreC) region of HBV genome were designed and chemically synthesized. (2) HBV-producing HepG2.2.15 cells were treated with or without siRNAs for 72 h. (3) HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay. (4) Intracellular viral DNA was quantified by real-time PCR (Polymerase Chain Reaction). (5) HBV viral mRNA was reverse transcribed and quantified by real-time PCR. (6) The change of cell cycle and apoptosis was determined by flow cytometry. Our data demonstrated that synthetic small interfering RNAs (siRNAs) targeting S and PreC gene could efficiently and specifically inhibit HBV replication and antigen expression. The expression of HBsAg and HBeAg and the replication of HBV could be specifically inhibited in a dose-dependent manner by siRNAs. Furthermore, our results showed that the combination of siRNAs targeting various regions could inhibit HBV replication and antigen expression in a more efficient way than the use of single siRNA at the same final concentration. No apoptotic change was observed in the cell after siRNA treatment. Our results demonstrated that siRNAs exerted robust and specific inhibition on HBV replication and antigen expression in a cell culture system and combination of siRNAs targeting different regions exhibited more potency.