The multifaceted role of the embryonic gene Cripto-1 in cancer, stem cells and epithelial-mesenchymal transition.
Cripto-1 (CR-1)/Teratocarcinoma-derived growth factor1 (TDGF-1) is a cell surface glycosylphosphatidylinositol (GPI)-linked glycoprotein that can function either in cis (autocrine) or in trans (paracrine). The cell membrane cis form is found in lipid rafts and endosomes while the trans acting form lacking the GPI anchor is soluble. As a member of the epidermal growth factor (EGF)/Cripto-1-FRL-1-Cryptic (CFC) family, CR-1 functions as an obligatory co-receptor for the transforming growth factor-β (TGF-β) family members, Nodal and growth and differentiation factors 1 and 3 (GDF1/3) by activating Alk4/Alk7 signaling pathways that involve Smads 2, 3 and 4. In addition, CR-1 can activate non-Smad-dependent signaling elements such as PI3K, Akt and MAPK. Both of these pathways depend upon the 78kDa glucose regulated protein (GRP78). Finally, CR-1 can facilitate signaling through the canonical Wnt/β-catenin and Notch/Cbf-1 pathways by functioning as a chaperone protein for LRP5/6 and Notch, respectively. CR-1 is essential for early embryonic development and maintains embryonic stem cell pluripotentiality. CR-1 performs an essential role in the etiology and progression of several types of human tumors where it is expressed in a population of cancer stem cells (CSCs) and facilitates epithelial-mesenchymal transition (EMT). In this context, CR-1 can significantly enhance tumor cell migration, invasion and angiogenesis. Collectively, these facts suggest that CR-1 may be an attractive target in the diagnosis, prognosis and therapy of several types of human cancer.


Can Anti-Müllerian Hormone Be a Reliable Biomarker for Assessing Ovarian Function in Women Postchemotherapy?
The predictive value of anti-Müllerian hormone (AMH) for ovarian dysfunction postchemotherapy is controversial. This study aimed to evaluate the value of serum AMH levels clinically and theoretically. We detected the serum estradiol, follicular stimulating hormone (FSH), luteinizing hormone (LH), and AMH levels in 144 premenopausal women with breast cancer receiving cyclophosphamide-based chemotherapy. The hormone levels before and postchemotherapy were compared; the correlations among the hormones and amenorrhea and menstrual recovery were analyzed. In addition, the serum AMH levels were detected randomly in 177 normal healthy women and 36 normal female C57BL/6J mice of different ages; meanwhile, the status of ovarian follicles was also examined. Furthermore, 72 Balb/c nude mice with breast cancer were randomly assigned to three groups that received different doses of cyclophosphamide (CTX) (control, 100 mg/kg, and 200 mg/kg), and the alterations in serum AMH levels and ovarian follicles were recorded and analyzed. Chemotherapy-induced amenorrhea was associated with prechemotherapy AMH levels, E2 levels, and FSH levels (P < 0.0001). The recovery of menstruation was associated with prechemotherapy AMH levels (P < 0.0001), but not with E2 and FSH levels (P > 0.05). In patients with breast cancer treated with chemotherapy, the serum AMH levels did not differ significantly between the pre- and post-chemotherapy periods in patients aged <35 years (P > 0.05), whereas a dramatic reduction was detected in patients aged >35 years (P < 0.0001). In healthy women, the serum AMH levels declined sharply after 35 years of age (P < 0.0001) and remained relatively stable at a younger age. Similar results were obtained in experiments using normal mice. The cancer-bearing mice exposed to 200 mg/kg CTX exhibited a significant decline in AMH levels and a remarkable decrease in the number of primordial and growing follicles (P < 0.0001). Our results indicate that AMH is an efficient marker for predicting postchemotherapy ovarian function exclusively in premenopausal female patients with breast cancer aged >35 years.


Primary structure of endoglin, an RGD-containing glycoprotein of human endothelial cells.
Endoglin is a major glycoprotein of human vascular endothelium. As observed with monoclonal antibody 44G4, the distribution of endoglin is restricted to endothelial cells in all tissues except bone marrow. cDNA clones were isolated from an endothelial cell lambda gt11 cDNA library using a rabbit antibody prepared against endoglin purified from placenta. Eleven antibody-positive and cross-hybridizing clones were obtained; reactivity with endothelial cell 3.4-kilobase mRNA transcript was observed. The N-terminal sequence of placental endoglin was determined and found within the deduced protein sequence, thus confirming the identity of the cDNA and revealing a partial signal peptide. Endoglin is a type I integral membrane protein of Mr = 68,051 with an extracellular region of 561 amino acids, a hydrophobic transmembrane domain, and a 47-residue cytoplasmic tail. There are four potential N-linked glycosylation sites in the N-terminal domain and a probable O-glycan domain rich in Ser and Thr residues proximal to the membrane-spanning domain. Data base searches revealed that endoglin is a novel protein. The sequence contains an RGD tripeptide (374-376), the first identified on a surface protein of endothelium. The presence of RGD, a key recognition structure in cellular adhesion, suggests a critical role for endoglin in the binding of endothelial cells to integrins and/or other RGD receptors.


Recent progress and application of the tetrahedral framework nucleic acid materials on drug delivery.
The application of DNA framework nucleic acid materials in the biomedical field has witnessed continual expansion. Among them, tetrahedral framework nucleic acids (tFNAs) have gained significant traction as the foremost biological vectors due to their superior attributes of editability, low immunogenicity, biocompatibility, and biodegradability. tFNAs have demonstrated promising results in numerous in vitro and in vivo applications. This review summarizes the latest research on tFNAs in drug delivery, including a discussion of the advantages of tFNAs in regulating biological behaviors, and highlights the updated development and advantageous applications of tFNAs-based nanostructures from static design to dynamically responsive design. tFNAs possess distinct biological regulatory attributes and can be taken up by cells without the requirement of transfection, differentiating them from other biological vectors. tFNAs can be easily physically/chemically modified and seamlessly incorporated with other functional systems. The static design of the tFNAs-based drug delivery system makes it versatile, reproducible, and predictable. Further use of the dynamic response mechanism of DNA to external stimuli makes tFNAs-based drug delivery more effective and specific, improving the uptake and utilization of the payload by the intended target. Dynamic targeting is poised to become the future primary approach for drug delivery.


Rhodium(iii)-catalyzed three-component cascade synthesis of 6H-benzo[c]chromenes through C-H activation.
A three-component cascade reaction is described that provides concise access to 6H-benzo[c]chromenes via Rh(iii)-catalyzed annulation of aryl ketone O-acyloximes, quinones and acetone. Acetone acts as a co-solvent and as a reactant. This reaction shows high efficiency, atom- and step-economy, good substrate scope, excellent functional group compatibility and gives the products in good yields.


Nobiletin, a citrus flavonoid, improves cognitive impairment and reduces soluble Aβ levels in a triple transgenic mouse model of Alzheimer's disease (3XTg-AD).
Alzheimer's disease (AD), the most common form of dementia among the elderly, is characterized by the progressive decline of cognitive function. Increasing evidence indicates that the production and accumulation of amyloid β (Aβ), particularly soluble Aβ oligomers, is central to the pathogenesis of AD. Our recent studies have demonstrated that nobiletin, a polymethoxylated flavone from citrus peels, ameliorates learning and memory impairment in olfactory-bulbectomized mice, amyloid precursor protein transgenic mice, NMDA receptor antagonist-treated mice, and senescence-accelerated mouse prone 8. Here, we present evidence that this natural compound improves cognitive impairment and reduces soluble Aβ levels in a triple transgenic mouse model of AD (3XTg-AD) that progressively develops amyloid plaques, neurofibrillary tangles, and cognitive impairments. Treatment with nobiletin (30 mg/kg) for 3 months reversed the impairment of short-term memory and recognition memory in 3XTg-AD mice. Our ELISA analysis also showed that nobiletin reduced the levels of soluble Aβ1-40 in the brain of 3XTg-AD mice. Furthermore, nobiletin reduced ROS levels in the hippocampus of 3XTg-AD as well as wild-type mice. These results suggest that this natural compound has potential to become a novel drug for the treatment and prevention of AD.


[Regulation of alkaline protease synthesis in Bacillus subtilis A-50].
The effect of glucose and amino acids on the synthesis of alkaline protease of the prototroph strain and auxotrophs of Bacillus subtilis A-50 was studied. There are both general and different from one another mechanisms of regulation for sporulation and protease synthesis as one of early stages of sporulation. Most auxotroph mutations pleiotropically decrease the level of protease synthesis and the efficiency of sporulation. At the same time the differences depending on sporulation and protease formation from glucose, amino acids and caseinoacids are observed. The increase in glucose concentration in the medium from 0.5 to 5% leads to intensification of protease synthesis and decrease of the sporulation efficiency. The suppression of alkaline protease formation in B. subtilis occurs when amino acid concentration is of the order of 1.5 mg/ml in the studied combination in the presence of 1--5% glucose. The strongest inhibitory effect was discovered for 2d, 3d and 4th amino acid groups in the presence of 5% glucose, whereas amino acids of the first group stimulate the protease synthesis in the presence of 2% glucose. The stimulatory effect of amino acids of the first groups is due to histidine and arginine.


[Molecular genetic diversity of the pea (Pisum sativum L.) from the collection of N.I. Vavilov Research Institute of Plant Industry as detected by AFLP analysis].
In this work, a pea selection obtained from the Vavilov Research Institute collection comprising 83 P. sativum samples, including representatives of three subspecies, was studied using the ?FL? labeling method. Local cultivars for different uses with maximum ecological and geographical diversity (including those from the centers of origin of the species) were predominantly chosen for the study; a number of their morphological and biological characteristics were evaluated. We obtained 382 polymorphic AFLP fragments; each sample was characterized by a unique set of these fragments. The genetic diversity of the studied material was evaluated, and a wide range of genetic differences in the investigated samples (0.07-0.27) was demonstrated. The affiliation of the samples to the certain subspecies was not confirmed by the obtained data; the ecogeographical differentiation of the samples was not reflected by the data. Factor analysis allowed us to identify the sample groups of European and Asian origin and the intermediate nature of most of the samples from the studied selection.


Protein profiles of cardiomyocyte differentiation in murine embryonic stem cells exposed to perfluorooctane sulfonate.
Perfluorooctane sulfonate (PFOS) is a persistent organic contaminant that may affect diverse systems in animals and humans, including the cardiovascular system. However, little is known about the mechanism by which it affects the biological systems. Herein, we used embryonic stem cell test procedure as a tool to assess the developmental cardiotoxicity of PFOS. The differentially expressed proteins were identified by quantitative proteomics that combines the stable isotope labeling of amino acids with high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. Results of the embryonic stem cell test procedure suggested that PFOS was a weak embryotoxic chemical. Nevertheless, a few marker proteins related to cardiovascular development (Brachyury, GATA4, MEF2C, α-actinin) were significantly reduced by exposure to PFOS. In total, 176 differential proteins were identified by proteomics analysis, of which 67 were upregulated and 109 were downregulated. Gene ontology annotation classified these proteins into 13 groups by molecular functions, 12 groups by cellular locations and 10 groups by biological processes. Most proteins were mainly relevant to either catalytic activity (25.6%), nucleus localization (28.9%) or to cellular component organization (19.8%). Pathway analysis revealed that 32 signaling pathways were affected, particularly these involved in metabolism. Changes in five proteins, including L-threonine dehydrogenase, X-ray repair cross-complementing 5, superoxide dismutase 2, and DNA methyltransferase 3b and 3a were confirmed by Western blotting, suggesting the reliability of the technique. These results revealed potential new targets of PFOS on the developmental cardiovascular system.


SUMOylation modulates the function of DDX19 in mRNA export.
Nuclear export of mRNAs is a critical regulatory step in eukaryotic gene expression. The mRNA transcript undergoes extensive processing, and is loaded with a set of RNA-binding proteins (RBPs) to form export-competent messenger ribonucleoprotein particles (mRNPs) in the nucleus. During the transit of mRNPs through the nuclear pore complex (NPC), the DEAD-box ATPase - DDX19 (herein referring to DDX19A and DDX19B) - remodels mRNPs at the cytoplasmic side of the NPC, by removing a subset of RNA-binding proteins to terminate mRNP export. This requires the RNA-dependent ATPase activity of DDX19 and its dynamic interactions with Gle1 and Nup214. However, the regulatory mechanisms underlying these interactions are unclear. We find that DDX19 gets covalently attached with a small ubiquitin-like modifier (SUMO) at lysine 26, which enhances its interaction with Gle1. Furthermore, a SUMOylation-defective mutant of human DDX19B, K26R, failed to provide a complete rescue of the mRNA export defect caused by DDX19 depletion. Collectively, our results suggest that SUMOylation fine-tunes the function of DDX19 in mRNA export by regulating its interaction with Gle1. This study identifies SUMOylation of DDX19 as a modulatory mechanism during the mRNA export process. This article has an associated First Person interview with the first author of the paper.


Sugar-sweetened beverages and risk of hypertension and CVD: a dose-response meta-analysis.
A number of prospective cohort studies have investigated the associations between consumption of sugar-sweetened beverages (SSB) and the risk of hypertension, CHD and stroke, but revealed mixed results. In the present study, we aimed to perform a dose-response meta-analysis of these prospective studies to clarify these associations. A systematic literature search was conducted using the PubMed and Embase databases up to 5 May 2014. Random- or fixed-effects models were used to calculate the pooled relative risks (RR) with 95 % CI for the highest compared with the lowest category of SSB consumption, and to conduct a dose-response analysis. A total of six prospective studies (240 726 participants and 80 411 incident cases of hypertension) from four publications on hypertension were identified. A total of four prospective studies (194 664 participants and 7396 incident cases of CHD) from four publications on CHD were identified. A total of four prospective studies (259 176 participants and 10 011 incident cases of stroke) from four publications on stroke were identified. The summary RR for incident hypertension was 1·08 (95 % CI 1·04, 1·12) for every additional one serving/d increase in SSB consumption. The summary RR for incident CHD was 1·17 (95 % CI 1·10, 1·24) for every serving/d increase in SSB consumption. There was no significant association between SSB consumption and total stroke (summary RR 1·06, 95 % CI 0·97, 1·15) for every serving/d increase in SSB consumption. The present meta-analysis suggested that a higher consumption of SSB was associated with a higher risk of hypertension and CHD, but not with a higher risk of stroke.


Ocular surface complications result from dysregulation of the OGF-OGFr signaling pathway in female diabetic rats.
Approximately 4.5 million women in the United States exhibit diabetes-associated ocular complications. The time course and magnitude of these complications, and their association with the dysregulation of the opioid growth factor (OGF)-OGF receptor (OGFr) signaling pathway are unknown. The present study investigated the onset and magnitude of ocular surface complications and the association with a dysregulated OGF-OGFr signaling pathway in diabetic female rats. Adult female Sprague-Dawley rats were injected with streptozotocin in order to establish a model of type 1 diabetes (T1D), and a subset received insulin (T1D-INS). Blood glucose, body weight, tear production and corneal sensitivity, as well as serum and tissue expression levels of OGF and OGFr, were assessed. Corneal epithelial wound healing was also evaluated. In a second study, female T1D rats were treated with topical naltrexone (NTX) to determine whether blockade of the OGF-OGFr signaling pathway by NTX altered development of corneal surface complications. Female T1D rats had elevated glucose levels and reduced body weight compared with control and T1D-INS rats. In both diabetic groups, tear production was decreased within 2 weeks and corneal sensitivity was decreased 2.5-fold within 5 weeks, while corneal epithelial wound healing was delayed only in T1D rats. Serum and tissue levels of OGF and OGFr were elevated in diabetes. Twice daily NTX treatment reversed most ocular surface complications in the diabetic female rats. The present data demonstrated a seminal discovery in female T1D rats, in which the onset and magnitude of diabetes-associated ocular surface complications were associated with dysregulation of the OGF-OGFr regulatory pathway. Blockade of the OGF-OGFr pathway with the opioid receptor antagonist NTX prevented the onset and/or decreased the magnitude of these deficits. The current data support the need for translational research on this therapeutic approach for diabetic human subjects.


Using an Endogenous CRISPR-Cas System for Genome Editing in the Human Pathogen Clostridium difficile.
The human enteropathogen Clostridium difficile constitutes a key public health issue in industrialized countries. Many aspects of C. difficile pathophysiology and adaptation inside the host remain poorly understood. We have recently reported that this bacterium possesses an active CRISPR-Cas system of subtype I-B for defense against phages and other mobile genetic elements that could contribute to its success during infection. In this paper, we demonstrate that redirecting this endogenous CRISPR-Cas system toward autoimmunity allows efficient genome editing in C. difficile We provide a detailed description of this newly developed approach and show, as a proof of principle, its efficient application for deletion of a specific gene in reference strain 630Δerm and in epidemic C. difficile strain R20291. The new method expands the arsenal of the currently limiting set of gene engineering tools available for investigation of C. difficile and may serve as the basis for new strategies to control C. difficile infections.IMPORTANCEClostridium difficile represents today a real danger for human and animal health. It is the leading cause of diarrhea associated with health care in adults in industrialized countries. The incidence of these infections continues to increase, and this trend is accentuated by the general aging of the population. Many questions about the mechanisms contributing to C. difficile's success inside the host remain unanswered. The set of genetic tools available for this pathogen is limited, and new developments are badly needed. C. difficile has developed efficient defense systems that are directed against foreign DNA and that could contribute to its survival in phage-rich gut communities. We show how one such defense system, named CRISPR-Cas, can be hijacked for C. difficile genome editing. Our results also show a great potential for the use of the CRISPR-Cas system for the development of new therapeutic strategies against C. difficile infections.


Detecting past male-mediated expansions using the Y chromosome.
Males and females display biological differences that lead to a higher variance of offspring number in males, and this is frequently exacerbated in human societies by mating practices, and possibly by past socio-cultural circumstances. This implies that the genetic record might contain the imprint of past male-mediated expansions, which can be investigated by analysing the male-specific region of the Y chromosome (MSY). Here, we review studies that have used MSY data to infer such expansions. Sets of short-tandem repeats define haplotypes of very low average frequencies, but in a few cases, high-frequency haplotypes are observed, forming the core of descent clusters. Estimates of the ages of such clusters, together with geographical information, have been used to propose powerful historical founders, including Genghis Khan, although without direct supporting evidence. Resequencing of multi-megabase segments of MSY has allowed the construction of detailed phylogenies in which branch lengths are proportional to time, leading to the identification of lineage expansions in the last few millennia as well as the more distant past. Comparisons with maternally-inherited mitochondrial DNA sequence data allow the male specificity of some of these expansions to be demonstrated. These include expansions in Europe in the last ~5000 years that may be associated with a cultural shift during the Bronze Age, as well as expansions elsewhere in the world for which explanations from archaeological evidence are not yet clear.


Preferential cytoplasmic location of FtsZ, a protein essential for Escherichia coli septation.
An ftsZ thermonull mutant has been constructed in which the ftsZ gene has been deleted from the Escherichia coli chromosome while maintaining a wild-type copy of the gene in a thermosensitive plasmid. Under conditions in which the ftsZ+ allele is unable to be replicated at the same pace as the chromosome, the cells become non-viable and grow as filaments, indicating that, contrary to other reports, FtsZ performs a function essential for cell survival. Antibodies raised against FtsZ have been used to detect the cellular location of FtsZ and its contents per cell. Fractionation experiments indicate that most of the total FtsZ present in the cell stays in the cytoplasm.


Efficacy of 3D-printed assisted percutaneous transhepatic one-step biliary fistulation combined with rigid choledochoscopy for intrahepatic bile duct stones.
This study evaluated the efficacy and safety of three-dimensional printing model-assisted percutaneous transhepatic one-step biliary fistulation (PTOBF) combined with rigid choledochoscopy for intrahepatic bile duct stones in patients with type I bile duct classification. The clinical data of 63 patients with a type I intrahepatic bile duct were reviewed from January 2019 to January 2023; 30 patients who underwent 3D printed model-assisted PTOBF combined with rigid choledochoscopy composed the experimental group and 33 patients who underwent simple PTOBF combined with rigid choledochoscopy composed the control group. Six indicators, including one-stage operation time and clearance rate, final removal rate, bleeding volume, channel size and complications, were observed and analyzed in the two groups. The one-stage and final removal rate in the experimental group was higher than that in the control group (P = 0.034, P = 0.014 versus control group). The time of one-stage operation, bleeding volume, and incidence of complications in the experimental group were significantly lower than those in the control group (P < 0.001, P = 0.039, P = 0.026 versus control group). Compared with simple PTOBF combined with rigid choledochoscopy, 3D printed model-assisted PTOBF combined with rigid choledochoscopy is a safer and more effective method for treating intrahepatic bile duct stones.


[Oxidizing agents and free radicals in biomedicine].
The reactions which involve oxidants and free radicals species have played an essential role at the beginning of aerobic life, and they are an intrinsic part in the regulation of the cellular processes. However, as a result of the derived toxic effects of these oxidative processes, antioxidants molecules appeared in the very early stages of the evolution and they are able to control the production of these reactants and their dangerous effects. Oxidants and antioxidants have a clear function in the cellular physiology. When this delicate balance change many biochemical and cellular reactions are altered, and that may cause different pathologic diseases. In addition, free radicals of oxygen are thought to play a growing role in the biological process of ageing (1). It is not unusual to find papers about these reactants in every topic of the biomedicine. The main oxidants and free radicals in the organisms are oxygen-related agents, which are globally called reactive oxygen intermediates (ROI). These unavoidable, useful and dangerous biological oxidants are well characterized, although the recent implications they received in some pathologies deserve, in our opinion, a general review.


The effects of encoding in hypnosis and post-hypnotic suggestion on academic performance.
This study examined the relationship between proactive learning in hypnosis, post-hypnotic suggestion, and academic performance. Participants (N = 56) were randomly assigned to a control group or a treatment group. The treatment group was hypnotized and read a passage while in hypnosis. Concurrently, they were given a post-hypnotic suggestion, which attempted to aid recognition and performance on a test immediately following the hypnosis session. Both groups completed a multiple-choice test based on the aforementioned passage. An analysis of covariance discerned the effect of proactive learning and post-hypnotic suggestion on test performance, while controlling for the variance introduced by scholastic aptitude as measured by the ACT. Results indicated that the hypnosis sessions predicted significantly impaired test performance.


Inhibition of the influenza virus RNA-dependent RNA polymerase by antisera directed against the carboxy-terminal region of the PB2 subunit.
The influenza virus RNA polymerase consists of a heterotrimeric complex of the PB1, PB2 and PA proteins, with the PB2 subunit responsible for recognizing 5' cap structures on the host cell RNAs used as primers for virus mRNA synthesis. To investigate further the role PB2 plays in mRNA synthesis, a set of polyclonal antisera raised against defined regions of the protein were tested for their ability to inhibit the virion transcriptase. All five sera were of sufficient titre to immunoprecipitate PB2 and four were capable of recognizing polymerase complexes containing PB1 and PA. However, only the serum raised against the carboxy terminus of PB2 (F5) substantially inhibited polymerase activity. This serum drastically reduced synthesis primed by globin mRNA, but only partially inhibited transcription primed by the dinucleotide ApG, or ApG and cap analogue. The preferential inhibition of globin-primed synthesis did not result from interference with cap recognition, as serum F5 did not reduce labelling of PB2 in a photoaffinity cap-binding assay. However, IgG and Fab fragments from F5 were found to inhibit virion endonuclease activity. This suggests that the C terminus of PB2 plays a crucial role in transcription initiation and implicates PB2 in endonuclease activity.


The MG-QOL15 Japanese version: validation and associations with clinical factors.
Our study aim was to produce a Japanese translation of the 15-item Myasthenia Gravis Quality-of-Life Scale (MG-QOL15), assess its reliability and validity, and examine clinical factors affecting self-perceived QOL in MG. We evaluated 327 consecutive patients with MG seen at six neurological centers. All patients completed the Japanese version of the MG-QOL15 (MG-QOL15-J), the Beck Depression Inventory-second edition (BDI-II), and a generic health-related QOL questionnaire, the SF-36. Disease severity was determined according to the Myasthenia Gravis Foundation of America (MGFA) quantitative MG score and the MG composite. The MG-QOL15-J exhibited adequate internal reliability, test-retest repeatability, and concurrent validity with SF-36, disease severity, and known-patient groups categorized by MGFA post-intervention status. Multivariate analysis revealed severity, dose of oral corticosteroids, and BDI-II as independent factors negatively affecting QOL. The MG-QOL15-J is anticipated to be a valuable clinical measure of QOL in Japanese patients with MG.


Development of a Trypanosoma cruzi strain typing assay using MS2 peptide spectral libraries (Tc-STAMS2).
Chagas disease also known as American trypanosomiasis is caused by the protozoan Trypanosoma cruzi. Over the last 30 years, Chagas disease has expanded from a neglected parasitic infection of the rural population to an urbanized chronic disease, becoming a potentially emergent global health problem. T. cruzi strains were assigned to seven genetic groups (TcI-TcVI and TcBat), named discrete typing units (DTUs), which represent a set of isolates that differ in virulence, pathogenicity and immunological features. Indeed, diverse clinical manifestations (from asymptomatic to highly severe disease) have been attempted to be related to T.cruzi genetic variability. Due to that, several DTU typing methods have been introduced. Each method has its own advantages and drawbacks such as high complexity and analysis time and all of them are based on genetic signatures. Recently, a novel method discriminated bacterial strains using a peptide identification-free, genome sequence-independent shotgun proteomics workflow. Here, we aimed to develop a Trypanosoma cruzi Strain Typing Assay using MS/MS peptide spectral libraries, named Tc-STAMS2. The Tc-STAMS2 method uses shotgun proteomics combined with spectral library search to assign and discriminate T. cruzi strains independently on the genome knowledge. The method is based on the construction of a library of MS/MS peptide spectra built using genotyped T. cruzi reference strains. For identification, the MS/MS peptide spectra of unknown T. cruzi cells are identified using the spectral matching algorithm SpectraST. The Tc-STAMS2 method allowed correct identification of all DTUs with high confidence. The method was robust towards different sample preparations, length of chromatographic gradients and fragmentation techniques. Moreover, a pilot inter-laboratory study showed the applicability to different MS platforms. This is the first study that develops a MS-based platform for T. cruzi strain typing. Indeed, the Tc-STAMS2 method allows T. cruzi strain typing using MS/MS spectra as discriminatory features and allows the differentiation of TcI-TcVI DTUs. Similar to genomic-based strategies, the Tc-STAMS2 method allows identification of strains within DTUs. Its robustness towards different experimental and biological variables makes it a valuable complementary strategy to the current T. cruzi genotyping assays. Moreover, this method can be used to identify DTU-specific features correlated with the strain phenotype.


Rac regulates integrin-mediated spreading and increased adhesion of T lymphocytes.
Leukocyte adhesion to the extracellular matrix (ECM) is tightly controlled and is vital for the immune response. Circulating lymphocytes leave the bloodstream and adhere to ECM components at sites of inflammation and lymphoid tissues. Mechanisms for regulating T-lymphocyte-ECM adhesion include (i) an alteration in the affinity of cell surface integrin receptors for their extracellular ligands and (ii) an alteration of events following postreceptor occupancy (e.g., cell spreading). Whereas H-Ras and R-Ras were previously shown to affect T-cell adhesion by altering the affinity state of the integrin receptors, no signaling molecule has been identified for the second mechanism. In this study, we demonstrated that expression of an activated mutant of Rac triggered dramatic spreading of T cells and their increased adhesion on immobilized fibronectin in an integrin-dependent manner. This effect was not mimicked by expression of activated mutant forms of Rho, Cdc42, H-Ras, or ARF6, indicating the unique role of Rac in this event. The Rac-induced spreading was accompanied by specific cytoskeletal rearrangements. Also, a clustering of integrins at sites of cell adhesion and at the peripheral edges of spread cells was observed. We demonstrate that expression of RacV12 did not alter the level of expression of cell surface integrins or the affinity state of the integrin receptors. Moreover, our results indicate that Rac plays a role in the regulation of T-cell adhesion by a mechanism involving cell spreading, rather than by altering the level of expression or the affinity of the integrin receptors. Furthermore, we show that the Rac-mediated signaling pathway leading to spreading of T lymphocytes did not require activation of c-Jun kinase, serum response factor, or pp70(S6 kinase) but appeared to involve a phospholipid kinase.


West Nile virus encephalitis in a renal transplant recipient: the role of intravenous immunoglobulin.
West Nile virus is a common viral infection in endemic areas. Although the disease has a benign course in immunocompetent individuals, it tends to run a more malignant course in immunocompromised patients such as solid-organ transplant recipients. In this study, a renal transplant recipient presented with fever, impaired speech, obtundation, and features suggestive of meningitis on cerebrospinal fluid examination. Although initial serological study results were negative, the patient was treated promptly with intravenous immunoglobulin (IVIG) based on a strong clinical suspicion of West Nile virus encephalitis. Therapy with IVIG was associated with complete recovery of neurological features, and the patient was discharged on day 7 after resolution of neurological complications. The favorable outcome of this patient may be related to early treatment with IVIG.


Expression of the Na+ dependent uridine transport system of rabbit small intestine: studies with mRNA-injected Xenopus laevis oocytes.
Xenopus laevis oocytes were used as an expression system to prove and characterize the carrier-mediated transport of uridine in the small intestine. Significant Na+ dependency was observed for the uptake of [3H]uridine by Xenopus laevis oocytes injected with poly(A)+RNA prepared from rabbit small intestinal mucosa. By contrast, the uptake of [3H]uridine was negligible in water-injected oocytes. There was no significant difference in the Na+ dependent uptake rates of [3H]uridine among oocytes expressed by using mRNA prepared by three different methods. The uptake of [3H]uridine by mRNA-injected oocytes was enhanced by increasing the culturing time after mRNA injection. Concentration dependency for uridine transport was observed with the Michaelis constant of 8.27 microM, which was comparable to that reported in the study using the brush-border membrane vesicles from rabbit small intestine (6.4 microM). Furthermore, the uptake of [3H]uridine was significantly inhibited by adenosine and thymidine, but not by adenine and uracil. Consequently, the transport system of uridine expressed in mRNA-injected oocytes is clarified to be similar to that functioning in the brush-border membrane of the small intestine.


Identification of extracellular N-acylhomoserine lactone acylase from a Streptomyces sp. and its application to quorum quenching.
N-acylhomoserine lactones (AHLs) play an important role in regulating virulence factors in pathogenic bacteria. Recently, the enzymatic inactivation of AHLs, which can be used as antibacterial targets, has been identified in several soil bacteria. In this study, strain M664, identified as a Streptomyces sp., was found to secrete an AHL-degrading enzyme into a culture medium. The ahlM gene for AHL degradation from Streptomyces sp. strain M664 was cloned, expressed heterologously in Streptomyces lividans, and purified. The enzyme was found to be a heterodimeric protein with subunits of approximately 60 kDa and 23 kDa. A comparison of AhlM with known AHL-acylases, Ralstonia strain XJ12B AiiD and Pseudomonas aeruginosa PAO1 PvdQ, revealed 35% and 32% identities in the deduced amino acid sequences, respectively. However, AhlM was most similar to the cyclic lipopeptide acylase from Streptomyces sp. strain FERM BP-5809, exhibiting 93% identity. A mass spectrometry analysis demonstrated that AhlM hydrolyzed the amide bond of AHL, releasing homoserine lactone. AhlM exhibited a higher deacylation activity toward AHLs with long acyl chains rather than short acyl chains. Interestingly, AhlM was also found to be capable of degrading penicillin G by deacylation, showing that AhlM has a broad substrate specificity. The addition of AhlM to the growth medium reduced the accumulation of AHLs and decreased the production of virulence factors, including elastase, total protease, and LasA, in P. aeruginosa. Accordingly, these results suggest that AHL-acylase, AhlM could be effectively applied to the control of AHL-mediated pathogenicity.


[Changes in various indices of thyroid function in obese adults during diet therapy].
Modifications in thyroid function caused by changes in diet have been reported in the literature though the results are often conflicting. The present study was conducted on 48 obese adults divided into two groups: the first over 60% and the second under 60% above their ideal weight. A control group of healthy subjects of normal weight and comparable age was also studied. The observation lasted 9 weeks during which a low calorie diet providing about 1250 kCal was administered. Mean basal levels of FT3, TT3, FT4 and TSH revealed no significant differences between the obese subjects and the control group. Mean TT4 was significantly higher in the obese and mean T3U significantly lower. During low calorie diet treatment the obese subjects produced no significant variations in the above parameters, apart from a significant increase in T3U. The study revealed no evidence of the low T3 syndrome that has long been reported in the literature.


A microfluidic sensor for detecting chlorophenols using cross-linked enzyme aggregates (CLEAs).
Chlorophenols have a strong medicinal smell and can be detected by the human nose at parts-per-million levels. Therefore, continuous monitoring of chlorophenols in water supplies is highly important. Herein, we reported a microfluidic sensor which can be used to detect 2,4-dichlorophenol (2,4-DCP) in real time with a limit of detection of around 0.1 ppm. The microfluidic sensor is a membrane-less galvanic cell which consists of two laminar flows running in parallel inside a straight channel. The sensor measures the potential difference between a solution containing 2,4-DCP and a reference solution containing acetate buffer. In a continuous-flow mode, the cell potential is proportional to the concentration of 2,4-DCP. To render specificity for the sensor, we incorporate a pre-treatment section where the incoming solution containing 2,4-DCP is split into two streams. One of the streams is brought into contact with cross-linked laccase aggregates (which catalyzes the hydrolysis of 2,4-DCP) and the second stream is taken as a reference solution. By comparing the potential difference between the two streams, we can determine the concentration of 2,4-DCP with high specificity. The microfluidic sensor platform is potentially useful for real-time detection of micropollutants present in aquatic systems with high sensitivity and specificity.


Differential effects of inappropriate empirical antibiotic therapy in adults with community-onset gram-positive and gram-negative aerobe bacteremia.
Bacteremia is associated with high morbidity and mortality, which contribute substantially to health care costs. A beneficial influence of appropriate empirical antimicrobial therapy (EAT) on patient outcome is evidenced; However, the evidence highlighting a comparison of clinical manifestations and of the effects of inappropriate EAT between Gram-positive and Gram-negative bacteremia is insufficient. In a retrospective 6-year cohort study, the total 2053 adults (Gram-positive, 566; Gram-negative 1487) presenting with community-onset monomicrobial aerobes bacteremia were recruited. Inappropriate EAT was defined as the first dose of an appropriate antimicrobial agent not being administered within the first 24 h after blood cultures were drawn. Although the bacteremia severity (a Pitt bacteremia score) at onset, comorbidity severity (the McCabe-Johnson classification), and 28-day mortality rate were similar in the two groups. Furthermore, after adjustment of independent predictors of 28-day mortality respectively recognized by the multivariate regression model in Gram-negative and Gram-positive groups, the Kaplan-Meier curve and Cox regression analysis revealed a significant difference (adjust odds ratio [AOR], 2.68; P < 0.001) between appropriate and inappropriate EAT in the Gram-negative group, but not in the Gram-positive group (AOR, 1.54; P = 0.06). Conclusively, patients with Gram-positive and Gram-negative bacteremia exhibited the similar presentation in bacteremia severity, but a greater impact of inappropriate EAT on survival of patients with Gram-negative aerobe bacteremia was evidenced.


Melem (2,5,8-triamino-tri-s-triazine), an important intermediate during condensation of melamine rings to graphitic carbon nitride: synthesis, structure determination by X-ray powder diffractometry, solid-state NMR, and theoretical studies.
Single-phase melem (2,5,8-triamino-tri-s-triazine) C(6)N(7)(NH(2))(3) was obtained as a crystalline powder by thermal treatment of different less condensed C-N-H compounds (e.g., melamine C(3)N(3)(NH(2))(3), dicyandiamide H(4)C(2)N(4), ammonium dicyanamide NH(4)[N(CN)(2)], or cyanamide H(2)CN(2), respectively) at temperatures up to 450 degrees C in sealed glass ampules. The crystal structure was determined ab initio by X-ray powder diffractometry (Cu K alpha(1): P2(1)/c (No. 14), a = 739.92(1) pm, b = 865.28(3) pm, c = 1338.16(4) pm, beta = 99.912(2) degrees, and Z = 4). In the solid, melem consists of nearly planar C(6)N(7)(NH(2))(3) molecules which are arranged into parallel layers with an interplanar distance of 327 pm. Detailed (13)C and (15)N MAS NMR investigations were performed. The presence of the triamino form instead of other possible tautomers was confirmed by a CPPI (cross-polarization combined with polarization inversion) experiment. Furthermore, the compound was characterized using mass spectrometry, vibrational (IR, Raman), and photoluminescence spectroscopy. The structural and vibrational properties of molecular melem were theoretically studied on both the B3LYP and the MP2 level. A structural optimization in the extended state was performed employing density functional methods utilizing LDA and GGA. A good agreement was found between the observed and calculated structural parameters and also for the vibrational frequencies of melem. According to temperature-dependent X-ray powder diffractometry investigations above 560 degrees C, melem transforms into a graphite-like C-N material.


Osteoporosis in anorexia nervosa.
Osteoporosis is common in anorexia nervosa. It places these patients at increased lifetime risk for fractures. Bone loss may never recover completely even once weight is restored. The strongest predictors of osteoporosis include low body weight and amenorrhea. Loss of bone density can occur rapidly and very early in the course of anorexia nervosa. The etiology of bone loss in the patient with anorexia nervosa is multifactorial. In addition to reduced estrogen and progesterone, excess cortisol levels and low levels of insulin growth factor (IGF-1), a correlate for bone formation, are observed. Dual energy x-ray absorptiometry screening is important to assess bone density. However, successful treatments to reverse bone loss, in those with anorexia nervosa, are lacking. Early diagnosis and treatment of anorexia nervosa are paramount to prevent initial weight loss and subsequent loss of bone.


Comparison of atraumatic restorative treatment and conventional restorative procedures in a hospital clinic: evaluation after 30 months.
The purpose of this study was to evaluate two glass-ionomer cements placed in the occlusal surfaces of permanent molar teeth, using two cavity preparation methods. Three dentists placed 149 restorations for 68 patients in a hospital clinic. Atraumatic restorative treatment or conventional cavity preparation methods were used for two encapsulated, high-strength conventional glass-ionomer cements: Fuji IX GP and Ketac-Molar. Non-gamma 2 amalgam alloy was used in conventional preparations for comparison. The restorative procedures were uneventful, but cavity preparations made with atraumatic restorative treatment hand instruments took approximately twice as long as did conventional rotary instrumentation. After 30 months, only one glass-ionomer cement restoration had failed. Both glass-ionomer cements showed high early losses of sealant material, but caries was not detected in the exposed fissures. Both glass-ionomer cements also showed relatively high restoration wear. At 30 months, the mean cumulative net occlusal wear was 119 +/- 12 mm for Fuji IX GP and 96 +/- 13 mm for Ketac-Molar; the difference was not statistically significant. Color matching improved significantly by 6 months; there was no significant difference in color match between the two glass-ionomer cements by 12 months. Minor surface tarnishing and marginal discrepancies were present in the amalgam restorations and increased with time. The occlusal restorations performed satisfactorily over periods of up to 30 months. However, the continued deterioration of the cements requires longer-term studies to be undertaken.


Alkali treatment of microrough titanium surfaces affects macrophage/monocyte adhesion, platelet activation and architecture of blood clot formation.
Titanium implants are most commonly used for bone augmentation and replacement due to their favorable osseointegration properties. Here, hyperhydrophilic sand-blasted and acid-etched (SBA) titanium surfaces were produced by alkali treatment and their responses to partially heparinized whole human blood were analyzed. Blood clot formation, platelet activation and activation of the complement system was analyzed revealing that exposure time between blood and the material surface is crucial as increasing exposure time results in higher amount of activated platelets, more blood clots formed and stronger complement activation. In contrast, the number of macrophages/monocytes found on alkali-treated surfaces was significantly reduced as compared to untreated SBA Ti surfaces. Interestingly, when comparing untreated to modified SBA Ti surfaces very different blood clots formed on their surfaces. On untreated Ti surfaces blood clots remain thin (below 15 mm), patchy and non-structured lacking large fibrin fiber networks whereas blood clots on differentiated surfaces assemble in an organized and layered architecture of more than 30 mm thickness. Close to the material surface most nucleated cells adhere, above large amounts of non-nucleated platelets remain entrapped within a dense fibrin fiber network providing a continuous cover of the entire surface. These findings might indicate that, combined with findings of previous in vivo studies demonstrating that alkali-treated SBA Ti surfaces perform better in terms of osseointegration, a continuous and structured layer of blood components on the blood-facing surface supports later tissue integration of an endosseous implant.


Effect of parental age and associated size on fecundity, growth and survival in the yellow seahorse Hippocampus kuda.
Seahorses, together with the pipefishes (Family Syngnathidae), are the only vertebrates in which embryonic development takes place within a specialised body compartment, the brood pouch, of the male instead of the female. Embryos develop in close association with the brood pouch epithelium in a manner that bears some resemblance to embryo-placental relationships in mammals. We have explored the hypothesis that parental body size and age should affect offspring postnatal growth and survival if brood pouch quality impacts upon prenatal embryonic nutrition or respiration. Using an aquarium population of the yellow seahorse, Hippocampus kuda, we show here that large parents produce offspring whose initial postnatal growth rates (weeks one to three) were significantly higher than those of the offspring of younger and smaller parents. Whereas 90% of offspring from the larger parents survived for the duration of the study (7 weeks), less that 50% of offspring from smaller parents survived for the same period. For the offspring of large parents, growth rates from individual males were negatively correlated with the number of offspring in the cohort (r=-0.82; P<0.05); this was not the case for offspring from small parents (r=0.048; P>0.9). Observations of embryos within the pouch suggested that when relatively few embryos are present they may attach to functionally advantageous sites and thus gain physiological support during gestation. These results suggest that male body size, and pouch size and function, may influence the future fitness and survival of their offspring.


[The clinical significance of leukocyte chemiluminescence in myocardial infarct].
A study was made of the luminol-dependent leukocyte chemiluminescence (LCL) of the whole blood in 45 patients with acute myocardial infarction (AMI). It has been shown that measurement of LCL during the first examination of the patient may play a significant part in the diagnosis of AMI. LCL monitoring is of independent importance for the control of resorption and reparative processes and for the diagnosis of the lingering course and threatened relapse of AMI. The authors provide evidence for the concept of the trigger inclusion of the leukocytic component into the chain of pathophysiological processes in AMI at hours 5-10 since its onset. Derangement of that mechanism may be one of the causes of the lingering and recurrent course of infarction. The treatment of AMI should be carried out with regard to the effect of therapeutic modalities on leukocyte function as well as to the phase of the pathophysiological process.


Increased 18F-FDG Uptake in Multiple Muscles in a Patient With Violent Cough.
Increased muscular FDG uptake could be due to various causes. Detailed analysis combined with history might be helpful for image interpretation. We present FDG PET/CT findings of a 69-year-old woman with lung cancer. The images demonstrated intense FDG uptake in multiple muscles, likely due to cough before the PET/CT study. To relieve the patient's cough, codeine was administrated. The second F-FDG PET/CT was performed 2 days later. The images showed that the abnormal muscular activity had become decreased, which was slightly lower than hepatic activity.


Current status of intestinal transplantation in East Asia.
Intestinal transplantation (ITx) activity remains low in East Asia. We conducted a multinational, retrospective study on patients who underwent ITx in Korea, Taiwan, and Japan, to provide an overview and to foresee future developments and collaborations in this region. Total 71 ITx were performed in 67 patients. Living donor ITx was most commonly conducted in Japan (n = 13). Despite the low caseload, all three countries demonstrated acceptable patient survival rates of approximately 70% at 5 years. Over 70% of recipients with a functioning graft were free from total parenteral nutrition. There is an urgent need to establish a nationwide and multinational registry of ITx recipients and patients with intestinal failure in East Asia. An efficient referral system to specialized intestinal rehabilitation and ITx centers and a multidisciplinary team approach is also warranted to provide state-of-the-art treatment for patients desperately waiting for a chance to survive.


Extracellular compartments in matrix morphogenesis: collagen fibril, bundle, and lamellar formation by corneal fibroblasts.
The regulation of collagen fibril, bundle, and lamella formation by the corneal fibroblasts, as well as the organization of these elements into an orthogonal stroma, was studied by transmission electron microscopy and high voltage electron microscopy. Transmission and high voltage electron microscopy of chick embryo corneas each demonstrated a series of unique extracellular compartments. Collagen fibrillogenesis occurred within small surface recesses. These small recesses usually contained between 5 and 12 collagen fibrils with typically mature diameters and constant intrafibrillar spacing. The lateral fusion of the recesses resulted in larger recesses and consequent formation of prominent cell surface foldings. Within these surface foldings, bundles that contained 50-100 collagen fibrils were formed. The surface foldings continued to fuse and the cell surface retracted, forming large surface-associated compartments in which bundles coalesced to form lamellae. High voltage electron microscopy of 0.5 micron sections cut parallel to the corneal surface revealed that the corneal fibroblasts and their processes had two major axes at approximately right angles to one another. The surface compartments involved in the production of the corneal stroma were aligned along the fibroblast axes and the orthogonality of the cell was in register with that of the extracellular matrix. In this manner, corneal fibroblasts formed collagen fibrils, bundles, and lamellae within a controlled environment and thereby determined the architecture of the corneal stroma by the configuration of the cell and its associated compartments.


Investigation of cyclooxygenase and signaling pathways involved in human platelet aggregation mediated by synergistic interaction of various agonists.
In the present study, the mechanism(s) of synergistic interaction of various platelet mediators such as arachidonic acid (AA) when combined with 5-hydroxytryptamine (5-HT) or adenosine diphosphate (ADP) on human platelet aggregation were examined. The results demonstrated that 5-HT had no or negligible effect on aggregation but it did potentiate the aggregation response of AA. Similarly, the combination of subeffective concentrations of ADP and AA exhibited noticeable rise in platelet aggregation. Moreover, the observed synergistic effect of AA with 5-HT on platelets was inhibited by different cyclooxygenase (COX) inhibitors, namely ibuprofen and celecoxib, with half maximal inhibitory effect (IC50) values of 18.0 ± 1.8 and 15.6 ± 3.4 μmol/L, respectively. Interestingly, the synergistic effect observed for AA with 5-HT was, also, blocked by the 5-HT receptor blockers cyproheptadine (IC50=22.0 ± 7 μmol/L), ketanserin (IC50=152 ± 23 μmol/L), phospholipase C (PLC) inhibitor (U73122; IC50=6.1 ± 0.8 μmol/L), and mitogen activated protein kinase (MAPK) inhibitor (PD98059; IC50=3.8 ± 0.5 μmol/L). Likewise, the synergism of AA and ADP was, also, attenuated by COX inhibitors (ibuprofen; IC50=20 ± 4 μmol/L and celecoxib; IC50=24 ± 7 μmol/L), PLC inhibitor (U73122; IC50=3.7 ± 0.3 μmol/L), and MAPK inhibitor (PD98059; IC50=2.8 ± 1.1 μmol/L). Our observed data demonstrate that the combination of subthreshold concentrations of agonists amplifies platelet aggregation and that these synergistic effects largely depend on activation of COX/thromboxane A2, receptor-operated Ca(2+) channels, Gq/PLC, and MAPK signaling pathways. Moreover, our data revealed that inhibition of COX pathways by using both selective and/or non-selective COX inhibitors blocks not only AA metabolism and thromboxane A2 formation, but also its binding to Gq receptors and activation of receptor-operated Ca(2+) channels in platelets. Overall, our results show that PLC and MAPK inhibitors proved to inhibit the synergistic activation of platelets by several/multiple agonists.


Osseointegration of a 3D Printed Stemmed Titanium Dental Implant: A Pilot Study.
In this pilot study, a 3D printed Grade V titanium dental implant with a novel dual-stemmed design was investigated for its biocompatibility in vivo. Both dual-stemmed (n = 12) and conventional stainless steel conical (n = 4) implants were inserted into the tibial metaphysis of New Zealand white rabbits for 3 and 12 weeks and then retrieved with the surrounding bone, fixed, dehydrated, and embedded into epoxy resin. The implants were analyzed using correlative histology, microcomputed tomography, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The histological presence of multinucleated osteoclasts and cuboidal osteoblasts revealed active bone remodeling in the stemmed implant starting at 3 weeks and by 12 weeks in the conventional implant. Bone-implant contact values indicated that the stemmed implants supported bone growth along the implant from the coronal crest at both 3- and 12-week time periods and showed bone growth into microporosities of the 3D printed surface after 12 weeks. In some cases, new bone formation was noted in between the stems of the device. Conventional implants showed mechanical interlocking but did have indications of stress cracking and bone debris. This study demonstrates the comparable biocompatibility of these 3D printed stemmed implants in rabbits up to 12 weeks.


Tea catechins have dual effect on mast cell degranulation induced by compound 48/80.
Green tea catechins are emerging as one of the most efficient and safest ingredient in health promoting food. We investigated catechin's effects on intracellular ROS generation in mast cell activation and degranulation. Compound 48/80, receptor mimetic basic secretagogues for mast cell, induced ROS generation dose-dependently with bell-shaped degranulation pattern in canine cutaneous mastocytoma cells (CM-MC). When intracellular ROS level was relatively low, catechins decreased both ROS and the degranulation. However, when intracellular ROS level was remarkably high, catechins decreased ROS level but increased the degranulation paradoxically. Gallocatechins showed the stronger effects than non-gallated catechins. Exogenous H(2)O(2) also shows dual effect on degranulation dose-dependently. EGCG shows the dual effect on the tyrosine and threonine phosphorylation depending on the concentration of compound 48/80. Particularly, 60 kDa protein tyrosine-phosphorylated by EGCG with 3 microg/ml of compound 48/80 might be a negative regulator for the degranulation. Taken together, there is an optimal level of ROS for the degranulation, and the catechins have a dual function by controlling ROS level.


Safety of transvenous atrial defibrillation: studies in the canine sterile pericarditis model.
It is recognized that a ventricular vulnerability period exists during which atrial shock delivery may induce a ventricular tachyarrhythmia. This study was designed to define the zone in which the ventricles are vulnerable to induction of ventricular tachyarrhythmia during delivery of atrial shocks in the sterile pericarditis canine model of atrial fibrillation. Two days after creation of sterile pericarditis, 24 dogs underwent either a four-part or five-part ventricular vulnerability protocol during which atrial shocks were delivered between transvenous catheters, one in the distal coronary sinus and one in the right atrial appendage. The protocol included part 1, shocks during induced atrial fibrillation; parts 2 through 4, shocks delivered synchronously with the last ventricular beat of one of the following three ventricular pacing protocols: constant ventricular rates (S1S1), short-long-short cycles (S1S2S3-V), and ventricular premature beats (S1); and part 5, shocks delivered synchronously with the last R wave resulting from an atrially paced short-long-short cycle (S1S2S3-A). Ventricular tachyarrhythmia was induced 122 times: 2 of 665 shocks in two dogs in part 1, 29 of 786 shocks in nine dogs in part 2, 67 of 734 shocks in 15 dogs in part 3, 24 of 919 shocks in five dogs in part 4, and none in part 5. All ventricular proarrhythmia resulted from shocks delivered during the T wave of a preceding ventricular beat. No episodes of ventricular tachyarrhythmia were induced by atrial shocks synchronized to R waves with the previous RR at intervals above the QT+60 ms interval (absolute interval >320 ms), with one exception, at the QT+100 ms interval (absolute interval 360 ms). With transvenous electrode catheters used to deliver atrial shocks, life-threatening ventricular rhythms were induced but were limited to a specific zone defined by the QT interval.


Hearing impairment in essential tremor.
To assess hearing in patients with essential tremor (ET) vs patients with Parkinson disease (PD) and normal controls. The authors assessed demographic and clinical information including use of hearing aids in 250 patients with ET, 127 patients with PD, and 127 normal controls. The authors administered the Nursing Home Hearing Handicap Index (NHHI), a validated measure of hearing disability. Regression techniques were used to adjust for factors such as age and sex. The authors assessed a complete audiologic evaluation in a subset of patients with ET. Patients with ET had worse adjusted NHHI scores when compared to patients with PD (p < 0.001), controls (p < 0.001), and both (p < 0.001). A higher percentage of patients with ET also used hearing aids (p < 0.0001). In the ET group, hearing loss was associated with tremor severity (p = 0.02) and tended to be associated with older age (p = 0.06), male sex (p = 0.06), and the absence of dystonia (p = 0.18). Audiology testing was consistent with high-frequency sensorineural hearing loss. Central processing was not disproportionally greater than peripheral loss. Patients with ET have increased hearing disability compared to patients with PD and normal controls, which correlates with tremor severity.


Opiate self-administration as a measure of chronic nociceptive pain in arthritic rats.
The study examined the validity of oral fentanyl self-administration (FSA) as a measure of the chronic nociceptive pain that develops in rats with adjuvant arthritis independently of acute noxious challenges. Arthritic rats self-administered more of a 0.008 mg/ml fentanyl solution (up to 3.4 g/rat per day) than non-arthritic controls (0.5 g/rat per day) and did so with a biphasic time course that reached peak during weeks 3 and 4 after inoculation with Mycobacterium butyricum. The time course paralleled both the disease process and the chronic pain. Continuous infusion of dexamethasone during weeks 3 and 4 via subcutaneous osmotic pumps at 0.0025-0.04 mg/rat per day disrupted the arthritic disease and decreased FSA to a level (i.e. by 65%) similar to that observed in non-arthritic rats. Continuous naloxone (2.5 mg/rat per day) decreased FSA (by 55%) in arthritic but not in non-arthritic animals. Continuous, subcutaneous infusion of fentanyl also decreased arthritic FSA in a manner that varied with dose at 0.04-0.16 mg/rat per day doses, but leveled off at 47% of controls with 0.31 mg/rat per day. The effects of continuous fentanyl on arthritic FSA occurred only with those doses and dose-dependent dynamics with which fentanyl also induced dependence in non-arthritic rats. The findings indicate that pain, rather than the rewarding or dependence-inducing action of fentanyl mediates FSA in arthritic rats. Paralleling patient-controlled analgesic drug intake, FSA offers a specific measure allowing the dynamic effects of neurobiological agents to be studied in this unique animal model of persistent nociceptive pain.


The partially diabetic pancreas: a histological study of a new animal model.
An animal model in which part of the pancreas was made diabetic due to almost total loss of insulin-secreting B cells, while the remainder of the gland remained normal, is described. In rabbits, a vascular clamp was placed across the junction of the body and tail of the pancreas, thus occluding the circulation to the tail. Alloxan (200 mg/kg) was injected i.v., and 4 min later dextrose (0.5 g/kg) was given by the same route. After a further 2 min the clamp was removed. Thirty-four animals were studied, 17 of which died in the first postoperative week of surgical complications or of alloxan-induced toxicity to the liver and kidneys. The survivors were killed between 4 and 12 weeks after surgery and were not metabolically diabetic. They had a virtually complete absence of B cells but a normal population of A, D, and PP cells in the head and body of the pancreas. The islets in the tail of the pancreas appeared entirely normal. This model appears suitable for studying the effects of locally produced insulin on pancreatic exocrine function in metabolically normal animals.


[Guidelines for treatment of missing anterior teeth].
The orthodontic treatment of patients with missing incisors should be directed to avoid prosthetic solutions. Specific rules have to be applied for the closure of spaces due to missing teeth to reduce the occurrence of papillae retraction and to place the substituting teeth in such a position that their crowns can be built up to look like the ones they replace. To realize these goals the mesiodistal angulations should deviate from the standard ones, a proper distribution of crown width should be achieved and finally a permanent retention with a palatally bonded thin multi-stranded wire should be installed in most cases.


Risk factors and predictors of carbapenem-resistant Pseudomonas aeruginosa and Acinetobacter baumannii mortality in critically ill bacteraemic patients over a 6-year period (2010-15): antibiotics do matter.
Purpose. Acinetobacter baumannii and Pseudomonas aeruginosa provoke serious infections, especially in intensive care unit (ICU) patients.Methodology. The risk factors and predictors of mortality for P. aeruginosa (n=84; 46 carbapenem-resistant) and A. baumannii (n=129; all carbapenem-resistant) bloodstream infections (BSIs) in an ICU were evaluated. Antibiotic susceptibility testing was performed using the agar disk diffusion method according to EUCAST guidelines. The minimum inhibitory concentration was determined by a gradient method (Etest). Multilocus sequence typing (MLST) was performed for P. aeruginosa during the carbapenem-resistant outbreak in 2014. Epidemiological data were collected from the patients' chart reviews.Results/Key findings. Hospitalization during the summer months, prior KPC-producing Klebsiella pneumoniae (KPC-Kp) BSI, and the administration of tigecycline, aminoglycosides and cortisone were independently associated with P. aeruginosa BSIs. MLST revealed the dissemination of clone ST227, including carbapenem-resistant P. aeruginosa strains. Hospitalization during the summer months, prior KPC-Kp BSI, and the administration of antibiotics, carbapenem and cortisone were independently associated with A. baumannii BSIs. The 30-day mortality rate for P. aeruginosa and A. baumannii BSI was 45.2 and 39.5 %, respectively. Sequential organ failure assessment (SOFA) score at onset, septic shock, age, and prior KPC-Kp BSI were significantly associated with P. aeruginosa BSI mortality. The administration of at least one active antibiotic was identified as a predictor of a good prognosis. Septic shock and simplified acute physiology score (SAPS) II at onset were independently associated with A. baumannii BSI mortality. The administration of at least one active antibiotic and colistin-vancomycin co-administration were identified as predictors of a good prognosis.Conclusion. KPC-Kp infection predisposes ICU patients to BSI by either A. baumannii or P. aeruginosa. The administration of at least one active antibiotic leads to better survival rates.


Resolution of internal carotid dissection with middle cerebral artery occlusion in pregnancy.
Introduction. Cervical artery dissection (CAD) is a common cause of stroke in younger patients. While the incidence of stroke in pregnancy is increasing, CAD remains a rare cause of ischemic stroke in the pregnant population, with only 30 cases described in the literature, most in the postpartum period. Methods. The case of a pregnant patient at 18 weeks of gestation presenting with CAD and ischemic stroke following intercourse is discussed. Discussion. CAD results from an intimal tear in the carotid artery, allowing accumulation of blood in the vessel wall. Stroke results from embolization of thrombogenic material in the wall. Etiology includes minor trauma, connective tissue disorders, or anatomic variations of the carotid artery. Most patients present with headache and/or neck pain, while ischemic symptoms are seen in at least 50% of patients. In the pregnant population, imaging with MRI or MRA of the head and neck aids in diagnosis. Once the diagnosis is made, patients are treated with either anticoagulation or antiplatelet medications. The optimal treatment in both pregnant and nonpregnant patients has not been well-studied. Conclusion. CAD is an important diagnosis to consider in a pregnant patient with persistent headache, especially if neurological symptoms are present. Imaging should be quickly obtained so treatment can be initiated.


Energy density of the diets of Japanese adults in relation to food and nutrient intake and general and abdominal obesity: a cross-sectional analysis from the 2012 National Health and Nutrition Survey, Japan.
The associations of dietary energy density with dietary intake and obesity have been largely unexplored in non-Western populations. The present cross-sectional study examined the associations using data from the 2012 National Health and Nutrition Survey, Japan. Dietary intake was assessed using a 1-d semi-weighed dietary record in 15 618 Japanese adults aged ≥20 years. Mean dietary energy density (calculated on the basis of foods only) was 5·98 (sd 1·20) kJ/g in men and 5·72 (sd 1·16) kJ/g in women. Dietary energy density was positively associated with intakes of bread, noodles (only men), meat, fats and oils, and sugar and confectionery but inversely with intakes of white rice (only men), potatoes, pulses, vegetables, fruits, and fish and shellfish. For nutrient intake, dietary energy density was positively associated with total fat and SFA but inversely associated with all other nutrients examined such as protein, carbohydrate, alcohol (only women), dietary fibre, and several vitamins and minerals, including Na. After adjustment for potential confounding factors, dietary energy density was positively associated with abdominal obesity (waist circumference ≥80 cm) in women (adjusted prevalence ratio between the extreme tertiles 1·07; 95 % CI 1·02, 1·12; P for trend=0·003). Dietary energy density was also positively but non-significantly associated with general obesity (BMI≥25 kg/m2) in women (P for trend=0·08). There were no such associations in men. In conclusion, lower energy density of the diets of Japanese adults was associated with favourable food and nutrient intake patterns, except for higher Na, and, in only women, a lower prevalence of abdominal obesity.


The Link4Health study to evaluate the effectiveness of a combination intervention strategy for linkage to and retention in HIV care in Swaziland: protocol for a cluster randomized trial.
Gaps in the HIV care continuum contribute to suboptimal individual health outcomes and increased risk of HIV transmission at the population level. Implementation science studies are needed to evaluate clinic-based interventions aimed at improving retention of patients across the continuum. Link4Health uses an unblended cluster site-randomized design to evaluate the effectiveness of a combination intervention strategy (CIS) as compared to standard of care on linkage to and retention in care among HIV-diagnosed adults in Swaziland. The CIS intervention targets a multiplicity of structural, behavioral, and biomedical barriers through five interventions: (1) point-of-care CD4 testing at time of HIV testing, (2) accelerated antiretroviral therapy (ART) initiation for eligible patients, (3) mobile phone appointment reminders, (4) care and prevention packages, and (5) non-cash financial incentives for linkage and retention. The unit of randomization is a network of HIV clinics inclusive of a secondary facility coupled with an affiliated primary facility. Ten study units were randomized based on implementing partner, geographic location, and historic volume of HIV patients. Target enrollment was 2200 individuals, each to be followed for 12 months. Eligibility criteria includes HIV-positive test, age >18 years, willing to receive HIV care at a clinic in the study unit and consent to study procedures. Exclusion criteria included previous HIV care in the past 6 months, planning to leave the community, and current pregnancy. The primary study outcome is linkage within 1 month and retention at 12 months after testing HIV positive. Secondary outcomes include viral load suppression at 12 months, time to ART eligibility and initiation, participant acceptability, and cost-effectiveness. The trial status is that study enrollment is complete and follow-up procedures are ongoing. Link4Health evaluates a novel and pragmatic combination intervention strategy to improve linkage to and retention in care among adults with HIV in Swaziland. If the strategy is found to be effective, this study has the potential to inform HIV service delivery in resource-limited settings. Clinicaltrials.gov NCT01904994.


Oral-facial-digital syndrome VII is oral-facial-digital syndrome I: a clarification.
We report on further clinical findings in the one single family in the literature classified as oral-facial-digital (OFD) type VII in order to demonstrate that the diagnosis in this kindred should, in fact, be OFD type I. The mother and the daughter described in the original report have since developed polycystic kidney disease. In addition, the daughter recently had a daughter of her own with central nervous system, oral and digital anomalies. Linkage studies have shown that all the affected women share the same haplotype across the previously identified region Xp22.2p22.3 to which OFD I maps. Although the pedigree was too small for a significant lod score, the combination of clinical and molecular information clearly shows that the disease in this family is OFD I. We report this family in order to clarify and simplify the classification of the oral-facial-digital syndrome spectrum and to recommend the removal of OFD VII from the classification system of the oral-facial-digital syndromes.


Healthcare reform in the USA, 2010: the rocky road ahead?
Historic legislation for healthcare reform in the United States was enacted in March 2010. Reforms in medical practice, payment for services, and access to care and insurance will be introduced by complex processes over time through 2019. The overriding goals of healthcare reform are cost containment and guaranteeing access to all Americans. The contentious political struggle that preceded the legislation is emblematic of the continuous struggle in American society to define who is worthy of services. Understanding the value framework for social and welfare provisions in American society is crucial to making sense of the piecemeal policy making characteristic of the development of healthcare over the past 50 years. Here some highlights of the reform and the complex organisation of American healthcare are discussed.


The effect of panretinal photocoagulation on confocal laser scanning ophthalmoscopy and stereo photographic parameters of optic disk topography in patients with diabetic retinopathy.
To determine the effect of panretinal photocoagulation on optic disk topographic parameters in non-glaucomatous patients with proliferative diabetic retinopathy. This was a prospective, single-center, observational study. Thirty-eight eyes of 26 patients with diabetes underwent panretinal photocoagulation for proliferative diabetic retinopathy. Stereoscopic disk photographs and optic nerve head parameters were evaluated using the Zeiss fundus camera and the confocal scanning laser ophthalmoscope (Heidelberg Retinal Tomograph), respectively, at baseline and 12 months after the completion of panretinal photocoagulation. Thirty-eight eyes of 26 patients (15 female) with a mean age of 53.7 (range 26-74) years were recruited. No significant difference was found between the stereo photography determined mean horizontal and vertical cup-to-disk ratio before and after panretinal photocoagulation treatment (p=0.461 and 0.839, respectively). The global values of the optic nerve head parameters analyzed with the HRT3 showed no significant change from baseline to 12 months, including the disk area, cup area, rim area, cup volume, rim volume, cup-to-disk area ratio, linear cup-to-disk ratio, mean cup depth, maximum cup depth, cup shape measure, height variation contour, mean retinal nerve fiber layer thickness, and cross-sectional area. Our results suggest that panretinal photocoagulation does not cause morphological optic disk changes in patients with diabetic proliferative retinopathy after 1 year of follow-up.


Identification of optimal measurement sets for complete flux elucidation in metabolic flux analysis experiments.
Metabolic flux analysis (MFA) methods use external flux and isotopic measurements to quantify the magnitude of metabolic flows in metabolic networks. A key question in this analysis is choosing a set of measurements that is capable of yielding a unique flux distribution (identifiability). In this article, we introduce an optimization-based framework that uses incidence structure analysis to determine the smallest (or most cost-effective) set of measurements leading to complete flux elucidation. This approach relies on an integer linear programming formulation OptMeas that allows for the measurement of external fluxes and the complete (or partial) enumeration of the isotope forms of metabolites without requiring any of these to be chosen in advance. We subsequently query and refine the measurement sets suggested by OptMeas for identifiability and optimality. OptMeas is first tested on small to medium-size demonstration examples. It is subsequently applied to a large-scale E. coli isotopomer mapping model with more than 17,000 isotopomers. A number of additional measurements are identified leading to maximum flux elucidation in an amorphadiene producing E. coli strain.


Early Head Tremor in Essential Tremor: A Case Series and Commentary.
Classically, the onset of head tremor in essential tremor (ET) patients follows that of hand tremor, such that there is a somatotopic spread of involved areas. Here we present a series of seven self-reportedly "unaffected" relatives of ET cases. These seven were clinically asymptomatic and had normal levels of arm tremor on examination, yet each evidenced a transient head wobble on examination. We estimate the prevalence of this phenotype within the two studies from which cases were ascertained. ET cases and their self-reportedly affected and unaffected relatives, enrolled in two family studies, underwent a medical history and videotaped neurological examination. In seven self-reportedly "unaffected" relatives, a transient and subtle head wobble was seen, always during sustained phonation, speech, or reading aloud. Total tremor score (a measure of arm tremor) ranged from 5 to 12 (i.e., mild tremor within the range of normal). The prevalence of this phenotype of early head tremor was 3.7% in one study and 23.1% in the other. We present a series of seven individuals who had early head tremor in an evolving transition state from normal to ET. These cases raise a number of broad clinical, phenotypic, and pathophysiological issues about ET.


Role of health-related quality of life measures in the routine care of people with multiple sclerosis.
Health-related quality of life instruments are expected to be of particular value in routine care of people with multiple sclerosis (MS), where they may facilitate the detection of disease aspects that would otherwise go unrecognised, help clinicians appreciate patient priorities particularly in terms of treatment goals, facilitate physician-patient communication, and promote shared decision-making. However, it appears that these instruments are little used routine clinical approaches to people with MS. To address this issue, I performed a bibliographic search of studies that evaluated the efficacy of generic or disease-specific health-related quality of life (HRQOL) instruments in MS clinical practice from clinicians' or patients' perspectives. I found only one cross-sectional study, which compared preferences for three instruments, and assessed acceptability in people with MS. Reasons for lack of transfer of HRQOL measurements to clinical practice may be cultural, methodological, or practical. With regard to MS, the proliferation of instruments seems to constitute a barrier, with no particular instrument having gained wide popularity or consensus. Other barriers are lack of resources for the administration, collection and storage of the data, and inability of clinicians to score, interpret, and use HRQOL instrument to guide clinical care. It is therefore important to refine existing tools, extending clinical validation to wider contexts and cultures. More studies assessing acceptability and clinicians' and patients' preferences for different instruments are also required.


Imaging of fistula in ano.
Fistula in ano is a common condition that often recurs despite seemingly adequate surgery, usually because of infection that was missed at surgery. It is now increasingly recognized that preoperative imaging can help identify infection that would have otherwise gone unidentified. In particular, magnetic resonance (MR) imaging findings have been shown to influence surgery and markedly diminish the chance of recurrence; thus, preoperative imaging will become increasingly routine in the future. In this article, the authors describe the pathogenesis, classification, and imaging of fistula in ano, with an emphasis on MR imaging. Most important, the authors describe how the radiologist is well placed to answer the surgical riddles that must be solved for treatment to be effective.


Induction of monocyte chemoattractant protein-1 and interleukin-10 by TGFbeta1 in melanoma enhances tumor infiltration and immunosuppression.
Melanoma progression is associated with the expression of different growth factors, cytokines, and chemokines. Because TGFβ1 is a pleiotropic cytokine involved not only in physiologic processes but also in cancer development, we analyzed in A375 human melanoma cells, the effect of TGFβ1 on monocyte chemoattractant protein-1 (MCP-1) and interleukin-10 (IL-10) expression, two known factors responsible for melanoma progression. TGFβ1 increased the expression of MCP-1 and IL-10 in A375 cells, an effect mediated by the cross-talk between Smad, PI3K (phosphoinositide 3-kinase)/AKT, and BRAF-MAPK (mitogen activated protein kinase) signaling pathways. Supernatants from TGFβ1-treated A375 cells enhanced MCP-1-dependent migration of monocytes, which, in turn, expressed high levels of TGF,β1, bFGF, and VEGF mRNA. Moreover, these supernatants also inhibited functional properties of dendritic cells through IL-10-dependent mechanisms. When using in vitro, the TGFβ1-blocking peptide P144, TGFβ1-dependent Smad3 phosphorylation, and expression of MCP-1 and IL-10 were inhibited. In vivo, treatment of A375 tumor-bearing athymic mice with P144 significantly reduced tumor growth, associated with a lower macrophage infiltrate and decreased intratumor MCP-1 and VEGF levels, as well as angiogenesis. Finally, in C57BL/6 mice with B16-OVA melanoma tumors, when administered with immunotherapy, P144 decreased tumor growth and intratumor IL-10 levels, linked to enhanced activation of dendritic cells and natural killer cells, as well as anti-OVA T-cell responses. These results show new effects of TGFβ1 on melanoma cells, which promote tumor progression and immunosuppression, strongly reinforcing the relevance of this cytokine as a molecular target in melanoma.


Comparative analysis of PacBio and ONT RNA sequencing methods for Nemopilema Nomurai venom identification.
Recent studies on marine organisms have made use of third-generation sequencing technologies such as Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT). While these specialized bioinformatics tools have different algorithmic designs and performance capabilities, they offer scalability and can be applied to various datasets. We investigated the effectiveness of PacBio and ONT RNA sequencing methods in identifying the venom of the jellyfish species Nemopilema nomurai. We conducted a detailed analysis of the sequencing data from both methods, focusing on key characteristics such as CD, alternative splicing, long-chain noncoding RNA, simple sequence repeat, transcription factor, and functional transcript annotation. Our findings indicate that ONT generally produced higher raw data quality in the transcriptome analysis, while PacBio generated longer read lengths. PacBio was found to be superior in identifying CDs and long-chain noncoding RNA, whereas ONT was more cost-effective for predicting alternative splicing events, simple sequence repeats, and transcription factors. Based on these results, we conclude that PacBio is the most specific and sensitive method for identifying venom components, while ONT is the most cost-effective method for studying venogenesis, cnidocyst (venom gland) development, and transcription of virulence genes in jellyfish. Our study has implications for future sequencing technologies in marine jellyfish, and highlights the power of full-length transcriptome analysis in discovering potential therapeutic targets for jellyfish dermatitis.


Why do so many petals have conical epidermal cells?
The conical epidermal cells found on the petals of most Angiosperm species are so widespread that they have been used as markers of petal identity, but their function has only been analysed in recent years. This review brings together diverse data on the role of these cells in pollination biology. The published effects of conical cells on petal colour, petal reflexing, scent production, petal wettability and pollinator grip on the flower surface are considered. Of these factors, pollinator grip has been shown to be of most significance in the well-studied Antirrhinum majus/bumble-bee system. Published data on the relationship between epidermal cell morphology and floral temperature were limited, so an analysis of the effects of cell shape on floral temperature in Antirrhinum is presented here. Statistically significant warming by conical cells was not detected, although insignificant trends towards faster warming at dawn were found, and it was also found that flat-celled flowers could be warmer on warm days. The warming observed is less significant than that achieved by varying pigment content. However, the possibility that the effect of conical cells on temperature might be biologically significant in certain specific instances such as marginal habitats or weather conditions cannot be ruled out. Conical epidermal cells can influence a diverse set of petal properties. The fitness benefits they provide to plants are likely to vary with pollinator and habitat, and models are now required to understand how these different factors interact.


The developmental high wire: Balancing resource investment in immunity and reproduction.
The strategic allocation of resources into immunity poses a unique challenge for individuals, where infection at different stages of development may result in unique trade-offs with concurrent physiological processes or future fitness-enhancing traits. Here, we experimentally induced an immune challenge in female Gryllus firmus crickets to test whether illness at discrete life stages differentially impacts fitness. We injected heat-killed Serratia marcescens bacteria into antepenultimate juveniles, penultimate juveniles, sexually immature adults, and sexually mature adults, and then measured body growth, instar duration, mating rate, viability of stored sperm, egg production, oviposition rate, and egg viability. Immune activation significantly impacted reproductive traits, where females that were immune challenged as adults had decreased mating success and decreased egg viability compared to healthy individuals or females that were immune challenged as juveniles. Although there was no effect of an immune challenge on the other traits measured, the stress of handling resulted in reduced mass gain and smaller adult body size in females from the juvenile treatments, and females in the adult treatments suffered from reduced viability of sperm stored within their spermatheca. In summary, we found that an immune challenge does have negative impacts on reproduction, but also that even minor acute stressors can have significant impacts on fitness-enhancing traits. These findings highlight that the factors affecting fitness can be complex and at times unpredictable, and that the consequences of illness are specific to when during an individual's life an immune challenge is induced.


A compact and high sensitivity positron detector using dual-layer thin GSO scintillators for a small animal PET blood sampling system.
For quantitative measurements of small animals such as mice or rats, a compact and high sensitivity continuous blood sampling detector is required because their blood sampling volume is limited. For this purpose we have developed and tested a new positron detector. The positron detector uses a pair of dual-layer thin gadolinium orthosilicate (GSO) scintillators with different decay times. The front layer detects the positron and the background gamma photons, and the back layer detects the background gamma photons. By subtracting the count rate of the latter from that of the former, the count rate of the positrons can be estimated. The GSO for the front layer has a Ce concentration of 1.5 mol% (decay time of 35 ns), and that for the back layer has a Ce concentration of 0.5 mol% (decay time of 60 ns). By using the pulse shape analysis, the count rate of these two GSOs can be discriminated. The thickness is 0.5 mm, which is thick enough to detect positrons while minimizing the detection of the background gamma photons. These two types of thin GSOs were optically coupled to each other and connected to a metal photomultiplier tube (PMT) through triangular light guides. The signal from the PMT was digitized by 100 MHz free-running A-D converters in the data acquisition system and digitally integrated at two different integration times for the pulse shape analysis. We obtained good separation of the pulse shape distributions of these two GSOs. The energy threshold level was decreased to 80 keV, increasing the sensitivity of the detector. The sensitivity of a small diameter plastic tube was 8.6% and 24% for the F-18 and C-11 positrons, respectively. The count rate performance was linear up to approximately 50 kcps. The background counts from the gamma photons could be precisely corrected. The time-activity curve (TAC) of the rat artery blood was successfully obtained and showed a good correlation with that measured using a well counter. With these results, we confirmed that the developed blood sampling detector is promising for quantitative measurement for an animal positron emission tomography system.


Improving signal and transit peptide predictions using AlphaFold2-predicted protein structures.
Many proteins contain cleavable signal or transit peptides that direct them to their final subcellular locations. Such peptides are usually predicted from sequence alone using methods such as TargetP 2.0 and SignalP 6.0. While these methods are usually very accurate, we show here that an analysis of a protein's AlphaFold2-predicted structure can often be used to identify false positive predictions. We start by showing that when given a protein's full-length sequence, AlphaFold2 builds experimentally annotated signal and transit peptides in orientations that point away from the main body of the protein. This indicates that AlphaFold2 correctly identifies that a signal is not destined to be part of the mature protein's structure and suggests, as a corollary, that predicted signals that AlphaFold2 folds with high confidence into the main body of the protein are likely to be false positives. To explore this idea, we analyzed predicted signal peptides in 48 proteomes made available in DeepMind's AlphaFold2 database (https://alphafold.ebi.ac.uk). Applying TargetP 2.0 and SignalP 6.0 to the 561,562 proteins in the database results in 95,236 being predicted to contain a cleavable signal or transit peptide. In 95.1% of these cases, the AlphaFold2 structure of the full-length protein is fully consistent with the prediction of TargetP 2.0 or SignalP 6.0. In the remaining 4.9% of cases where the AlphaFold2 structure does not appear consistent with the prediction, the signal is often only predicted with low confidence. The potential false positives identified here may be useful for training even more accurate signal prediction methods.


Stage of change for making an informed decision about Medicare health plans.
To assess the applicability of the transtheoretical model of change (TTM) to informed choice in the Medicare population. Two hundred and thirty-nine new Medicare enrollees randomly selected from the Center for Medicare and Medicaid Services' October 2001 Initial Enrollee File, a repository of data for persons who are going to turn 65 and become entitled to enroll in Medicare in the next 3 months. Study participants completed TTM measures of stage of change, decisional balance, and self-efficacy for informed choice, as well as measures of Medicare knowledge, perceived knowledge, and information seeking. Model testing was conducted to determine whether well-established relationships between stage of change, decisional balance, and self-efficacy replicate for informed choice in the Medicare population, and whether Medicare knowledge and information-seeking increase across the stages. Survey data were collected using mail surveys with telephone follow-up for nonresponders. Predicted relationships were established between stage of change for informed choice and decisional balance, self-efficacy, Medicare knowledge, and information seeking. The amount of variance accounted for by stage of change for informed choice was larger than that found for smoking cessation, where the TTM has had its greatest successes. The methods and findings lay the groundwork for development of TTM-based interventions for Medicare beneficiaries, and provide a prototype for the application of the TTM to informed decision making among other types of consumers who are being asked to take more responsibility for their health care.


Replication forks blocked by protein-DNA complexes have limited stability in vitro.
There are many barriers that replication forks must overcome in order to duplicate a genome in vivo. These barriers include damage to the template DNA and proteins bound to this template. If replication is halted by such a block, then the block must be either removed or bypassed for replication to continue. If continuation of replication employs the original fork, avoiding the need to reload the replication apparatus, then the blocked replisome must retain functionality. In vivo studies of Escherichia coli replication forks suggest that replication forks blocked by protein-DNA complexes retain the ability to resume replication upon removal of the block for several hours. Here we tested the functional stability of replication forks reconstituted in vitro and blocked by lac repressor-operator complexes. Once a fork comes to a halt at such a block, it cannot continue subsequently to translocate through the block until addition of IPTG induces repressor dissociation. However, the ability to resume replication is retained only for 4-6 min regardless of the topological state of the template DNA. Comparison of our in vitro data with previous in vivo data suggests that either accessory factors that stabilise blocked forks are present in vivo or the apparent stability of blocked forks in vivo is due to continual reloading of the replication apparatus at the site of the block.


Number of somatic mutations in the mitochondrial D-loop region indicates poor prognosis in breast cancer, independent of TP53 mutation.
The objective of this study was to investigate whether somatic mutations in the mitochondrial DNA (mtDNA) D-loop region correlate with known prognostic factors, namely, age, tumor size, lymph node status, metastasis, tumor-node-metastasis stage, lymphovascular invasion, and status of the progesterone receptor, estrogen receptor, ERBB2 (alias HER2/neu), and TP53 proteins (as determined by immunohistochemistry) and to investigate their relationship, if any, to TP53 mutations in human breast cancer. Thirty breast tumors without BRCA mutation, along with adjacent nontumorous tissues, were genotyped for the mtDNA D-loop region and for the promoter as well as the coding region of the TP53 gene. Clinicopathological parameters were recorded and assessed. In all, 17 somatic mtDNA D-loop mutations were identified, in 13 of 30 tumor samples (43%); two mutations were novel: 544C>T and 16510A>C. Four TP53 mutations were found in six tumor samples (20%), and two (c.437G>A and c.706T>C) were novel. Only progesterone receptor status correlated with the number of somatic mtDNA D-loop mutations (likelihood chi-square test; P < 0.05). Somatic mutations in the mtDNA D-loop and in TP53 were independent of each other (Fisher's exact test; P > 0.05). These results suggest that the number of somatic mtDNA D-loop mutations may be an indicator of poor prognosis through a mechanism independent of TP53.