Fragmentation of Human Neutrophil α-Defensin 4 to Combat Multidrug Resistant Bacteria.
The occurrence and spread of multidrug-resistant bacteria is a prominent health concern. To curb this urgent threat, new innovative strategies pursuing novel antimicrobial agents are of the utmost importance. Here, we unleashed the antimicrobial activity of human neutrophil peptide-4 (HNP-4) by tryptic digestion. We identified a single 11 amino acid long fragment (HNP-41 - 11) with remarkable antimicrobial potential, exceeding that of the full length peptide on both mass and molar levels. Importantly, HNP-41 - 11 was equally bactericidal against multidrug-resistant and non-resistant strains; a potency that was further enhanced by N- and C-terminus modifications (acetylation and amidation, respectively). These observations, combined with negligible cytotoxicity not exceeding that of the full length peptide, presents proteolytic digestion of innate host-defense-peptides as a novel strategy to overcome the current health crisis related to antibiotic-resistant bacteria.


The relationship between the size of a muscle afferent volley and the cerebral potential it produces.
This study examined the relationship between the size of an afferent neural input produced by electrical stimulation of the posterior tibial nerve at the ankle and the size of the early components of the evoked cerebral potential. For five of six subjects the first peak of the afferent neural volley recorded in the popliteal fossa was uncontaminated by either motor efferents or cutaneous afferents. This was established by measuring the conduction times of motor fibres in the posterior tibial nerve and cutaneous fibres in the sural and posterior tibial nerves over the ankle to popliteal fossa segment. It is likely therefore that the first peak of the afferent volley contained predominantly, if not exclusively, activity in rapidly conducting afferents from the small muscles of the foot. The size of the two earliest components of the cerebral potential did not increase in direct proportion to the size of the afferent volley which produced it. The early components of the cerebral potential reached a maximum when the responsible muscle afferent volley was less than 50% of its maximum.


MicroRNA profiling of human primary macrophages exposed to dengue virus identifies miRNA-3614-5p as antiviral and regulator of ADAR1 expression.
Due to the high burden of dengue disease worldwide, a better understanding of the interactions between dengue virus (DENV) and its human host cells is of the utmost importance. Although microRNAs modulate the outcome of several viral infections, their contribution to DENV replication is poorly understood. We investigated the microRNA expression profile of primary human macrophages challenged with DENV and deciphered the contribution of microRNAs to infection. To this end, human primary macrophages were challenged with GFP-expressing DENV and sorted to differentiate between truly infected cells (DENV-positive) and DENV-exposed but non-infected cells (DENV-negative cells). The miRNAome was determined by small RNA-Seq analysis and the effect of differentially expressed microRNAs on DENV yield was examined. Five microRNAs were differentially expressed in human macrophages challenged with DENV. Of these, miR-3614-5p was found upregulated in DENV-negative cells and its overexpression reduced DENV infectivity. The cellular targets of miR-3614-5p were identified by liquid chromatography/mass spectrometry and western blot. Adenosine deaminase acting on RNA 1 (ADAR1) was identified as one of the targets of miR-3614-5p and was shown to promote DENV infectivity at early time points post-infection. Overall, miRNAs appear to play a limited role in DENV replication in primary human macrophages. The miRNAs that were found upregulated in DENV-infected cells did not control the production of infectious virus particles. On the other hand, miR-3614-5p, which was upregulated in DENV-negative macrophages, reduced DENV infectivity and regulated ADAR1 expression, a protein that facilitates viral replication.


MutS recognition of exocyclic DNA adducts that are endogenous products of lipid oxidation.
The ability of the methyl-directed mismatch repair system to recognize and repair the exocyclic adducts propanodeoxyguanosine (PdG) and pyrimido[1,2-alpha]purin-10(3H)-one (M(1)G), the major adduct derived from the endogenous mutagen malondialdehyde, has been assessed both in vivo and in vitro. Both adducts were site-specifically incorporated into M13MB102 DNA, and the adducted genomes were electroporated into wild-type or mutS-deficient Escherichia coli strains. A decrease in mutations caused by both adducts was observed in mutS-deficient strains, suggesting that MutS was binding to the adducts and blocking repair by nucleotide excision repair. This hypothesis was supported by the differences in mutation frequency observed when hemimethylated genomes containing PdG on the (-)-strand were electroporated into a uvrA(-) strain. The ability of purified MutS to bind to PdG- or M(1)G-containing 31-mer duplexes in vitro was assessed using both surface plasmon resonance and gel shift assays. MutS bound to M(1)G:T-containing duplexes with similar affinity to a G:T mismatch but less strongly to M(1)G:C- and PdG-containing duplexes. Dissociation from each of the adduct-containing duplexes occurred at a faster rate than from a G:T mismatch. The present results indicate that MutS can bind to exocyclic adducts resulting from endogenous DNA damage and trigger their removal by mismatch repair or protect them from removal by nucleotide excision repair.


Safety Assessment of PEG-150 Pentaerythrityl Tetrastearate as Used in Cosmetics.
The Cosmetic Ingredient Review Expert Panel (Panel) reviewed the safety of polyethylene glycol (PEG)-150 pentaerythrityl tetrastearate, which functions as a viscosity-increasing agent-aqueous in cosmetic products and is being used at concentrations up to 5%. Given the chemical structure (large molecule), skin penetration is not likely. The available toxicity data and the low ingredient use concentrations suggest that systemic toxicity would not be likely even if percutaneous absorption were to occur. Additionally, the negative human repeated insult patch test data on the undiluted ingredient were deemed sufficient for evaluating skin irritation and sensitization potential. The Panel concluded that PEG-150 pentaerythrityl tetrastearate is safe in cosmetics in the present practices of use and concentration described in this safety assessment.


The association between length of emergency department boarding and mortality.
Emergency department (ED) boarding has been associated with several negative patient-oriented outcomes, from worse satisfaction to higher inpatient mortality rates. The current study evaluates the association between length of ED boarding and outcomes. The authors expected that prolonged ED boarding of admitted patients would be associated with higher mortality rates and longer hospital lengths of stay (LOS). This was a retrospective cohort study set at a suburban academic ED with an annual ED census of 90,000 visits. Consecutive patients admitted to the hospital from the ED and discharged between October 2005 and September 2008 were included. An electronic medical record (EMR) system was used to extract patient demographics, ED disposition (discharge, admit to floor), ED and hospital LOS, and in-hospital mortality. Boarding was defined as ED LOS 2 hours or more after decision for admission. Descriptive statistics were used to evaluate the association between length of ED boarding and hospital LOS, subsequent transfer to an intensive care unit (ICU), and mortality controlling for comorbidities. There were 41,256 admissions from the ED. Mortality generally increased with increasing boarding time, from 2.5% in patients boarded less than 2 hours to 4.5% in patients boarding 12 hours or more (p < 0.001). Mean hospital LOS also showed an increase with boarding time (p < 0.001), from 5.6 days (SD ± 11.4 days) for those who stayed in the ED for less than 2 hours to 8.7 days (SD ± 16.3 days) for those who boarded for more than 24 hours. The increases were still apparent after adjustment for comorbid conditions and other factors. Hospital mortality and hospital LOS are associated with length of ED boarding.


Biochemical characterization of the Ran-RanBP1-RanGAP system: are RanBP proteins and the acidic tail of RanGAP required for the Ran-RanGAP GTPase reaction?
RanBP type proteins have been reported to increase the catalytic efficiency of the RanGAP-mediated GTPase reaction on Ran. Since the structure of the Ran-RanBP1-RanGAP complex showed RanBP1 to be located away from the active site, we reinvestigated the reaction using fluorescence spectroscopy under pre-steady-state conditions. We can show that RanBP1 indeed does not influence the rate-limiting step of the reaction, which is the cleavage of GTP and/or the release of product P(i). It does, however, influence the dynamics of the Ran-RanGAP interaction, its most dramatic effect being the 20-fold stimulation of the already very fast association reaction such that it is under diffusion control (4.5 x 10(8) M(-1) s(-1)). Having established a valuable kinetic system for the interaction analysis, we also found, in contrast to previous findings, that the highly conserved acidic C-terminal end of RanGAP is not required for the switch-off reaction. Rather, genetic experiments in Saccharomyces cerevisiae demonstrate a profound effect of the acidic tail on microtubule organization during mitosis. We propose that the acidic tail of RanGAP is required for a process during mitosis.


Role of vitamin D glucosiduronate in calcium homeostasis.
Evidence has been presented suggesting the presence of vitamin D(3) 3beta-glucosiduronate and 1,25-dihydroxyvitamin D(3) glucosiduronate in rat bile. To evaluate the role of vitamin D glucosiduronates in calcium and phosphorus homeostasis, we synthesized vitamin D(3) 3beta-glucosiduronate and tested its biological activity in calcium- and vitamin D-deficient rats. After the intravenous administration of vitamin D(3) 3beta-glucosiduronate to rats maintained on a low calcium diet, there was an increase in duodenal calcium transport and an increase in serum calcium. Vitamin D(3) 3beta-glucosiduronate, however, was less active than equimolar amounts of vitamin D(3). At doses of less than 0.65-1 nmol per rat, the conjugate exhibited no activity. When vitamin D(3) 3beta-glucosiduronate was administered to vitamin D-deficient rats, 25-hydroxyvitamin D was detected in the serum; the increase in serum 25-hydroxyvitamin D levels was less than that observed after the administration of an equimolar amount of vitamin D(3). Vitamin D(3) 3beta-glucosiduronate showed no detectable activity in the induction of calcium binding protein in chick embryonic duodena, a system in which no endogenous steroid beta-glucuronidase activity is detectable. These data demonstrate that vitamin D(3) 3beta-glucosiduronate is biologically active in vivo and that the observed activity is due to hydrolysis of the conjugate to vitamin D(3). As vitamin D(3) 3beta-glucosiduronate is excreted in the bile of rats, it is possible that this conjugate is reutilized in vivo after hydrolysis to free vitamin D(3). These results suggest the existence of a mechanism for reutilization of the biliary products of vitamin D(3).


A novel method for classifying starch digestion by modelling the amylolysis of plant foods using first-order enzyme kinetic principles.
Studying starch amylolysis kinetics in vitro is valuable for predicting the postprandial glycaemic response to starch intake. Prediction of starch amylolysis behaviour is challenging however, because of the many physico-chemical factors which influence amylolysis. The Logarithm of Slope (LOS) method for analysis of digestibility curves using first-order enzyme kinetics can identify and quantify nutritionally important starch fractions. The early stages of in vitro amylolysis of hydrothermally processed chickpea and durum wheat with variable degrees of structural integrity were studied. The end-point product concentration (C∞) and the pseudo first-order digestibility rate constant k, obtained from LOS analysis, were then used to compute predictive digestibility curves for evaluation of the model performance. LOS analysis enabled rapid identification of nutritionally important starch-fractions. It was clear that purified starches and flours were digested by a single-phase process, but starch amylolysis in macroparticles occurred by a two-phase system that reflected differences in substrate accessibility. The model gave an excellent fit to data obtained from a range of heterogeneous materials. It provides a rigorous means of studying the mechanisms of starch amylolysis in samples of varying complexity, and we strongly recommend its use for the rapid and accurate predictions of amylolysis. Such predictions have implications for prevention and management of type 2 diabetes mellitus and obesity.


A novel link between circPDE3B and ferroptosis in esophageal squamous cell carcinoma progression.
To unravel whether ferroptosis involves with the actions by circPDE3B-mediated facilitation of esophageal squamous cell carcinoma (ESCC) progression. Human ESCC tissues and cell lines were prepared for the evaluation of ferroptosis. Cellular iron, ROS, GSH, and MDA levels were measured to assess ferroptosis. Flow cytometry was employed to analyze apoptosis and cell cycle. Subcellular fractionation and fluorescence in situ hybridization (FISH) were conducted to validate the localization of circPDE3B. RNA pull-down, RNA immunoprecipitation (RIP), and luciferase assay were subjected to identify the molecular mechanisms. Nude mouse xenograft model was carried out to evaluate the function of circPDE3B/SLC7A11/CBS in vivo. Increased circPDE3B in human ESCC specimens was positively correlated with ferroptosis-related molecules, SLC7A11 and CBS. Functionally, circPDE3B knockdown triggered ferroptosis, apoptosis, and cell cycle arrest in ESCC cells. Whereas, these effects were obviously blocked by miR-516b-5p inhibitor. Mechanistically, not only circPDE3B sponged miR-516b-5p to upregulate CBS, but also directly bound with HNRNPK to stabilize SLC7A11. In mice, depletion of circPDE3B restrained ESCC growth, while this was abolished by overexpression of CBS or SLC7A11. In summary, circPDE3B promotes ESCC progression by suppressing ferroptosis through recruiting HNRNPK/SLC7A11 and miR-516b-5p/CBS axes.


Postrenal transplant malignancy: Incidence, risk factors, and prognosis.
The newer and potent immunosuppressive agents have successfully reduced the risk of rejection after kidney transplantation, but the development of cardiovascular diseases, infections, and malignancy is major factors limiting their success. Posttransplantation malignancy is the second most common cause of death in renal transplant recipients after cardiovascular disease; it is expected that mortality due to malignancy may become the most common cause of death within the next two decades. This study is designed to evaluate the incidence, risk factors, and types of malignancies occurring after renal transplantation and their impact on patient and graft survival. A total of 2288 patients underwent living donor renal allotransplantation in the Urology and Nephrology Center, Mansoura University, during the period between 1975 and 2011. Among these patients, 100 patients developed posttransplantation malignancy. Patients were categorized into five major groups according to their type of malignancy; Kaposi's sarcoma (KS), non-Kaposi's skin tumors (non-KS), posttransplant lymphoproliferative disorders (PTLD), solid tumors, and genitourinary and reproductive system (GU and RS). Overall, the incidence of cancer in renal transplant recipients was 4%. There were 83 male (83%) and 17 female patients (17%). The most frequent cancer was KS seen in 33 patients (33%). The lowest median time to development of cancer was observed in KS (35 months). The highest median time to development of cancer was observed in PTLD (133 months). The best graft survival was observed in PTLD and the worst in non-KS tumors. The best patient survival was observed in KS and the worst in GU and RS tumors. Azathioprine-based regimen was associated with a higher rate of cancer. The number of patients who died was 65 (65%). Our results indicate that the occurrence of malignancy has an important impact on short- and long-term graft and patient survival.


Characterization of crackles from patients with fibrosis, heart failure and pneumonia.
The spectral analysis of crackles sounds has been carried out based on the assumption that they are stationary signals, and the majority of the work on the crackles was accomplished before the publication of the Computerized Respiratory Sound Analysis (CORSA) guidelines. This works characterizes crackles acquired from patients with fibrosis, heart failure, and pneumonia, breathing at a constant rate, with a system developed according to the CORSA guidelines. Their maximum frequency was obtained by applying discrete pseudo Wigner-Ville distribution, suitable for non-stationary signals, and an objective method to estimate the maximum frequency, the modified geometric method. The effects of the breathing rate and the tidal volume on the spectra of the crackles were also investigated. The role of the high-pass filter cutoff frequency of the acquisition system on the characteristics of the acquired crackles was also assessed in this present study. Higher high-pass filter cutoff frequency allows for higher amplification which modifies the maximum frequency and the 2CD index. It is shown that the crackles acquired according to the CORSA guidelines have higher frequencies and shorter 2CD indexes than those previously reported, highlighting the need for the standardization and detailed report of the acquisition setup when quantifying lung sounds. The results pointed out that the maximum frequency and the 2CD indexes may allow crackles generated by fibrosis to be distinguished from the ones generated by the heart failure and pneumonia. It is not possible, however, by means of these two indexes, to differentiate between pneumonia and heart failure crackles.


Advanced ovarian yolk sac tumor: upfront surgery or neoadjuvant chemotherapy followed by interval debulking?
To compare surgery and survival outcomes between neoadjuvant chemotherapy and primary debulking surgery in patients with advanced ovarian yolk sac tumor. In this retrospective cohort analysis, patients with stage III to IV ovarian yolk sac tumor or mixed germ cell tumors containing yolk sac tumor elements, and who underwent surgery at Peking Union Medical College Hospital between January 2011 and December 2021, were identified. Patient characteristics, treatment, and survival data were analyzed between the two groups. A total of 40 patients were enrolled: 19 patients received neoadjuvant chemotherapy followed by interval surgery, and 21 patients were treated with primary debulking surgery. After neoadjuvant chemotherapy, the surgical conditions of patients were improved. All patients achieved cytoreduction to R0 or R1 at interval surgery. No statistical difference was found in 3-year disease-free survival and overall survival between the neoadjuvant chemotherapy group and the primary debulking surgery group (log rank p=0.4 and 0.94). Patients had less blood loss (328.4 vs 1285.7 mL, p=0.029), lower transfusion volume (1044.4 vs 3066.7 mL, p=0.011), and fewer peri-operative complications (15.8% vs 47.6%, p=0.032) at the interval debulking surgery after neoadjuvant chemotherapy compared with patients who underwent primary debulking surgery. For patients with advanced-stage ovarian yolk sac tumor, neoadjuvant chemotherapy followed by interval surgery is an alternative option, especially for those who cannot tolerate the primary debulking surgery because of high tumor burden and vulnerable status.


Taming Tumor Glycolysis and Potential Implications for Immunotherapy.
Immune evasion and deregulation of energy metabolism play a pivotal role in cancer progression. Besides the coincidence in their historical documentation and concurrent recognition as hallmarks of cancer, both immune evasion and metabolic deregulation may be functionally linked as well. For example, the metabolic phenotype, particularly tumor glycolysis (aerobic glycolysis), impacts the tumor microenvironment (TME), which in turn acts as a major barrier for successful targeting of cancer by antitumor immune cells and other therapeutics. Similarly, in the light of recent research, it has been known that some of the immune sensitive antigens that are downregulated in cancer may also be restored or induced by cellular/metabolic stress. For instance, cancer cells downregulate the cell surface ligands such as MHC class I chain-related (MIC) protein-(A/B) that are normally upregulated in disease/pathological conditions. Noteworthy, the MHC class I chain-related protein A and B (MIC-A/B) are recognized by natural killer (NK) cells for immune elimination. Interestingly, MIC-A/B is stress inducible as demonstrated by oxidative stress and other cellular-stress factors. Consequently, stimulation of metabolic stress has also been shown to sensitize cancer cells to NK cell-mediated cytotoxicity. Taken together, data from recent reports imply that dysregulation of tumor glycolysis could facilitate induction of immune sensitive surface ligands leading to increased efficacy of antitumor immunotherapeutics. Nonetheless, dysregulated tumor glycolysis may also impact the TME and alter it from acidic, low pH into a therapeutically desirable TME that can enhance the effective infiltration of antitumor immune cells. In this mini-review, targeting tumor glycolysis has been discussed to evaluate its potential implications to enhance and/or facilitate anticancer immunity.


Human Cytomegalovirus Uses a Host Stress Response To Balance the Elongation of Saturated/Monounsaturated and Polyunsaturated Very-Long-Chain Fatty Acids.
Stress and virus infection regulate lipid metabolism. Human cytomegalovirus (HCMV) infection induces fatty acid (FA) elongation and increases the abundance of lipids with very-long-chain FA (VLCFA) tails. While reprogramming of metabolism can be stress related, the role of stress in HCMV reprogramming of lipid metabolism is poorly understood. In this study, we engineered cells to knock out protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) in the ER stress pathway and measured lipid changes using lipidomics to determine if PERK is needed for lipid changes associated with HCMV infection. In HCMV-infected cells, PERK promotes increases in the levels of phospholipids with saturated FA (SFA) and monounsaturated FA (MUFA) VLCFA tails. Further, PERK enhances FA elongase 7 (ELOVL7) protein levels, which elongates SFA and MUFA VLCFAs. Additionally, we found that increases in the elongation of polyunsaturated fatty acids (PUFAs) associated with HCMV infection were independent of PERK and that lipids with PUFA tails accumulated in HCMV-infected PERK knockout cells. Additionally, the protein levels of ELOVL5, which elongates PUFAs, are increased by HCMV infection through a PERK-independent mechanism. These observations show that PERK differentially regulates ELOVL7 and ELOVL5, creating a balance between the synthesis of lipids with SFA/MUFA tails and PUFA tails. Additionally, we found that PERK was necessary for virus replication and the infectivity of released viral progeny. Overall, our findings indicate that PERK-and, more broadly, ER stress-may be necessary for the membrane biogenesis needed to generate infectious HCMV virions.IMPORTANCE HCMV is a common herpesvirus that establishes lifelong persistent infections. While infection is asymptomatic in most people, HCMV causes life-threatening illnesses in immunocompromised people, including transplant recipients and cancer patients. Additionally, HCMV infection is a leading cause of congenital disabilities. HCMV replication relies on lipid synthesis. Here, we demonstrated that the ER stress mediator PERK controls FA elongation and the cellular abundance of several types of lipids following HCMV infection. Specifically, PERK promotes FA elongase 7 synthesis and phospholipids with saturated/monounsaturated very-long-chain FA tails. Overall, our study shows that PERK is an essential host factor that supports HCMV replication and promotes lipidome changes caused by HCMV infection.


Characterization of a germline Vk gene encoding cationic anti-DNA antibody and role of receptor editing for development of the autoantibody in patients with systemic lupus erythematosus.
We found previously that cationic anti-DNA autoantibodies (autoAbs) have nephritogenic potential and usage of a specific germline Vk gene, A30, has major influences on cationic charge of the autoAb in human lupus nephritis. In the present study, we have characterized A30 germline Vk gene using cosmid cloning technique in patients with SLE. A30 gene locus locates in less than 250 kb from the Ck region, and the cationic anti-DNA mRNA used the upstream Jk2 gene, indicating that cationic anti-DNA mRNA is a product of primary gene rearrangement. By using PCR technique, we found that A30 gene locus in the genome was defective in eight out of nine SLE patients without nephritis. In contrast, all nine patients with lupus nephritis had intact A30 gene. The presence and absence of A30 gene was associated with the development of lupus nephritis or not (P < 0.01, by Fisher's exact test, two-sided). It was thus suggested that absence of functional A30 gene may rescue from developing lupus nephritis in the patients. A30 is reported to be a potentially functional but rarely expressed Vk gene in humans. It is possible that normal B cells edit primarily rearranged A30 gene with autoreactive potentials by receptor editing mechanism for changing the affinity of the B cell Ag receptor to avoid self-reactivity, whereas SLE B cells may have a defect in this mechanism. Indeed, we found that normal B cells edit A30-Jk2 gene in their genome possibly by inversion mechanism, whereas SLE B cells contain rearranged A30-Jk2-Ck gene in the genome and express A30-associated mRNA, suggesting that receptor editing mechanism is also defective in patients with SLE. Our study suggests that polymorphism of Ig Vk locus, and failure of receptor editing may contribute to the development of pathogenic anti-DNA responses in humans.


Survival by histology among patients with bone and soft tissue sarcoma who undergo metastasectomy: protocol for a systematic review and meta-analysis.
Metastasectomy is performed on a select cohort of patients with advanced and/or recurrent bone and soft tissue sarcomas because of the potential for long term relapse free and overall survival associated with the procedure. However, the evidence supporting metastasectomy is difficult to summarize without a systematic examination of existing literature. The objective of this systematic review will be to examine survival among both adults and children with advanced and recurrent bone and STS who undergo metastasectomy. We designed and registered a study protocol for a systematic review and meta-analysis. We will include data from survival studies (e.g., randomized trials, cohort studies, routine case registries, and case control) conducted in children and adults with advanced and recurrent bone and soft tissue sarcoma who undergo metastasectomy. The primary outcome will be overall survival. Secondary outcomes will be 30-day post-operative mortality, recurrence-free survival, time off systemic therapy, and patient-reported outcomes including quality of life end points where available. Literature searches will be performed in multiple electronic databases including Ovid MEDLINE ® (1946 to present), Ovid EMBASE (1974 to present), Web of Science, and Cochrane Library. Grey literature will be identified through searching references, conference abstracts, Papers First, and Google Scholar. Two investigators will independently screen all citations, full-text articles, and abstract data. Full-text articles selected for analysis will be assessed for quality and risk of bias. If feasible, we will conduct a random effects meta-analysis. Estimates will be stratified according to histology comparing survival based on organ of metastasectomy. Additional analysis will be conducted to explore the potential sources of heterogeneity according to various patient, disease, and treatment characteristics (e.g., metastasis status, age, disease burden, and concomitant interventions). This systematic review and meta-analysis will identify, evaluate, and integrate data on survival of metastasectomy of bone and soft tissue sarcoma by organ of metastasis. Our findings may have implications for clinicians, patients, and their families when considering selection for resection of oligometastatic disease in de novo, or recurrent bone and soft tissue sarcoma. Implications for future research will be identified to improve the outcomes of these complex patients. PROSPERO CRD42019126906.


Enzymatic properties of human 25-hydroxyvitamin D3 1alpha-hydroxylase coexpression with adrenodoxin and NADPH-adrenodoxin reductase in Escherichia coli.
We have cloned human 25-hydroxyvitamin D3 1alpha-hydroxylase cDNAs from normal subjects and patients with pseudovitamin D-deficient rickets (PDDR), and expressed the cDNAs in Escherichia coli JM109 cells. Kinetic analysis of normal 1alpha-hydroxylase in the reconstituted system revealed that Km values for 25(OH)D3 and (24R), 25(OH)2D3 were 2.7 and 1.1 microM, respectively. The lower Km value and higher Vmax/Km value for (24R),25(OH)2D3 indicated that it is a better substrate than 25(OH)D3 for 1alpha-hydroxylase. These results are quite similar to those of mouse 1alpha-hydroxylase. To establish a highly sensitive in vivo system, 1alpha-hydroxylase, adrenodoxin and NADPH-adrenodoxin reductase were coexpressed in E. coli cells. The recombinant E. coli cells showed remarkably high 1alpha-hydroxylase activity, suggesting that the electrons were efficiently transferred from NADPH-adrenodoxin reductase through adrenodoxin to 1alpha-hydroxylase in E. coli cells. Using this system, the activities of four mutants of 1alpha-hydroxylase, R107H, G125E, R335P and P382S, derived from patients with PDDR were examined. Although no significant reduction in expression of these mutants was observed, none showed detectable activity. These results strongly suggest that the mutations found in the patients with PDDR completely abolished 1alpha-hydroxylase activity by replacement of one amino acid residue.


Osteopontin is not critical for otoconia formation or balance function.
Unlike the structural and mechanical role of bone crystals, the inertial mass of otoconia crystals provides a shearing force to stimulate the mechanoreceptors of the utricle and saccule (the gravity receptor organ) under the stimuli of linear motion. It is not clear whether otoconia, composed primarily of CaCO3 and glycoproteins, go through similar calcification processes as bone. We have recently shown that otoconin-90 (Oc90) regulates the growth of otoconia crystals as osteopontin does bone crystals. Here, we analyzed the role of this non-collagenous bone matrix protein, osteopontin, in otoconia formation and balance function utilizing its knockout mice, whose inner ear phenotype has not been examined. Despite the presence of the protein in wild-type otoconia and vestibular hair cells, morphological, ultrastructural, and protein and calcium composition analyses of osteopontin null otoconia show that the protein is not needed for crystal formation, and no evidence of compensatory protein deposition is found. Employment of a wide spectrum of balance behavioral tests demonstrates that the protein is not critical for balance function either, which is confirmed by the normal function of the gravity receptor organ directly measured with linear vestibular-evoked potentials (VsEPs). When compared with findings on other otoconins, the data manifest a hierarchy of importance of proteins in crystallization and indicate mechanistic similarities and differences between bone and otoconia calcification.


Plasma Cell-Free DNA Methylomics of Bipolar Disorder With and Without Rapid Cycling.
Rapid cycling (RC) burdens bipolar disorder (BD) patients further by causing more severe disability and increased suicidality. Because diagnosing RC can be challenging, RC patients are at risk of rapid decline due to delayed suitable treatment. Here, we aimed to identify the differences in the circulating cell-free DNA (cfDNA) methylome between BD patients with and without RC. The cfDNA methylome could potentially be developed as a diagnostic test for BD RC. We extracted cfDNA from plasma samples of BD1 patients (46 RC and 47 non-RC). cfDNA methylation levels were measured by 850K Infinium MethylationEPIC array. Principal component analysis (PCA) was conducted to assess global differences in methylome. cfDNA methylation levels were compared between RC groups using a linear model adjusted for age and sex. PCA suggested differences in methylation profiles between RC groups (p = 0.039) although no significant differentially methylated probes (DMPs; q > 0.15) were found. The top four CpG sites which differed between groups at p < 1E-05 were located in CGGPB1, PEX10, NR0B2, and TP53I11. Gene set enrichment analysis (GSEA) on top DMPs (p < 0.05) showed significant enrichment of gene sets related to nervous system tissues, such as neurons, synapse, and glutamate neurotransmission. Other top notable gene sets were related to parathyroid regulation and calcium signaling. To conclude, our study demonstrated the feasibility of utilizing a microarray method to identify circulating cfDNA methylation sites associated with BD RC and found the top differentially methylated CpG sites were mostly related to the nervous system and the parathyroid.


A Simple Combined Use of CRISPR-Cas9 and Cre-LoxP Technologies for Generating Conditional Gene Knockouts in Mammalian Cells.
Gene knockout technologies have contributed fundamentally to our understanding of the cellular functions of various genes. Two prevalent systems used for the efficient elimination of the expression of specific genes are the Cre-LoxP system and the CRISPR-Cas9 system. Here, we present a simple method that combines the use of CRISPR-Cas9 and Cre-LoxP for the conditional deletion of essential genes in mammalian cells. First, an inducible Cre recombinase is stably expressed in the cells. Next, CRISPR-Cas9 is used to knock out an essential gene, whose function is complemented by stable expression of a FLAG-tagged version of the same protein encoded from a floxed transcription unit containing silent mutations, making it refractory to the CRISPR-Cas9 guide. This FLAG-tagged protein can be deleted by activating the expressed Cre protein, enabling evaluation of the cellular consequences of its deletion. We have further used this system to evaluate the ability of phylogenic homologues and of potential mutants to cover functionally for the deleted gene.


Paediatric biobanks: what makes them so unique?
Paediatric biobanks store and organise the biological material of children. They are an invaluable resource for the study of the development, health and behaviour of children. International norms for the management of adult biobanks exist, but paediatric biobanks require distinct policies to account for the needs of children, their general incapacity, and their intellectual development throughout the life of the biobank. Because of their particular nature we revisit the issues of consent, the return of research results, and privacy, and discuss how each could be modulated in the paediatric context. We recognize that such modifications entail further financial and logistical complications but maintain that it is essential that paediatric biobanks consider these issues and adapt their biobanks management policies accordingly, rather than extrapolate the current adult-based norms and jeopardise the rights of child participants.


Administration of DL-2-amino-5-phosphonovaleric acid (AP5) induces transient inhibition of reminder-activated memory retrieval in day-old chicks.
DL-2-Amino-5-phosphonovaleric acid (50 microM) administered immediately after a visual reminder presented to day-old chickens between 7.5 min and 24 h following a single trial passive avoidance learning task produced transient losses of memory on retention test, an effect not observed in the absence of a reminder or when the reminder was given 48 h post learning. The duration of the transient deficits decreased with increasing interval between training and the reminder trial. The time of onset of memory loss after the reminder trial appeared to increase with increasing interval between the training and the reminder trials. The results suggest that, for a period of at least up to 24 h after passive avoidance training, retrieval of memory may lead to processes which are sensitive to inhibition by the NMDA receptor antagonist AP5, with the duration of sensitivity post retrieval decreasing as the period of memory consolidation increases. The results extend previously reported findings and suggest the possibility that consolidation of a stable memorial representation of a learning experience may take over several days and may entail the concurrent laying down of a stable retrieval mechanism.


Effects of adopting a smoke-free policy in state psychiatric hospitals.
The aim of this study was to investigate how adopting a smoke-free policy in state psychiatric hospitals affected key factors, including adverse events, smoking cessation treatment options, and specialty training for clinical staff about smoking-related issues. Hospitals were surveyed in 2006 and 2008 about their smoking policies, smoking cessation aids, milieu management, smoking cessation treatment options, and aftercare planning and referrals for smoking education. Comparisons were made between hospitals that went smoke-free between the two time periods (N=28) and those that did not (N=42). Among hospitals that changed to a smoke-free policy, the proportion that reported adverse events decreased by 75% or more in three areas: smoking or tobacco use as a precursor to incidents that led to seclusion or restraint, smoking-related health conditions, and coercion or threats among patients and staff. Hospitals that did not adopt a smoke-free policy cited several barriers, including resistance from staff, patients, and advocates. Although staff were concerned that implementing a smoke-free policy would have negative effects, this was not borne out. Findings indicated that adopting a smoke-free policy was associated with a positive impact on hospitals, as evidenced by a reduction in negative events related to smoking. After adoption of a smoke-free policy, fewer hospitals reported seclusion or restraint related to smoking, coercion, and smoking-related health conditions, and there was no increase in reported elopements or fires. For hospitals adopting a smoke-free policy in 2008, there was no significant difference between 2006 and 2008 in the number offering nicotine replacement therapies or clinical staff specialty training. Results suggest that smoking cessation practices are not changing in the hospital as a result of a change in policy.


Ready…aim…fire into the lumen: a new role for enterocyte microvilli in gut host defense.
Recent studies from our laboratory revealed that enterocyte brush border microvilli release small vesicles laden with host defense machinery into the intestinal lumen. In this addendum, we introduce a multi-faceted model for the function of these lumenal vesicles in the gut; we also consider some of the important unanswered questions that must be addressed in order to develop our understanding of this novel aspect of innate intestinal immunity.


Stranger in a foreign land.
Undergoing surgery can be a very daunting process for patients, especially if the surgery is major. As perioperative nurses or anesthesia providers, it is easy to become comfortable with the environment and forget that, for patients, it can be similar to being in a foreign country where everything, including the language, is scary and intimidating. I learned to be more sensitive to my patients when I found myself in an unfamiliar situation and saw some similarities to what my patients were experiencing.


Intrasession and Intersession Reliability of Quadriceps' and Hamstrings' Electromyography During a Standardized Hurdle Jump Test With Single Leg Landing.
The objective of this study was to develop a standardized test to determine quadriceps and hamstrings muscle activation in a position emulating a noncontact anterior cruciate ligament injury. We assessed the intrasession and intersession reliability of surface electromyography (EMG) of the dominant leg after single-leg landing from a standardized hurdle jump. Eighteen subjects (10 males, 8 females) participated in 4 repeated sessions. During each session, individuals performed 3 successful jumps over a hurdle set to 75% of their maximal countermovement jump height and landed on their dominant leg. A jump was only considered successful if the individual could maintain the landing position for longer than 2 seconds after initial ground contact. In one of the 4 sessions, subjects were tested again after a 4-minute rest. The activation of the vastus lateralis (VL), vastus medialis (VM), and biceps femoris (BF), were examined by quantifying the root mean squared (RMS) EMG for 2 seconds immediately after the initial contact. Data from all 3 successful jumps were used to generate intraclass correlation coefficients (ICC), which were then used to determine intrasession and intersession reliability of surface EMG for each muscle. Intrasession reliability was excellent with ICC values of 0.96, 0.94, and 0.93 for the VL, VM, and BF, respectively. Additionally, intersession ICCs were 0.92 (VL), 0.95 (VM), and 0.94 (BF). The standardized hurdle jump with single-leg landing seemed to be a reliable technique for measuring muscle activation for 3 muscles that contribute to knee stabilization.


Bullying prevalence across contexts: a meta-analysis measuring cyber and traditional bullying.
Bullying involvement in any form can have lasting physical and emotional consequences for adolescents. For programs and policies to best safeguard youth, it is important to understand prevalence of bullying across cyber and traditional contexts. We conducted a thorough review of the literature and identified 80 studies that reported corresponding prevalence rates for cyber and traditional bullying and/or aggression in adolescents. Weighted mean effect sizes were calculated, and measurement features were entered as moderators to explain variation in prevalence rates and in traditional-cyber correlations within the sample of studies. Prevalence rates for cyber bullying were lower than for traditional bullying, and cyber and traditional bullying were highly correlated. A number of measurement features moderated variability in bullying prevalence; whereas a focus on traditional relational aggression increased correlations between cyber and traditional aggressions. In our meta-analytic review, traditional bullying was twice as common as cyber bullying. Cyber and traditional bullying were also highly correlated, suggesting that polyaggression involvement should be a primary target for interventions and policy. Results of moderation analyses highlight the need for greater consensus in measurement approaches for both cyber and traditional bullying.


[Study of a multifield equivalent cardiac generator].
The electric field of the isolated dog heart immersed into a liquid volumetric conductor was subjected to a thorough investigation. Measurements and subsequent calculations showed that in precordial leads the quantitative description of the cardiac generator by a multipole equivalent generator of the 1st order (dipole) is rough approximation. Addition of a multipole component of the 2nd order (quadripole) to the equivalent generator increases but relatively little the accuracy of description. It is suggested that in this problem the multipole expansion proves to be a slowly convergent series. For this reason, application of multipole equivalent cardiac generator fixed in the electrical center of the heart in the clinical electrocardiography is to be considered as having no prospect.


Outcome of modern shunt therapy in patients with idiopathic normal pressure hydrocephalus 6 years postoperatively.
Shunt therapy in idiopathic normal pressure hydrocephalus (iNPH) can improve symptoms in 84% of patients 1 year postoperatively. Therefore, implantation of a ventriculoperitoneal shunt (VPS) prevents or at least prolongs the time during which patients are not dependent on care from others because of gait disorder or dementia. In this study, the medium-term results of modern shunt therapy were evaluated. The authors retrospectively studied outcome in iNPH patients in whom VPSs were implanted. At yearly follow-up examinations over at least 6 years, clinical symptoms were recorded according to the NPH recovery rate (NPH-RR): (preoperative Kiefer Scale [KS] score - postoperative KS score)/(preoperative KS score × 10). Time and cause of death over this period were also analyzed. Among the 147 patients treated for iNPH in the period between 1997 and 2006, 69 had died at the time of the authors' survey. Sixty-one patients reached the 6-year examination. The median age was 64 years (range 33-83 years) at the time of VPS implantation and 73 years (range 41-91 years) at the time of this study. Six years after shunt implantation, the median KS score was significantly lower than the preoperative score (4.3 vs 8.1 points). Fifty-nine percent of 61 patients had an excellent outcome, 15% had satisfactory benefit, and 26% had unsatisfactory results 6 years postoperatively. Three of the 147 patients who underwent implantation of a VPS died of cerebral diseases 4-10 years postoperatively. Implantation of a VPS is a safe procedure and can improve symptoms in 74% of patients with iNPH in the longer term.


The role of team goal monitoring in the curvilinear relationship between team efficacy and team performance.
In this research, we apply a team self-regulatory perspective to build and test theory focusing on the relationships between team efficacy and 2 key team performance criteria: a performance behavior (i.e., team effort) and a performance outcome (i.e., objective team sales). We theorize that rather than having a linear association, the performance benefits of team efficacy reach a point of inflection, reflective of too much of a good thing. Further, in an effort to establish a boundary condition of the inverted-U shaped relationship we predict, we also test the moderating role played by team goal monitoring in the nonmonotonic relationship between team efficacy and team performance. The results from a lagged field test, in which we collect multisource data from 153 technology sales teams, reveal a significant curvilinear association that is moderated by team goal monitoring behavior. Implications for theory and practice are discussed.


Rates and indicators for amniotomy during labor--a descriptive cross sectional study between primigravidas and gravida 2 and above.
Artificial rupture of membranes (Amniotomy) is a common obstetric intervention. Its rates and indications had been subjected to criticism in medical literature. The current practices recommend to reduce its rate and keep the birthing process as natural as possible. This observational study aimed to describe the rates and indicators for practice of artificial rupture of membranes (Amniotomy) during normal labor and to determine if any significant differences existed between women who have had one pregnancy (PG) and women who have already delivered two or more children (G2 and above) on this obstetric interventions: artificial rupture of membranes (ARM). There were no PG participants with ruptured membranes whereas slightly more than half of the G2 and above participants (n = 88) had ruptured membranes. The most frequent cause for ARM was active management of latent phase of labor (PG n = 20 and G2 and above n = 9). Furthermore, slow progress of labor (PG n = 17 and G2 and above n = 7) and concerns with fetal heart rate (PG n = 13 and G2 and above n = 5) had the next highest number of occurrences. Results from the proportions tests revealed that there was one significant difference between gravidity groups on the frequency of APH (p = 0.039). That is, G2 and above participants had amniotomy done for APH (5 of 32 = 15.63%) significantly more often than PG participants (4 of 89 = 4.49%). And although not statistically significant (p = 0.084), there were 21 cases within the PG group where ARM was performed for no specific reason (21 of 89 = 23.6%) compared to three cases within the G2 and above group (3 of 32 = 9.4%). Although ARM is a commonly performed procedure during labor, there is not much difference between its indications between PG and G2 and above. The only significantly different indication was antepartum hemorrhage which was higher in G2 and above. Amniotomy was also performed without any clear indication in 26.4% of PG and 9.4% of G2 and above. Considering ARM as obstetric intervention efforts should be done to reduce its rates. There is a need for arranging normal labor workshops to revise the indications and reviewing the rates after these workshops to reduce the rates of ARM.


Oncogenes Activate an Autonomous Transcriptional Regulatory Circuit That Drives Glioblastoma.
Efforts to identify and target glioblastoma (GBM) drivers have primarily focused on receptor tyrosine kinases (RTKs). Clinical benefits, however, have been elusive. Here, we identify an SRY-related box 2 (SOX2) transcriptional regulatory network that is independent of upstream RTKs and capable of driving glioma-initiating cells. We identified oligodendrocyte lineage transcription factor 2 (OLIG2) and zinc-finger E-box binding homeobox 1 (ZEB1), which are frequently co-expressed irrespective of driver mutations, as potential SOX2 targets. In murine glioma models, we show that different combinations of tumor suppressor and oncogene mutations can activate Sox2, Olig2, and Zeb1 expression. We demonstrate that ectopic co-expression of the three transcription factors can transform tumor-suppressor-deficient astrocytes into glioma-initiating cells in the absence of an upstream RTK oncogene. Finally, we demonstrate that the transcriptional inhibitor mithramycin downregulates SOX2 and its target genes, resulting in markedly reduced proliferation of GBM cells in vivo.


Direct Comparison of Chol-siRNA Polyplexes and Chol-DsiRNA Polyplexes Targeting STAT3 in a Syngeneic Murine Model of TNBC.
RNA interference (RNAi) molecules have tremendous potential for cancer therapy but are limited by insufficient potency after intravenous (IV) administration. We previously found that polymer complexes (polyplexes) formed between 3'-cholesterol-modified siRNA (Chol-siRNA) or DsiRNA (Chol-DsiRNA) and the cationic diblock copolymer PLL[30]-PEG[5K] greatly increase RNAi potency against stably expressed LUC mRNA in primary syngeneic murine breast tumors after daily IV dosing. Chol-DsiRNA polyplexes, however, maintain LUC mRNA suppression for ~48 h longer after the final dose than Chol-siRNA polyplexes, which suggests that they are the better candidate formulation. Here, we directly compared the activities of Chol-siRNA polyplexes and Chol-DsiRNA polyplexes in primary murine 4T1 breast tumors against STAT3, a therapeutically relevant target gene that is overexpressed in many solid tumors, including breast cancer. We found that Chol-siSTAT3 polyplexes suppressed STAT3 mRNA in 4T1 tumors with similar potency (half-maximal ED50 0.3 mg/kg) and kinetics (over 96 h) as Chol-DsiSTAT3 polyplexes, but with slightly lower activity against total Stat3 protein (29% vs. 42% suppression) and tumor growth (11.5% vs. 8.6% rate-based T/C ratio) after repeated IV administration of equimolar, tumor-saturating doses every other day. Thus, both Chol-siRNA polyplexes and Chol-DsiRNA polyplexes may be suitable clinical candidates for the RNAi therapy of breast cancer and other solid tumors.


Early phase trial designs and endpoints for targeted therapies in rare genotype subsets.
The primary goal of phase I dose-finding trials has been historically to understand the safety profile of a new treatment/regimen and establish the maximum tolerated dose (MTD) for further investigation in a specific tumor type. These designs hinged on the assumption that the highest safe dose is also likely to be the most efficacious dose. In the current era of stratified medicine and targeted therapies, these assumptions and designs are quickly becoming obsolete. Newer approaches to dose-finding designs are emerging with a focus on not just safety, but also an efficacy (clinical- or biomarker-based) measure to guide the determination of the "optimal" dose (not necessarily the MTD) model, as opposed to rule-based designs. These designs allow for flexibility in trial conduct, with improved precision in estimating the dose to take forward for further testing, patient selection/enrichment strategies to maximize the chance of therapeutic effect, and expansion cohorts to further understand the safety/efficacy of the agent and patient subsets most likely to benefit from the agent under investigation. Although advances in technology have enabled the possibility of using these newer approaches, the acceptance and integration of these approaches in practice can only be accelerated if it is developed in concert with a clinical paradigm.


Heat treatment as a universal technical solution for silcrete use? A comparison between silcrete from the Western Cape (South Africa) and the Kalahari (Botswana).
Heat treatment was one of the first transformative technologies in the southern African Middle Stone Age (MSA), with many studies in the Cape coastal zone of South Africa identifying it as an essential step in the preparation of silcrete prior to its use in stone tool manufacture. To date, however, no studies have investigated whether heat treatment is necessary for all silcrete types, and how geographically widespread heat treatment was in the subcontinent. The aim of this study is to investigate experimentally whether heat treatment continued further north into the Kalahari Desert of Botswana and northernmost South Africa, the closest area with major silcrete outcrops to the Cape. For this we analyse the thermal transformations of silcrete from both regions, proposing a comprehensive model of the chemical, crystallographic and 'water'-related processes taking place upon heat treatment. For the first time, we also explore the mobility of minor and trace elements during heat treatment and introduce a previously undescribed mechanism-steam leaching-causing depletion of a limited number of elements. The results of this comparative study reveal the Cape and Kalahari silcrete to respond fundamentally differently to heat treatment. While the former can be significantly improved by heat, the latter is deteriorated in terms of knapping quality. These findings have important implications for our understanding of the role of fire as a technical solution in MSA stone tool knapping, and for the extension of its use in southern Africa. Silcrete heat treatment-at least in the form we understand it today-may have been a strictly regional phenomenon, confined to a narrow zone along the west and south coast of the Cape. On the basis of our findings, silcrete heat treatment should not be added as a new trait on the list of behaviours that characterise the MSA of the southern African subcontinent.


Enrichment devices for green turtles (Chelonia mydas) reared in captivity programs.
Environmental enrichment supports the well-being and welfare of captive animals. In the current study, the most suitable form of enrichment device for captive green turtles (Chelonia mydas) was investigated, to support head-start programs rearing turtles for release into their natural habitat. Fifteen-day-old turtles (113-114 g initial weight, n = 75) were randomly distributed into 15 experimental plastic tanks, comprising 5 treatments across three pools of each condition. The turtles in the experimental groups were exposed to four forms of enrichment devices (RS, ring shape; HSQS, hollow square shape; SS, sphere shape; CS, cylinder shape), and their outcomes related to growth, feed utilization, behavior, reduction of injury from conspecifics, and several health parameters were compared to those of a control group. At the end of the 10-week trial, the growth and feed utilization parameters did not differ across the five groups (p > .05). Of the turtles in the experimental treatments, those in the RS treatment spent more time interacting with the enrichment device, followed by the HSQS group. The percentage of wounds suffered through biting was significantly reduced in the groups exposed to enrichment devices, notably in the turtles exposed to the SS device, followed by the RS device. Significant differences between experimental groups in the specific activities of the major intestinal protein-digesting enzymes (trypsin and chymotrypsin) were observed. There were no effects noted in the hematological parameters and the main carapace elemental profile as compared to the control treatment. These findings suggest that the RS device is most appropriate in enriching the environment of juvenile green turtles in captivity programs, as well as in zoos or aquaria.


KITE-BCI: A brain-computer interface system for functional electrical stimulation therapy.
Integrating brain-computer interface (BCI) technology with functional electrical stimulation therapy (FEST) is an emerging strategy for upper limb motor rehabilitation after spinal cord injury (SCI). Despite promising results, the combined use of these technologies (BCI-FEST) in clinical practice is minimal. To address this issue, we developed KITE-BCI, a BCI system specifically designed for clinical application and integration with dynamic FEST. In this paper, we report its technical features and performance. In addition, we discuss the differences in distributions of the BCI- and therapist-triggered stimulation latencies. Two single-arm 40-session interventional studies to test the feasibility of BCI-controlled FEST for upper limb motor rehabilitation in individuals with cervical SCI. Rehabilitation programs within the University and Lyndhurst Centres of the Toronto Rehabilitation Institute - University Health Network, Toronto, Canada. Five individuals with sub-acute (< 6 months post-injury) SCI at the C4-C5 level, AIS B-D, and three individuals with chronic (> 24 months post-injury) SCI at C4 level, AIS B-C. We measured BCI setup duration, and to characterize the performance of KITE-BCI, we recorded BCI sensitivity, defined as the percentage of successful BCI activations out of the total number of cued movements. The overall BCI sensitivities were 74.46% and 79.08% for the sub-acute and chronic groups, respectively. The average KITE-BCI setup duration across the two studies was 11 min and 13 s. KITE-BCI demonstrates a clinically viable single-channel BCI system for integration with FEST resulting in a versatile technology-enhanced upper limb motor rehabilitation strategy after SCI.


Timing of prescription of angiotensin-converting enzyme inhibitors/angiotensin receptor blockers in patients hospitalized for acute heart failure with reduced/mildly reduced ejection fraction: a retrospective analysis.
Although angiotensin-converting enzyme inhibitors (ACEis) and angiotensin II receptor blockers (ARBs) play critical roles in the treatment of heart failure with reduced or mildly reduced ejection fraction (HFrEF/HFmrEF; left-ventricular ejection fraction ≤ 50%), the ideal timing for initiation in patients with acute heart failure (AHF) is unclear. We sought to clarify the timing and safety of ACEi/ARB prescription relative to hemodynamic stabilization (pre or post) in patients hospitalized with acute HFrEF/HFmrEF. This was a retrospective, observational analysis of electronic data of patients hospitalized for AHF at 17 Japanese hospitals. Among 9107 patients hospitalized with AHF, 2648 had HFrEF/HFmrEF, and 83.0% met the hemodynamic stabilization criteria within 10 days of admission. During hospitalization, 63.5% of patients with HFrEF/HFmrEF were prescribed an ACEi/ARB, 79.4% of which were prescribed pre-stabilization. In a multivariable analysis, patients treated with an ACEi/ARB pre-stabilization were more likely to have comorbid hypertension, diabetes mellitus, or ischemic heart disease. ACEi/ARB prescription timing was not associated with adverse events, including hypotension and renal impairment, and early prescription was associated with a lower incidence of subsequent worsening of HF. In clinical practice, more hospitalized patients with AHF received an ACEi/ARB before compared with after hemodynamic stabilization, and no safety concerns were observed. Moreover, early prescription may be associated with a lower incidence of worsening HF.


The effects of quality of care on costs: a conceptual framework.
The quality of health care and the financial costs affected by receiving care represent two fundamental dimensions for judging health care performance. No existing conceptual framework appears to have described how quality influences costs. We developed the Quality-Cost Framework, drawing from the work of Donabedian, the RAND/UCLA Appropriateness Method, reports by the Institute of Medicine, and other sources. The Quality-Cost Framework describes how health-related quality of care (aspects of quality that influence health status) affects health care and other costs. Structure influences process, which, in turn, affects proximate and ultimate outcomes. Within structure, subdomains include general structural characteristics, circumstance-specific (e.g., disease-specific) structural characteristics, and quality-improvement systems. Process subdomains include appropriateness of care and medical errors. Proximate outcomes consist of disease progression, disease complications, and care complications. Each of the preceding subdomains influences health care costs. For example, quality improvement systems often create costs associated with monitoring and feedback. Providing appropriate care frequently requires additional physician visits and medications. Care complications may result in costly hospitalizations or procedures. Ultimate outcomes include functional status as well as length and quality of life; the economic value of these outcomes can be measured in terms of health utility or health-status-related costs. We illustrate our framework using examples related to glycemic control for type 2 diabetes mellitus or the appropriateness of care for low back pain. The Quality-Cost Framework describes the mechanisms by which health-related quality of care affects health care and health status-related costs. Additional work will need to validate the framework by applying it to multiple clinical conditions. Applicability could be assessed by using the framework to classify the measures of quality and cost reported in published studies. Usefulness could be demonstrated by employing the framework to identify design flaws in published cost analyses, such as omitting the costs attributable to a relevant subdomain of quality.


Targeting Human Transmission Biology for Malaria Elimination.
Malaria remains one of the leading causes of death worldwide, despite decades of public health efforts. The recent commitment by many endemic countries to eliminate malaria marks a shift away from programs aimed at controlling disease burden towards one that emphasizes reducing transmission of the most virulent human malaria parasite, Plasmodium falciparum. Gametocytes, the only developmental stage of malaria parasites able to infect mosquitoes, have remained understudied, as they occur in low numbers, do not cause disease, and are difficult to detect in vivo by conventional methods. Here, we review the transmission biology of P. falciparum gametocytes, featuring important recent discoveries of genes affecting parasite commitment to gametocyte formation, microvesicles enabling parasites to communicate with each other, and the anatomical site where immature gametocytes develop. We propose potential parasite targets for future intervention and highlight remaining knowledge gaps.


MicroRNAs act sequentially and asymmetrically to control chemosensory laterality in the nematode.
Animal microRNAs (miRNAs) are gene regulatory factors that prevent the expression of specific messenger RNA targets by binding to their 3' untranslated region. The Caenorhabditis elegans lsy-6 miRNA (for lateral symmetry defective) is required for the left/right asymmetric expression of guanyl cyclase (gcy) genes in two chemosensory neurons termed ASE left (ASEL) and ASE right (ASER). The asymmetric expression of these putative chemoreceptors in turn correlates with the functional lateralization of the ASE neurons. Here we find that a mutation in the die-1 zinc-finger transcription factor disrupts both the chemosensory laterality and left/right asymmetric expression of chemoreceptor genes in the ASE neurons. die-1 controls chemosensory laterality by activating the expression of lsy-6 specifically in ASEL, but not in ASER, where die-1 expression is downregulated through two sites in its 3' untranslated region. These two sites are complementary to mir-273, a previously uncharacterized miRNA, whose expression is strongly biased towards ASER. Forced bilateral expression of mir-273 in ASEL and ASER causes a loss of asymmetric die-1 expression and ASE laterality. Thus, an inverse distribution of two sequentially acting miRNAs in two bilaterally symmetric neurons controls laterality of the nematode chemosensory system.


Recent Insights into the Role of Unfolded Protein Response in ER Stress in Health and Disease.
Unfolded stress response (UPR) is a conserved cellular pathway involved in protein quality control to maintain homeostasis under different conditions and disease states characterized by cell stress. Although three general schemes of and genes induced by UPR are rather well-established, open questions remain including the precise role of UPR in human diseases and the interactions between different sensor systems during cell stress signaling. Particularly, the issue how the normally adaptive and pro-survival UPR pathway turns into a deleterious process causing sustained endoplasmic reticulum (ER) stress and cell death requires more studies. UPR is also named a friend with multiple personalities that we need to understand better to fully recognize its role in normal physiology and in disease pathology. UPR interacts with other organelles including mitochondria, and with cell stress signals and degradation pathways such as autophagy and the ubiquitin proteasome system. Here we review current concepts and mechanisms of UPR as studied in different cells and model systems and highlight the relevance of UPR and related stress signals in various human diseases.


Regulation of the dicarboxylic acid part of the citric acid cycle in Bacillus subtilis.
The regulation of alpha-ketogluterate dehydrogenase, succinate dehydrogenase, fumarase, malate dehydrogenase, and malic enzyme has been studied in Bacillus subitilis. The levels of these enzymes increase rapidly during late exponential phase in a complex medium and are maximal 1 to 2 h after the onset of sporulation. Regulation of enzyme synthesis has been studied in the wild type and different citric acid cycle mutants by adding various metabolites to the growth medium. Alpha-ketoglutarate dehydrogenase is induced by glutamate or alpha-ketoglutarate; succinate dehydrogenase is repressed by malate; and fumarase and malic enzyme are induced by fumarate and malate, respectively. The addition of glucose leads to repression of the citric acid cycle enzymes whereas the level of malic enzyme is unaffected. Studies on the control of enzyme activities in vitro have shown that alpha-ketoglutarate dehydrogenase and succinate dehydrogenase are inhibited by oxalacetate. Enzyme activities are also influenced by the energy level, expressed as the energy charge of the adenylate pool. Isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, and malic enzyme are inhibited at high energy charge values, whereas malate dehydrogenase is inhibited at low energy charge. A survey of the regulation of the citric acid cycle in B.subtilis, based on the present work and previously reported results, is presented and discussed.


NMDAR-dependent long-term depression is associated with increased short term plasticity through autophagy mediated loss of PSD-95.
Long-term depression (LTD) of synaptic strength can take multiple forms and contribute to circuit remodeling, memory encoding or erasure. The generic term LTD encompasses various induction pathways, including activation of NMDA, mGlu or P2X receptors. However, the associated specific molecular mechanisms and effects on synaptic physiology are still unclear. We here compare how NMDAR- or P2XR-dependent LTD affect synaptic nanoscale organization and function in rodents. While both LTDs are associated with a loss and reorganization of synaptic AMPARs, only NMDAR-dependent LTD induction triggers a profound reorganization of PSD-95. This modification, which requires the autophagy machinery to remove the T19-phosphorylated form of PSD-95 from synapses, leads to an increase in AMPAR surface mobility. We demonstrate that these post-synaptic changes that occur specifically during NMDAR-dependent LTD result in an increased short-term plasticity improving neuronal responsiveness of depressed synapses. Our results establish that P2XR- and NMDAR-mediated LTD are associated to functionally distinct forms of LTD.


Inducible caspase 9 suicide gene to improve the safety of allodepleted T cells after haploidentical stem cell transplantation.
Addback of donor T cells following T cell-depleted stem cell transplantation (SCT) can accelerate immune reconstitution and be effective against relapsed malignancy. After haploidentical SCT, a high risk of graft-versus-host disease (GVHD) essentially precludes this option, unless the T cells are first depleted of alloreactive precursor cells. Even then, the risks of severe GVHD remain significant. To increase the safety of the approach and thereby permit administration of larger T cell doses, we used a suicide gene, inducible caspase 9 (iCasp9), to transduce allodepleted T cells, permitting their destruction should administration have adverse effects. We made a retroviral vector encoding iCasp9 and a selectable marker (truncated CD19). Even after allodepletion (using anti-CD25 immunotoxin), donor T cells could be efficiently transduced, expanded, and subsequently enriched by CD19 immunomagnetic selection to >90% purity. These engineered cells retained antiviral specificity and functionality, and contained a subset with regulatory phenotype and function. Activating iCasp9 with a small-molecule dimerizer rapidly produced >90% apoptosis. Although transgene expression was downregulated in quiescent T cells, iCasp9 remained an efficient suicide gene, as expression was rapidly upregulated in activated (alloreactive) T cells. We have demonstrated the clinical feasibility of this approach after haploidentical transplantation by scaling up production using clinical grade materials.


scTEA-db: a comprehensive database of novel terminal exon isoforms identified from human single cell transcriptomes.
The usage of alternative terminal exons results in messenger RNA (mRNA) isoforms that differ in their 3' untranslated regions (3' UTRs) and often also in their protein-coding sequences. Alternative 3' UTRs contain different sets of cis-regulatory elements known to regulate mRNA stability, translation and localization, all of which are vital to cell identity and function. In previous work, we revealed that ∼25 percent of the experimentally observed RNA 3' ends are located within regions currently annotated as intronic, indicating that many 3' end isoforms remain to be uncovered. Also, the inclusion of not yet annotated terminal exons is more tissue specific compared to the already annotated ones. Here, we present the single cell-based Terminal Exon Annotation database (scTEA-db, www.scTEA-db.org) that provides the community with 12 063 so far not yet annotated terminal exons and associated transcript isoforms identified by analysing 53 069 publicly available single cell transcriptomes. Our scTEA-db web portal offers an array of features to find and explore novel terminal exons belonging to 5538 human genes, 110 of which are known cancer drivers. In summary, scTEA-db provides the foundation for studying the biological role of large numbers of so far not annotated terminal exon isoforms in cell identity and function.


HIV transmission through breastmilk: the science behind the understanding of current trends and future research.
Breastmilk protects the infant from many diseases and many short- term and long- term benefits accrue. At the same time it is also known that breastfeeding acts as a vehicle for some infective agents. It is now accepted that breastmilk transmission of Human Immunodeficiency Virus- 1 (HIV-1) is an important mode of paediatric infection . Despite this fact, many researchers have observed that corresponding to the volume of milk consumed by the infant, maternal transmission via breastmilk is still comparatively low. Some have noted the long latency period of breastmilk HIV transmission with evidence of numerous anti-HIV factors in breastmilk. Although there are accepted standard guidelines on infant feeding in mothers who are HIV positive in many countries, it maybe equally important to realize gaps in our knowledge of mother- to -child HIV transmission. From an evolutionary perspective, the role of the mammary epithelial cell (MEC) and of breastmilk , in contributing to and possibly in influencing HIV-1 transmission is intriguing. The presence of HIV-1 or of other viruses in maternal milk seem to be a requisite to spur immunological defenses to optimize necessary protection to the infant. This article reviews some aspects of the science of HIV transmission through breastmilk and reflects the concept -based understanding of current policies on HIV and breastfeeding. At the same time, it highlights uncertainties in this field and the urgency for future research in this direction. Accepting current notions of breastmilk HIV transmission, greater deliberation by research may throw more light on why breastfeeding with its abundant advantages is fraught with the hazards of transmission of a deadly disease.


Mercury advisories and household health trade-offs.
The conventional economic wisdom is that improving consumer information will enhance welfare. Yet, some scientists speculate that the Food and Drug Administration's prominent mercury in fish advisory may have harmed public health. Lower mercury intakes reduce neurological toxicity risks. However, since seafood is the predominant dietary source of healthful omega-3 fatty acids, reduced fish consumption may have significant offsetting health impacts. We explore this risk trade-off using a rich panel of household-level seafood consumption data. To control for confounding factors, we use a non-parametric changes-in-changes approach. We find strong evidence that while the advisory reduced mercury loadings, it did so at the expense of substantial reductions in healthful omega-3s. We find this response pattern even for consumers with low fish consumption. Using advisory response patterns as inputs into a prominent risk assessment model, the central estimate is that net benefits from the advisory were negative.


Effective training practices for non-specialist providers to promote high-quality mental health intervention delivery: A narrative review with four case studies from Kenya, Ethiopia, and the United States.
Mental health needs and disparities are widespread and have been exacerbated by the COVID-19 pandemic, with the greatest burden being on marginalized individuals worldwide. The World Health Organization developed the Mental Health Gap Action Programme to address growing global mental health needs by promoting task sharing in the delivery of psychosocial and psychological interventions. However, little is known about the training needed for non-specialists to deliver these interventions with high levels of competence and fidelity. This article provides a brief conceptual overview of the evidence concerning the training of non-specialists carrying out task-sharing psychosocial and psychological interventions while utilizing illustrative case studies from Kenya, Ethiopia, and the United States to highlight findings from the literature. In this article, the authors discuss the importance of tailoring training to the skills and needs of the non-specialist providers and their roles in the delivery of an intervention. This narrative review with four case studies advocates for training that recognizes the expertise that non-specialist providers bring to intervention delivery, including how they promote culturally responsive care within their communities.


Automated simultaneous analysis of monomethyl and mercuric Hg in biotic samples by Hg-thiourea complex liquid chromatography following acidic thiourea leaching.
A simple leaching procedure has been validated for quantitative isolation of both monomethyl (CH3Hg+) and inorganic (Hg(II)) mercuryfrom fresh or dried biotic tissue for simultaneous analysis via separation and quantification with Hg-thiourea complex liquid chromatography cold vapor atomic fluorescence spectrometry (HgTU/LC-CVAFS). The leaching solution comprises thiourea, hydrochloric acid, and glacial acetic acid and works by protonating thiol binding sites and forming water-soluble cationic CH3HgS=C(NH2)2+ and Hg[S=C(NH2)2]2(2+) complexes, which are easily separated from the solid matrix. The isolated complexes are preconcentrated online by either thiol resin trapping or a new iodide-complex polydivinylbenzene resin trapping (I-PDVB). The I-PDVB trapping involves only one reagent addition, requires no pH adjustments, and is quantitative over a large range of volumes and flow rates. The chromatography system can use either ion chromatography or a new ion-pairing reversed phase separation coupled to cold vapor generation and atomic fluorescence detection. The system allows quantitative sample introduction and yields absolute detection limits of 0.4 pg and 0.7 pg, for CH3Hg+ and Hg(II) respectively, enabling relative detection limits as low as 4 and 7 pg g(-1) with 100 mg samples, and yields % CV routinely less than 5% with well homogenized samples. Accuracy for both forms of mercury has been validated with multiple biotic reference materials and by comparison of the sum of CH3Hg+ and Hg(II) with total Hg on a variety of different biotic sample types (n = 49). The system can be calibrated with either aqueous standards or leached reference materials.


Epidemiology of alcohol use in the general population of Togo.
Alcohol use is responsible for a high level of mortality and morbidity throughout the world. The WHO global strategy recommends that the detrimental effects of alcohol use be reduced. The objective of this paper was to investigate, using data from the 2010 Togo STEPS survey, alcohol use and other health-related factors in the general population of Togo. This epidemiological investigation using the STEPwise approach was undertaken from December 1st, 2010, to January 23rd, 2011, throughout the five regions of Togo. Togo is a low-income country (World Bank) located in West Africa. The study involved 4800 people aged 15 to 64 who were representative of the population of Togo and who were selected using the one-stage cluster sampling method. The sample was young and predominantly male. Approximately one-third of the respondents were alcohol abstainers, with the majority of these being women. Approximately the same proportion of current drinkers (daily consumption) by gender was observed. The reported daily average consumption of alcohol was 13 g of pure alcohol for men and 9 g for women. The mean number of heavy drinking days over the previous 30 days was higher for men (3 days), and this included 37.5% of the men who drink. We suggest a comparative analysis of the prevalence of harmful alcohol use in Togo and the WHO African region.


Electrocardiographic evidence of myocardial salvage after thrombolysis in acute myocardial infarction.
There is a need for a simple clinical measurement that will indicate the extent of myocardial salvage after successful thrombolysis. This study examined whether coronary artery reperfusion reduced the infarct size as assessed electrocardiographically after thrombolytic treatment. The sum of the (sigma) ST segment area in leads showing ST segment elevation in the 12 lead electrocardiogram at presentation was used as an index of potential myocardial injury (initial ischaemic index). The evolved infarct size at 48 h was assessed by a QRS scoring system. Two groups of patients, both admitted with anterior myocardial infarction within 6 h of onset, were studied. Group 1 (n = 35) received analgesia only and group 2 (n = 33) received thrombolytic treatment either by the intracoronary (streptokinase, n = 13) or intravenous route (anistreplase, n = 20). Reperfusion was assessed angiographically. The mean (SD) potential infarct size assessed by the initial ischaemic index was similar in both groups (group 1, sigma ST area = 115 (60) mm2 and group 2 = 126 (77 mm2). The QRS score representing evolved infarct size was significantly lower in the treated group (4.1 (2.5] than in group 1 (7.8 (2.6]. The 95% confidence intervals for QRS scores based on the admission sigma ST area from patients with successful reperfusion were applied to a third set of patients (n = 22) to test the ability of the admission ST area (myocardial injury) to predict the QRS score accurately. While patients with successful reperfusion had significantly lower QRS scores than those who did not (4.5 (3.1) versus 9.3 (3.4)), the wide confidence intervals caused by inter-individual variability precluded an accurate prediction of the QRS score in an individual from the sigma ST area at time of presentation. There was no difference in infarct size in patients treated early (</= 3 h) (QRS score 4.2(2.8)) or later (3-6 h) (4.1(2.1)). This study provides evidence that sequential electrocardiographic changes are reduced in patients with anterior infarction who achieve reperfusion after thrombolytic treatment and that this benefit is shown with treatment given up to six hours after infarct onset. None the less, the relation between the initial ischaemic index and the evolved QRS score has wide confidence intervals, reflecting inter-individual variability, and does not allow the prediction of a QRS score in an individual patient.


Pulmonary injury caused by free fatty acid: evaluation of steroid and albumin therapy.
Intrapulmonary free fatty acid (FFA) release has been suggested as a pathogenetic mechanism in respiratory failure caused by acute pancreatitis and fat embolism. Utilizing the isolated perfused ventilated canine pulmonary lobe, we evaluated the effects of FFA infusion and its subsequent modification by albumin and steroid therapy. In control lobes perfused for a 4-hour period, there was minimal weight gain (11 gm), intrapulmonary shunting did not occur, and compliance remained within normal limits. When 1 ml of oleic acid was infused into the pulmonary artery lobe weight tripled (188 gm), intrapulmonary shunting (20%) developed, and compliance was significantly decreased compared to controls. When 30 gm of human salt-poor albumin was added to the perfusate immediately after FFA infusion, the lobe response was similar to that of untreated oleic acid lobes. In contrast, when 400 mg of methylprednisolone succinate was added to the perfusate immediately after FFA infusion, lobe weight gain was significantly reduced (94 gm) compared to oleic acid lobes, intrapulmonary shunting did not occur, and compliance was within normal limits. This study suggests that steroids may be of benefit in the treatment of respiratory insufficiency secondary to acute pancreatitis and fat embolism.


Deorphanizing NUDIX hydrolases from Trypanosoma: tantalizing links with metabolic regulation and stress tolerance.
An explosion of sequence information in the genomics era has thrown up thousands of protein sequences without functional assignment. Though our ability to predict function based on sequence alone is improving steadily, we still have a long way to go. Proteins with common evolutionary origins carry telling sequence signatures, which ought to reveal their biological roles. These sequence signatures have allowed us to classify proteins into families with similar structures, and possibly, functions. Yet, evolution is a perpetual tinkerer, and hence, sequence signatures alone have proved inadequate in understanding the physiological activities of proteins. One such enigmatic family of enzymes is the NUDIX ( nu cleoside di phosphate linked to a moiety X ) hydrolase family that has over 80000 members from all branches of the tree of life. Though MutT, the founding member of this family, was identified in 1954, we are only now beginning to understand the diversity of substrates and biological roles that these enzymes demonstrate. In a recent article by Cordeiro et al. in Bioscience Reports [Biosci. Rep. (2019)], two members of this protein family from the human pathogen Trypanosoma brucei were deorphanized as being polyphosphate hydrolases. The authors show that of the five NUDIX hydrolases coded by the T. brucei genomes, TbNH2 and TbNH4, show in vitro hydrolytic activity against inorganic polyphosphate. Through classical biochemistry and immunostaining microscopy, differences in their substrate specificities and sub-cellular localization were revealed. These new data provide a compelling direction to the study of Trypanosome stress biology as well as our understanding of the NUDIX enzyme family.


International Society for Extracellular Vesicles: first annual meeting, April 17-21, 2012: ISEV-2012.
Extracellular micro- and nano-scale membrane vesicles produced by different cells are recognised as an essential entity of physiological fluids in a variety of organisms and function as mediators of intercellular communication employed for the regulation of multiple systemic and local processes. In the last decade, an exponential amount of experimental work was dedicated to exploring the biogenesis and secretion mechanisms, physiological and pathological functions and potential applications of the extracellular vesicles (EVs). Noteworthy is the large heterogeneity of in vitro and in vivo models applied, technical approaches developed in these studies and the diversity of designations assigned to different or similar types of EVs. Hence, there is a clear necessity for a uniform nomenclature and standardisation of methods to isolate and characterise these vesicles. In April 2012, the first meeting of the International Society for Extracellular Vesicles (ISEV) took place bringing together this exponentially grown scientific community. The University of Gothenburg (Krefting Research Centre) together with the Interim Board of the Society created in September 2011 (Jan Lötvall, Clotilde Théry, Xandra Breakefield, Marca Wauben, Yong Song Gho, Lawrence Rajendran, Graça Raposo, Douglas Taylor, Margareta Sjöstrand and Esbjörn Telemo) organised this fantastic event that counted 488 registered and contributing participants. This meeting report provides a retrospective summary of the broad spectrum of ISEV-2012 sessions. Again, we emphasise novel findings, discussions and decisions met by the community during the meeting.


A Contemporary Systematic Approach to Assessing the Patient with Heart Failure with Reduced Ejection Fraction: Multimodal Noninvasive and Invasive Evaluation.
Heart failure with reduced ejection fraction (HFrEF) is a progressive clinical syndrome commonly associated with left ventricle dilatation and characterized by reduced cardiac output, secondary pulmonary and systemic venous congestion, and inadequate peripheral oxygen delivery. It is common yet complex and requires synthesis of evidence-based guidelines along with strong clinical acumen. The following is a review of an illustrative case that highlights the important clinical considerations in diagnosis, assessment, and management of HFrEF commonly encountered in practice. Explanations provided highlight of the relevant pathophysiology of HFrEF as well as detailed explanations of interpretation of examinations and both noninvasive and invasive assessment in heart failure. The example provided would hopefully serve as a potential point of reference for trainees as well as healthcare practitioners for patients with HFrEF.


Extramedullary hematopoiesis in the uterine isthmus: a case report and review of the literature.
Extramedullary hematopoiesis (EH) is the ectopic production of myeloid, erythroid and megakaryocytic elements. In postfetal life it usually occurs in conditions with hyperactive, depleted or infiltrated marrow; it is extremely rare in the genital tract. We report a case of EH in the uterine isthmus (UI) which was found incidentally in a 40-year-old patient who presented with a right ovarian cyst and a history of a right modified radical mastectomy for infiltrating invasive lobular carcinoma one year earlier. The patient underwent total abdominal hysterectomy with bilateral salpingo-oophorectomy. Microscopic examination of UI revealed foci of hematopoiesis, consisting of white blood cell precursors confirmed by positive chloroacetate-esterase staining. Bone marrow biospy showed diffuse infiltration secondary to breast cancer. Further work-up, including a bone scan, showed multiple metastases suggesting that the high degree of bone marrow infiltration by neoplasmatic cells had stimulated the EH. In conclusion, the unusual finding of EH in UI heralded widespread infiltration of bone marrow.


Cyclosporine a inhibits apoptosis of rat gingival epithelium.
The use of cyclosporine A (CsA) induces hyperplasia of the gingival epithelium in a site-specific response manner, but the molecular mechanism via which the lesion occurs is unclear. The present research aims to investigate the site-specific effect of CsA on the apoptosis of gingival epithelium associated with gingival hyperplasia. Forty Wistar rats were divided into CsA-treated and non-treated groups. Paraffin-embedded sections of mandibular first molars were selected for hematoxylin and eosin staining, immunohistochemistry analyses of bcl-2 and caspase-3, and the staining of terminal deoxynucleotidyl transfer-mediated dUTP nick-end labeling (TUNEL). The area of the whole gingival epithelium and the length of rete pegs were measured, and the number of bcl-2- and caspase-3-positive cells in the longest rete peg were counted. The analysis of variance for factorial designs and Fisher least significant difference test for post hoc analysis were used to determine the significance levels. In CsA-treated rats, bcl-2 expression was significantly upregulated, whereas caspase-3 expression was downregulated, along with a reduced number of TUNEL-positive cells. The site-specific distribution of bcl-2 was consistent with the site-specific hyperplasia of the gingival epithelium in CsA-treated rats. CsA inhibited gingival epithelial apoptosis via the mitochondrial pathway and common pathway. The antiapoptotic protein bcl-2 might play a critical role in the pathogenesis of the site-specific hyperplasia of gingival epithelium induced by CsA. There were mechanistic differences in the regulation of apoptosis for cells in the attached gingival epithelium, free gingival epithelium, and junctional epithelium.


Proliferating Pilar Tumors: Can Immunohistochemistry Differentiate Benign and Malignant Forms?
Proliferating pilar tumor (PPT) is an adnexal tumor of purported differentiation toward the follicular outer root sheath. Immunohistochemistry has been suggested to differentiate between benign and malignant forms. Eleven benign (PPT) and 9 malignant PPT lesions were reviewed; Ki67, p27, and p53 were applied. The staining intensity (strong, moderate, weak, and negative), positive cell numbers, and marker indexes (%) were scored using image-analysis software (ViraSoft). Overall, there was no significant correlation between Ki67 and p53 and histopathological features. However, malignant PPTs had significantly lower numbers of p27-positive cells (P = 0.030). Our study includes the largest group of patients in whom image analysis of p53, Ki67, and p27 has been used to try to separate benign from malignant lesions. Although there were no significant differences regarding Ki67 and p53, malignant lesions have a statistically lower expression of p27. Further studies may be needed to determine the clinical usefulness of image analysis in this differential diagnosis.


[Sexual behaviour among school adolescents in Córdoba].
To know sexual behaviour among school teenagers and to determine different risk behaviors and groups with specific needs of health education. Cross-sectional study. High-schools from Córdoba. 893 teenagers students selected by random sample. Anonymous self-rated survey about sexual behaviour and uses of contraceptives methods. The average age was 17.16 years (95% CI, 17.05-17.26), 48% males. 22.9% have had completed sexual relations; there were mainly girls (P=.001). The average age for the first relation was 16.64 years (95% CI, 16.45-16.82), with no difference between gender. Among girls, first sexual intercourse use to be with their regular partners (P<.001). 48% of the girls have had one or more sexual intercourse per week, that was only a 19.5% among boys (P<.001). 82% had used a contraceptive method at the first time, and the condom was the method more frequently used. 20% of school teenagers have had complete sexual intercourse, most of them before 18 years. Girls have more frequently sexual relations, usually with their regular partners. Condom is the contraceptive method more frequently used.


Toll-like receptors: lessons to learn from normal and malignant human B cells.
The humoral immune system senses microbes via recognition of specific microbial molecular motifs by Toll-like receptors (TLRs). These encounters promote plasma cell differentiation and antibody production. Recent studies have demonstrated the importance of the TLR system in enhancing antibody-mediated defense against infections and maintaining memory B cells. These results have led the way to the design of vaccines that target B cells by engaging TLRs. In hematologic malignancies, cells often retain B cell-specific receptors and associated functions. Among these, TLRs are currently exploited to target different subclasses of B-cell leukemia, and TLR agonists are currently being evaluated in clinical trials. However, accumulating evidence suggests that endogenous TLR ligands or chronic infections promote tumor growth, thus providing a need for further investigations to decipher the exact function of TLRs in the B-cell lineage and in neoplastic B cells. The aim of this review is to present and discuss the latest advances with regard to the expression and function of TLRs in both healthy and malignant B cells. Special attention will be focused on the growth-promoting effects of TLR ligands on leukemic B cells and their potential clinical impact.


The hospital role of family physicians.
This paper examines the hospital role of a random sample of all general and family physicians in the state of Washington. Of the 287 physicians in our sample, 81% admitted at least one patient to hospital during the two-week study period, and the average physician admitted six patients. The majority of the admitting diagnoses fell within the realm of internal medicine. Residency-trained and board-certified respondents were more likely to admit patients to hospital, and residency-trained and rural practitioners were much less likely to refer patients to other physicians for hospital care than their urban counterparts. Residency-trained physicians, in particular, were much more likely to practice hospital obstetrics than those without formal residency training. These data demonstrate the broad and significant hospital role of the family physician. Given the increased scope and intensity of hospital practice of residency-trained family physicians, and barring major changes in admitting privileges or new regulatory constraints, we predict that this group of physicians will continue to have a significant inpatient role in the United States.


The clinical-care focused psychological interview (CLiC): a structured tool for the assessment of cancer patients' needs.
Integrating the psychosocial perspective in oncology is warranted. Here, we introduce a structured psychological intervention, the clinical-care focused psychological interview (CLiC), to address patients' needs in the relationship with health professionals, clinical pathway and decision-making process. The perceived utility and feasibility of the CLiC were evaluated in a preliminary sample of 30 patients who were candidates to undergo a radical cystectomy at the European Institute of Oncology, Milan, Italy. Patients reported a very high satisfaction with the interview, because it prepared them for their upcoming surgery by gathering more information about their clinical pathway and allowed them to discover the information they still needed. 30% stated that CLiC helped them to reorganise their thoughts and 36.7% understood the role of psychological intervention in the clinical pathway. Only 20% considered the CLiC useful in helping to build their relationship with the clinical staff. Before an invasive surgery such as radical cystectomy, patients' need for information regarding the upcoming surgery seems to prevail. Knowing the future consequences and adjusting toward the disease could be paramount for patients in facing uncertainty and they might feel that creating a relationship with physicians could be a secondary issue. However, our data show that a structured psychological intervention such as the CLiC interview can collect important information for patients and healthcare professionals to develop real patient-centred care.