Oral contraceptives and oral antibiotics: interactions and advice in an accident and emergency setting.
(1) To determine what advice, if any, would be given by accident and emergency (A&E) doctors to women who were taking the combined oral contraceptive pill (OCP) if they had been issued with broad spectrum antibiotics and (2) after an audit programme had been instigated, whether appropriate advice was given to such women. A questionnaire was circulated to 12 doctors working in the Exeter A&E department to assess their level of knowledge in prescribing antibiotics to women taking the OCP. Notes of women aged 15-50 who had been prescribed broad spectrum antibiotics were examined to see if a contraceptive history had been taken. If the patient was found to be taking the combined OCP it was noted whether documented advice had been given about using an additional form of contraception. Six months later after two education sessions had been held, prescriptions and notes were examined. A patient education leaflet was produced to be given to these women, indicating what additional precautions should be taken after having been prescribed antibiotics. The A&E department of a busy district general hospital. Women aged 15-50 who had been issued with broad spectrum antibiotics. The level of knowledge in regard to contraceptive advice given to women taking the OCP among doctors working in an A&E department was poor. However, after educational sessions and the production of a patient information leaflet, there was an improvement in women receiving correct advice. The clinical significance of drug interactions between oral contraceptives and antibiotics indicates the importance of asking a full contraceptive drug history of any woman of childbearing age and documenting this in the notes. Regular audit of this topic is needed to keep it at the front of doctors' minds.


Aldosterone regulation of gene transcription leading to control of ion transport.
Aldosterone, like other steroid hormones, initiates its effects by binding to intracellular receptors; these receptors are then able to control the transcription of several genes. The products of these genes eventually modulate the activity of ionic transport systems located in the apical and the basolateral membrane of specialized epithelial cells, thereby modulating the excretion of Na+ and K+ ions. Considerable progress has been made recently in understanding these mechanisms and the structure of the proteins involved in these processes. A novel principle has been discovered to explain the selective effect of aldosterone on its target epithelia. These tissues exclude competing glucocorticoid hormones by the activity of the 11 beta-hydroxysteroid dehydrogenase to allow aldosterone, an enzyme-resistant steroid, to bind to its receptors. Aldosterone induces numerous changes in the activity of membrane ion transport systems and enzymes and cell morphology. Although the enhancement of Na,K-ATPase synthesis and the increase of the number of active Na+ channels in the apical membrane appear as both direct and primary effects, the mechanisms of the other effects remain to be determined. The knowledge of the primary structure of several elements of the aldosterone response system (e.g., mineralocorticoid receptor and Na,K-ATPase) allows us to understand abnormal regulation of Na+ balance at the molecular level and, potentially, to identify genetic alterations responsible for these defects.


Regulation of medical foods: toward a rational policy.
Medical foods are enterally administered formulas used as complete or supplemental nutrition support in the treatment of diseases and clinical disorders of hospitalized or incapacitated adult patients. Because clinical trials have failed to demonstrate a consistent benefit of these products on morbidity and mortality rates, it has been difficult to reach consensus on their primary role. Are medical foods components of supportive care or should they be considered pharmacologic treatment? To date, the US Congress has defined medical foods, but the Food and Drug Administration has not developed regulatory statutes for them beyond those designed to ensure the sanitary manufacture of all processed foods. Before effective regulations can be developed, medical foods need to be defined further in order to distinguish them from each other and from other classes of nutritional products such as parenteral nutrients or infant formulas. Because we believe that the role of medical foods is in supportive care rather than pharmacologic therapy, we argue that these products should be subject to regulations similar to those that apply to conventional foods.


A naturally occurring G11S mutation in the 3C-like protease from the SARS-CoV-2 virus dramatically weakens the dimer interface.
The 3C-like protease (3CLpro ) is crucial to the replication of SARS-CoV-2, the causative agent of COVID-19, and is the target of several successful drugs including Paxlovid and Xocova. Nevertheless, the emergence of viral resistance underlines the need for alternative drug strategies. 3CLpro only functions as a homodimer, making the protein-protein interface an attractive drug target. Dimerization is partly mediated by a conserved glycine at position 11. However, some naturally occurring SARS-CoV-2 sequences contain a serine at this position, potentially disrupting the dimer. We have used concentration-dependent activity assays and mass spectrometry to show that indeed the G11S mutation reduces the stability of the dimer by 600-fold. This helps to set a quantitative benchmark for the minimum potency required of any future protein-protein interaction inhibitors targeting 3CLpro and raises interesting questions regarding how coronaviruses bearing such weakly dimerizing 3CLpro enzymes are capable of replication. This article is protected by copyright. All rights reserved.


Determinants of turnover among nursing department employees.
A causal model of turnover, or quitting, among hospital nursing department employees was evaluated. This model includes job satisfaction, organizational commitment, and intent to leave as intervening variables that mediate 13 determinants of turnover. The sample consisted of 841 female nursing department employees selected from five hospitals in a western state. Attitudinal and background data were obtained through a mail questionnaire survey, and turnover was monitored for 18 months following the survey. Intent to leave had a strong direct effect on turnover while kinship responsibility, job satisfaction, and organizational commitment had indirect effects on turnover through intent to leave. Task repetitiveness, autonomy, promotional opportunities, and fairness of rewards were important determinants of jobs satisfaction and thus provide a mechanism whereby hospital management may enhance commitment to the organization while reducing turnover.


Production of cytokines by bone marrow cells obtained from patients with multiple myeloma.
Previous work with continuously cultured multiple myeloma lines suggested that cytokine production by tumor cells may mediate some of the medical complications of this disease. To further investigate this issue, we assayed freshly obtained bone marrow (BM) cells from myeloma patients for the in vitro production of cytokines and the presence of cytokine RNA. Production of cytokine protein was assessed by bioassays with the aid of specific neutralizing anticytokine antibodies. These assays detected interleukin-1 (IL-1) and tumor necrosis factor (TNF) secretion by myeloma BM cells, which was significantly greater than secretion from similarly processed BM cells of control individuals. In contrast, lymphotoxin and interleukin-2 (IL-2) production could not be detected. The levels of IL-1 and TNF produced in vitro peaked at 24 hours of culture and correlated with stage and the presence (or absence) of extensive osteolytic bone disease. Northern blot analysis demonstrated the presence of IL-1 beta and TNF RNA in uncultured myeloma BM cells but no detectable IL-1 alpha or lymphotoxin RNA. In addition, the amount of cytokine RNA correlated with protein production, being significantly greater in patients' BM cells than in control marrow. These data suggest a role for IL-1 beta and/or TNF in the pathophysiology of multiple myeloma and argue against a role for lymphotoxin or IL-2.


Effect of Radiofrequency Denervation on Pain Intensity Among Patients With Chronic Low Back Pain: The Mint Randomized Clinical Trials.
Radiofrequency denervation is a commonly used treatment for chronic low back pain, but high-quality evidence for its effectiveness is lacking. To evaluate the effectiveness of radiofrequency denervation added to a standardized exercise program for patients with chronic low back pain. Three pragmatic multicenter, nonblinded randomized clinical trials on the effectiveness of minimal interventional treatments for participants with chronic low back pain (Mint study) were conducted in 16 multidisciplinary pain clinics in the Netherlands. Eligible participants were included between January 1, 2013, and October 24, 2014, and had chronic low back pain, a positive diagnostic block at the facet joints (facet joint trial, 251 participants), sacroiliac joints (sacroiliac joint trial, 228 participants), or a combination of facet joints, sacroiliac joints, or intervertebral disks (combination trial, 202 participants) and were unresponsive to conservative care. All participants received a 3-month standardized exercise program and psychological support if needed. Participants in the intervention group received radiofrequency denervation as well. This is usually a 1-time procedure, but the maximum number of treatments in the trial was 3. The primary outcome was pain intensity (numeric rating scale, 0-10; whereby 0 indicated no pain and 10 indicated worst pain imaginable) measured 3 months after the intervention. The prespecified minimal clinically important difference was defined as 2 points or more. Final follow-up was at 12 months, ending October 2015. Among 681 participants who were randomized (mean age, 52.2 years; 421 women [61.8%], mean baseline pain intensity, 7.1), 599 (88%) completed the 3-month follow-up, and 521 (77%) completed the 12-month follow-up. The mean difference in pain intensity between the radiofrequency denervation and control groups at 3 months was -0.18 (95% CI, -0.76 to 0.40) in the facet joint trial; -0.71 (95% CI, -1.35 to -0.06) in the sacroiliac joint trial; and -0.99 (95% CI, -1.73 to -0.25) in the combination trial. In 3 randomized clinical trials of participants with chronic low back pain originating in the facet joints, sacroiliac joints, or a combination of facet joints, sacroiliac joints, or intervertebral disks, radiofrequency denervation combined with a standardized exercise program resulted in either no improvement or no clinically important improvement in chronic low back pain compared with a standardized exercise program alone. The findings do not support the use of radiofrequency denervation to treat chronic low back pain from these sources. trialregister.nl Identifier: NTR3531.


Use of a humanoid robot for auditory psychophysical testing.
Tasks in psychophysical tests can at times be repetitive and cause individuals to lose engagement during the test. To facilitate engagement, we propose the use of a humanoid NAO robot, named Sam, as an alternative interface for conducting psychophysical tests. Specifically, we aim to evaluate the performance of Sam as an auditory testing interface, given its potential limitations and technical differences, in comparison to the current laptop interface. We examine the results and durations of two voice perception tests, voice cue sensitivity and voice gender categorisation, obtained from both the conventionally used laptop interface and Sam. Both tests investigate the perception and use of two speaker-specific voice cues, fundamental frequency (F0) and vocal tract length (VTL), important for characterising voice gender. Responses are logged on the laptop using a connected mouse, and on Sam using the tactile sensors. Comparison of test results from both interfaces shows functional similarity between the interfaces and replicates findings from previous studies with similar tests. Comparison of test durations shows longer testing times with Sam, primarily due to longer processing times in comparison to the laptop, as well as other design limitations due to the implementation of the test on the robot. Despite the inherent constraints of the NAO robot, such as in sound quality, relatively long processing and testing times, and different methods of response logging, the NAO interface appears to facilitate collecting similar data to the current laptop interface, confirming its potential as an alternative psychophysical test interface for auditory perception tests.


Induction by dopamine D1 receptor agonist ABT-431 of dyskinesia similar to levodopa in patients with Parkinson disease.
Dyskinesias are a frequent adverse effect of long-term levodopa therapy. The relative contribution of dopamine D(1) and D(2) receptor function to the pathophysiology of levodopa-induced dyskinesias remains a matter of controversy. To establish whether a selective D(1) dopamine agonist induces more or less dyskinesia than levodopa in primed dyskinetic patients with Parkinson disease. We studied ABT-431, the prodrug of a fully selective D(1) agonist, in 20 subjects with advanced Parkinson disease and a fluctuating response to levodopa complicated by dyskinesias. Eight patients were studied in a double-blind, randomized design (French centers); 12, in an open, randomized design (US centers). We assessed and compared the antiparkinsonian (Unified Parkinson's Disease Rating Scale) and dyskinetic (response induced by an acute challenge of a suprathreshold dose of levodopa and by 4 different ascending doses (5, 10, 20, and 40 mg) of ABT-431 during the 6 hours after the challenge. The separate analysis of the double-blind and open data led to the same findings, ie, the antiparkinsonian and dyskinetic responses induced by ABT-431 were dose related. At the most effective doses (20 and 40 mg), ABT-431 exhibited similar antiparkinsonian benefit and produced similar dyskinesias as levodopa. Dopamine D(1) agonists can induce a full antiparkinsonian response but do not support previous hypotheses suggesting that D(1) agonists are more or less likely to produce dyskinesias than levodopa.


Rapid onset of transmission-reducing antibodies in javanese migrants exposed to malaria in papua, indonesia.
Transmission of Plasmodium falciparum malaria is initiated by sexual stages in the mosquito. Anti-Pfs48/45 and anti-Pfs230 sexual stage antibodies that are ingested together with parasites can reduce parasite development and subsequently malaria transmission. Acquisition of sexual stage immunity was studied in a cohort of 102 non-immune Javanese individuals migrating to hyperendemic Papua Indonesia. Seroprevalence of antibodies against Pfs48/45 and Pfs230 and functional transmission-reducing activity (TRA) were measured upon arrival and at 6, 12, and 24 months. Asexual parasitemia and gametocytemia were assessed every two weeks. The TRA and seroreactivity increased with the number of P. falciparum infections. The longitudinally sustained association between TRA and antibodies against Pfs48/45 (odds ratio [OR] = 3.74, 95% confidence interval [CI] = 1.51-9.29) and Pfs230 (OR = 3.72, 95% CI = 1.36-10.17) suggests that functional transmission reducing immunity is acquired after limited exposure to infection.


Priming of human neutrophils with N-formyl-methionyl-leucyl-phenylalanine by a calcium-independent, pertussis toxin-insensitive pathway.
Resting neutrophils may be "primed" to augmented effector function, eg, superoxide (O2-) production in the respiratory burst, upon a second stimulation with a variety of soluble agonists including formylated methionyl-leucyl-phenylalanine (FMLP) and phorbol myristate acetate (PMA). At priming concentrations of FMLP (5 x 10(-9) mol/L) that did not initiate O2- generation, two metabolic activities were noted: (1) approximately a threefold increase in the baseline intracellular calcium (Ca++i) level, that was not dependent on extracellular Ca++, and (2) a rapid rise in intracellular pH that was blocked by 5-(N,N-dimethyl) amiloride (DA), that had no effect on the Ca++i response to priming. Furthermore, there were no significant increases in inositol metabolites in cells primed and stimulated with FMLP compared with cells receiving the stimulating dose of FMLP alone and pretreatment with pertussis toxin (PT) (before the addition of the priming -5 x 10(-9) mol/L dose of FMLP), whereas abolishing the response to FMLP during the second stage of stimulation, had (1) no effect on FMLP-primed cells subsequently stimulated with PMA, and (2) only partially ablated the rise in Ca++i initiated with FMLP. That FMLP priming involved distinctive processes to those of the well characterized FMLP-coupled Ca++-dependent activation cascade was shown by the full priming effect attained in a Ca++-free buffer, which did not sustain an O2- response to a second-stage FMLP stimulation, but sustained a primed response to PMA. These data demonstrate that FMLP primes human neutrophils by a Ca++-independent and PT-insensitive pathway, offering a functional model for studying heterogeneous FMLP receptor-coupled reactions.


Psychometric Features of Dizziness Handicap Inventory (DHI): Development and Standardization in Gujarati Language.
Dizziness is a sensation of spatial disorientation of rotating or non-rotating in nature, mostly due to Oto-neurological insults. A symptom-specific dizziness handicap inventory may aid in understanding the severity of the condition and its impact on the quality of life. Therefore, the Dizziness Handicap Inventory-English version (DHI-E) was used as the most reliable subjective tool to assess the effect of dizziness in various aspects of life and so was translated into several languages. Hence, the present study aimed at developing and standardizing the self-administering Dizziness Handicap Inventory - Gujarati version (DHI-G). A cross-sectional survey design was performed. 50 participants, i.e. 18 males and 32 females aged between 15 to 55 years with complaints of vertigo/dizziness were recruited for the study. The questionnaire was re-administered to thirty of the total participants after 10 days of initial administration for examining test-retest reliability. DHI-G achieved an overall alpha score of 0.92 suggesting good internal consistency and the score of 0.84, 0.82 and 0.81 on three subscales i.e. physical, functional and emotional respectively. Intra class correlation (ICC) revealed good test-retest reliability with a score of 0.81. DHI-G can be used as a reliable and valid tool in the clinical and research setting extensively.


Dual stem cell therapy synergistically improves cardiac function and vascular regeneration following myocardial infarction.
Since both myocardium and vasculature in the heart are excessively damaged following myocardial infarction (MI), therapeutic strategies for treating MI hearts should concurrently target both so as to achieve true cardiac repair. Here we demonstrate a concomitant method that exploits the advantages of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) and human mesenchymal stem cell-loaded patch (hMSC-PA) to amplify cardiac repair in a rat MI model. Epicardially implanted hMSC-PA provide a complimentary microenvironment which enhances vascular regeneration through prolonged secretion of paracrine factors, but more importantly it significantly improves the retention and engraftment of intramyocardially injected hiPSC-CMs which ultimately restore the cardiac function. Notably, the majority of injected hiPSC-CMs display adult CMs like morphology suggesting that the secretomic milieu of hMSC-PA constitutes pleiotropic effects in vivo. We provide compelling evidence that this dual approach can be a promising means to enhance cardiac repair on MI hearts.


Low dead-space syringes for preventing HIV among people who inject drugs: promise and barriers.
This review examines evidence regarding the differential effects of high dead-space syringes (HDSS) and low dead-space syringes (LDSS) on HIV transmission among people who inject drugs (PWID). It also identifies areas for additional research and examines potential barriers to interventions that promote LDSS. Results of laboratory experiments and cross-sectional bio-behavioral surveys provide circumstantial evidence that the probability of HIV transmission associated with sharing LDSS is less than the probability of HIV transmission associated with sharing HDSS. Mathematical models suggest that LDSS may prevent injection-related HIV epidemics among PWID. Circumstantial evidence suggests that LDSS may substantially reduce HIV transmission among PWID, who share syringes. Additional research that links LDSS to reductions in HIV incidence is needed. Most currently available LDSS are 1 ml or smaller and have fixed needles. These cannot be used by PWID 'injecting' larger volumes of fluid and they may be rejected by PWID, who prefer syringes with detachable needles. Nonetheless, LDSS represent a potentially promising intervention that deserves serious consideration.


A 1-year follow-up study of capacity to love and work: What components of borderline personality disorder most impair interpersonal and vocational functioning?
For individuals with borderline personality disorder (BPD), both the reduction in symptoms and the improvement of vocational and interpersonal function (psychosocial function) are important for recovery. Research suggests that some components of BPD make it harder to achieve functional recovery; however, findings are varied and inconclusive. The present study assesses recovery over time in BPD, investigates which symptoms make it harder to function and explores the relationships between these symptoms. One hundred ninety-nine consecutively recruited individuals in psychological treatment for personality disorder were studied over 12 months. Measures of BPD symptom severity at intake were used to predict improvements in social and vocational function at follow-up. Exploratory modelling was conducted to understand the relationships between symptoms and function. Following 12 months of treatment, symptoms and functioning improved. Those who experienced more severe emptiness, impulsivity and self-harm had worse outcomes. A relationship between chronic emptiness at intake and impaired vocational outcome (days out of work) at follow-up was found, mediated by severity of impulsivity and frequency of self-harm. Chronic emptiness is associated with dysfunctional behaviours such as impulsivity and self-harm, and poor psychosocial improvement. Interventions targeting chronic emptiness in those most vulnerable may improve functional outcomes. © 2018 John Wiley & Sons, Ltd.


A transcriptomic snapshot of early molecular communication between Pasteuria penetrans and Meloidogyne incognita.
Southern root-knot nematode Meloidogyne incognita (Kofoid and White, 1919), Chitwood, 1949 is a key pest of agricultural crops. Pasteuria penetrans is a hyperparasitic bacterium capable of suppressing the nematode reproduction, and represents a typical coevolved pathogen-hyperparasite system. Attachment of Pasteuria endospores to the cuticle of second-stage nematode juveniles is the first and pivotal step in the bacterial infection. RNA-Seq was used to understand the early transcriptional response of the root-knot nematode at 8 h post Pasteuria endospore attachment. A total of 52,485 transcripts were assembled from the high quality (HQ) reads, out of which 582 transcripts were found differentially expressed in the Pasteuria endospore encumbered J2 s, of which 229 were up-regulated and 353 were down-regulated. Pasteuria infection caused a suppression of the protein synthesis machinery of the nematode. Several of the differentially expressed transcripts were putatively involved in nematode innate immunity, signaling, stress responses, endospore attachment process and post-attachment behavioral modification of the juveniles. The expression profiles of fifteen selected transcripts were validated to be true by the qRT PCR. RNAi based silencing of transcripts coding for fructose bisphosphate aldolase and glucosyl transferase caused a reduction in endospore attachment as compared to the controls, whereas, silencing of aspartic protease and ubiquitin coding transcripts resulted in higher incidence of endospore attachment on the nematode cuticle. Here we provide evidence of an early transcriptional response by the nematode upon infection by Pasteuria prior to root invasion. We found that adhesion of Pasteuria endospores to the cuticle induced a down-regulated protein response in the nematode. In addition, we show that fructose bisphosphate aldolase, glucosyl transferase, aspartic protease and ubiquitin coding transcripts are involved in modulating the endospore attachment on the nematode cuticle. Our results add new and significant information to the existing knowledge on early molecular interaction between M. incognita and P. penetrans.


Characterization of neural cell adhesion sites: point contacts are the sites of interaction between integrins and the cytoskeleton in PC12 cells.
As developing or regenerating neurons grow, their axons seek cues in the extracellular matrix that are recognized by integrin receptors. To understand the regulation and structure of neural integrin complexes, we have examined the association of two functionally important integrins, alpha 1 beta 1 and alpha 3 beta 1, within PC12 cells. Detergent-resistant cytoskeletal ghosts were prepared from PC12 cells and examined by immunoblotting. In cells maintained in suspension the alpha 1, alpha 3, and beta 1 integrin subunits were solubilized by Triton X-100 detergent. In contrast, when cells were grown on collagen or laminin about 50% of the alpha 1 and beta 1 subunits were retained with the cytoskeleton, but alpha 3 remained soluble. Confocal immunofluorescence microscopy of whole cells demonstrated that all three integrin subunits were expressed in a punctate pattern on the cell surface in point contacts. Point contacts were also found to be the predominant adhesion structure of dorsal root ganglion neurons. After detergent extraction of PC12 cells, the point contacts remained only at the cell-substrate interface. Vinculin, which is found consistently in focal contacts on non-neural cells, showed only a partial colocalization with the point contacts, being expressed mainly at the tips of filopodia and the periphery of cell bodies. Talin showed no obvious codistribution with beta 1 integrin immunoreactivity in point contacts. Immunoreactivity to p125FAK was not detected in PC12 cells, although astrocytes, which have both focal contacts and point contacts have p125FAK only at focal contacts. These observations, together with previous data (Turner et al., 1989; Tawil et al., 1993), suggest that point contacts are functional adhesion sites and are structurally distinct from focal contacts found in non-neuronal cells.


Treatment Modalities for Acne.
Acne is a common inflammatory skin disease which affects the pilosebaceous units of the skin. It can have severe psychological effects and can leave the patient with severe skin scarring. There are four well-recognized pathological factors responsible for acne which is also the target for acne therapy. In this review, different treatment options are discussed, including topical (i.e., retinoids, and antibiotics) and systemic (i.e., retinoids, antibiotics, and hormonal) treatments. Since the general public has been showing an increasing interest in more natural and generally safer treatment options, the use of complementary and alternative medicines (CAM) for treating acne was also discussed. The use of physical therapies such as comedone extraction, cryoslush therapy, cryotherapy, electrocauterization, intralesional corticosteroids and optical treatments are also mentioned. Acne has been extensively researched with regards to the disease mechanism as well as treatment options. However, due to the increasing resistance of Propionibacterium acnes towards the available antibiotics, there is a need for new treatment methods. Additionally, the lack of necessary evidence on the efficacy of CAM therapies makes it necessary for researchers to investigate these treatment options further.


Graphene oxide-carbon nanotube hybrid assemblies: cooperatively strengthened OH···O[double bond, length as m-dash]C hydrogen bonds and the removal of chemisorbed water.
Owing to their great significance for energy storage and sensing applications, multi-layer papers consisting of graphene oxide-carbon nanotube (GO-CNT) hybrid sheets were prepared by in situ exfoliation of graphite oxide in the presence of oxidized CNTs (oCNTs). For the first time we elucidate the influence of oCNTs on chemisorbed water (CW), i.e. the water molecules inherently bound to the oxygen functional groups (OFGs) of graphene oxide (GO) and responsible for irreversible structural damage upon thermal reduction processes. We show that oCNTs self-assemble onto GO sheets during the liquid phase processing steps by forming cooperatively strengthened OH···O[double bond, length as m-dash]C hydrogen bonds between the carboxylic groups of the oCNTs and OFGs of GO. At oCNT amounts of about 10 to 15 wt% this leads to the displacement of considerable amounts of CW without altering the original chemical composition of GO. The thermally reduced GO-CNT (rGO-CNT) papers reveal improved sp2 character and an enhancement of the specific capacitance by 75% with respect to thermally reduced GO (rGO), largely due to the effective removal of CW by oxidized CNTs. These findings disclose the relevance of the cooperative hydrogen bonding phenomena in graphene oxide paper/film electrodes for the development of improved electrochemical energy storage and sensing devices.


Clinical and molecular practice of European thoracic pathology laboratories during the COVID-19 pandemic. The past and the near future.
This study evaluated the consequences in Europe of the COVID-19 outbreak on pathology laboratories orientated toward the diagnosis of thoracic diseases. A survey was sent to 71 pathology laboratories from 21 European countries. The questionnaire requested information concerning the organization of biosafety, the clinical and molecular pathology, the biobanking, the workload, the associated research into COVID-19, and the organization of education and training during the COVID-19 crisis, from 15 March to 31 May 2020, compared with the same period in 2019. Questionnaires were returned from 53/71 (75%) laboratories from 18 European countries. The biosafety procedures were heterogeneous. The workload in clinical and molecular pathology decreased dramatically by 31% (range, 3%-55%) and 26% (range, 7%-62%), respectively. According to the professional category, between 28% and 41% of the staff members were not present in the laboratories but did teleworking. A total of 70% of the laboratories developed virtual meetings for the training of residents and junior pathologists. During the period of study, none of the staff members with confirmed COVID-19 became infected as a result of handling samples. The COVID-19 pandemic has had a strong impact on most of the European pathology laboratories included in this study. Urgent implementation of several changes to the organization of most of these laboratories, notably to better harmonize biosafety procedures, was noted at the onset of the pandemic and maintained in the event of a new wave of infection occurring in Europe.


Developmental Expression of SULT1C4 Transcript Variants in Human Liver: Implications for Discordance Between SULT1C4 mRNA and Protein Levels.
The cytosolic sulfotransferases (SULTs) metabolize a variety of xenobiotic and endogenous substrates. Several SULTs are expressed in the fetus, implying that these enzymes have important functions during human development. We recently reported that while SULT1C4 mRNA is abundant in prenatal human liver specimens, SULT1C4 protein is barely detectable. Two coding transcript variants (TVs) of SULT1C4 are indexed in GenBank, TV1 (full-length) and TV2 (lacking exons 3 and 4). The purpose of this study was to evaluate expression of the individual TVs as a clue for understanding the discordance between mRNA and protein levels. Reverse-transcription polymerase chain reaction was initially performed to identify TVs expressed in intestinal and hepatic cell lines. This analysis generated fragments corresponding to TV1, TV2, and a third variant that lacked exon 3 (E3DEL). Using reverse-transcription quantitative polymerase chain reaction assays designed to quantify TV1, TV2, or E3DEL individually, all three TVs were more highly expressed in prenatal than postnatal specimens. TV2 levels were ∼fivefold greater than TV1, while E3DEL levels were minimal. RNA sequencing (RNA-seq) analysis of another set of liver specimens confirmed that TV1 and TV2 levels were highest in prenatal liver, with TV2 higher than TV1. RNA-seq also detected a noncoding RNA, which was also more abundant in prenatal liver. Transfection of HEK293T cells with plasmids expressing individual Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-tagged SULT1C4 isoforms demonstrated that TV1 produced much more protein than did TV2. These data suggest that the lack of correspondence between SULT1C4 mRNA and protein levels in human liver is likely attributable to the inability of the more abundant TV2 to produce stable protein. SIGNIFICANCE STATEMENT: Cytosolic sulfotransferases (SULTs) metabolize a variety of xenobiotic and endogenous substrates, and several SULTs are highly expressed in the fetus, implying that they have important functions during human development. SULT1C4 is highly expressed in prenatal liver at the mRNA level but not the protein level. This study provides an explanation for this discordance by demonstrating that the predominant SULT1C4 transcript is a variant that produces relatively little protein.


Contributions of CYP2C9/CYP2C19 genotypes and drug interaction to the phenytoin treatment in the Korean epileptic patients in the clinical setting.
We examined the contribution of CYP2C9 and CYP2C19 genotypes and drug interactions to the phenytoin metabolism among 97 Korean epileptic patients to determine if pharmacogenetic testing could be utilized in routine clinical practice. The CYP2C9 polymorphism is a wellknown major genetic factor responsible for phenytoin metabolism. The CYP219 polymorphism, with a high incidence of variant alleles, has a minor influence on phenytoin treated Koran patients. Using a multiple regression model for evaluation of the CYP2C9 and CYP2C19 genotypes, together with other non-genetic variables, we explained 39.6% of the variance in serum phenytoin levels. Incorporation of genotyping for CYP2C9 and CYP2C19 into a clinical practice may be of some help in the determination of phenytoin dosage. However, because concurrent drug treatment is common in patients taking phenytoin and many environmental factors are likely to play a role in drug metabolism, these factors may overwhelm the relevance of CYP polymorphisms in the clinical setting. Further investigations with an approach to dose assessment that includes comprehensive interpretation of both pharmacogenetic and pharmacokinetic data along with understanding of the mechanism of drug interactions in dosage adjustment is warranted.


The expansion of mechanistic and organismic diversity associated with non-ribosomal peptides.
Non-ribosomal peptides are a group of secondary metabolites with a wide range of bioactivities, produced by prokaryotes and lower eukaryotes. Recently, non-ribosomal synthesis has been detected in diverse microorganisms, including the myxobacteria and cyanobacteria. Peptides biosynthesized non-ribosomally may often play a primary or secondary role in the producing organism. Non-ribosomal peptides are often small in size and contain unusual or modified amino acids. Biosynthesis occurs via large modular enzyme complexes, with each module responsible for the activation and thiolation of each amino acid, followed by peptide bond formation between activated amino acids. Modules may also be responsible for the enzymatic modification of the substrate amino acid. Recent analysis of biosynthetic gene clusters has identified novel integrated, mixed and hybrid enzyme systems. These diverse mechanisms of biosynthesis result in the wide variety of non-ribosomal peptide structures and bioactivities seen today. Knowledge of these biosynthetic systems is rapidly increasing and methods of genetically engineering these systems are being developed. In the future, this may lead to rational drug design through combinatorial biosynthesis of these enzyme systems.


OH(3) (-) and O(2)H(5) (-) double Rydberg anions: predictions and comparisons with NH(4) (-) and N(2)H(7) (-).
A low barrier in the reaction pathway between the double Rydberg isomer of OH(3) (-) and a hydride-water complex indicates that the former species is more difficult to isolate and characterize through anion photoelectron spectroscopy than the well known double Rydberg anion (DRA), tetrahedral NH(4) (-). Electron propagator calculations of vertical electron detachment energies (VEDEs) and isosurface plots of the electron localization function disclose that the transition state's electronic structure more closely resembles that of the DRA than that of the hydride-water complex. Possible stabilization of the OH(3) (-) DRA through hydrogen bonding or ion-dipole interactions is examined through calculations on O(2)H(5) (-) species. Three O(2)H(5) (-) minima with H(-)(H(2)O)(2), hydrogen-bridged, and DRA-molecule structures resemble previously discovered N(2)H(7) (-) species and have well separated VEDEs that may be observable in anion photoelectron spectra.


Opportunities and Challenges for Personal Heat Exposure Research.
Environmental heat exposure is a public health concern. The impacts of environmental heat on mortality and morbidity at the population scale are well documented, but little is known about specific exposures that individuals experience. The first objective of this work was to catalyze discussion of the role of personal heat exposure information in research and risk assessment. The second objective was to provide guidance regarding the operationalization of personal heat exposure research methods. We define personal heat exposure as realized contact between a person and an indoor or outdoor environment that poses a risk of increases in body core temperature and/or perceived discomfort. Personal heat exposure can be measured directly with wearable monitors or estimated indirectly through the combination of time-activity and meteorological data sets. Complementary information to understand individual-scale drivers of behavior, susceptibility, and health and comfort outcomes can be collected from additional monitors, surveys, interviews, ethnographic approaches, and additional social and health data sets. Personal exposure research can help reveal the extent of exposure misclassification that occurs when individual exposure to heat is estimated using ambient temperature measured at fixed sites and can provide insights for epidemiological risk assessment concerning extreme heat. Personal heat exposure research provides more valid and precise insights into how often people encounter heat conditions and when, where, to whom, and why these encounters occur. Published literature on personal heat exposure is limited to date, but existing studies point to opportunities to inform public health practice regarding extreme heat, particularly where fine-scale precision is needed to reduce health consequences of heat exposure. https://doi.org/10.1289/EHP556.


Comparison of two doses of recombinant human bone morphogenetic protein in absorbable collagen sponges for bone healing in dogs.
To determine the effects of 2 doses of recombinant human bone morphogenetic protein-2 in an absorbable collagen sponge (rhBMP-2/ACS) on bone healing in dogs. 27 adult dogs. Dogs underwent a mid-diaphyseal (1-mm) tibial osteotomy (stabilized with external skeletal fixation) and received an ACS containing 0.28 mg (0.2 mg/mL) or 0.56 mg (0.4 mg/mL) of rhBMP-2 or no treatment (control dogs). All dogs were examined daily; bone healing was assessed via radiography and subjective lameness evaluation every 2 weeks. After euthanasia at 8 weeks, tibiae were evaluated biomechanically and histologically. Control dogs required antimicrobial treatment for pin-site-related complications more frequently than did rhBMP-2/ACS-treated dogs. At 4 and 6 weeks, weight bearing was greater in dogs treated with rhBMP-2/ACS (0.2 mg/mL) than in control dogs, albeit not significantly. Compared with control treatment, both doses of rhBMP-2/ACS accelerated osteotomy healing at 4, 6, and 8 weeks, and the 0.2 mg/mL dose enhanced healing at 2 weeks; healing at 6 weeks was greater for the lower-dose treatment than for the higher-dose treatment. Histologically, healing at 8 weeks was significantly improved for both rhBMP-2/ACS treatments, compared with control treatment. Among groups, biomechanical variables did not differ, although less osteotomy-site failures occurred in rhBMP-2/ACS-treated groups. In dogs that underwent tibial osteotomy, rhBMP-2/ACS (0.2 mg/mL) appeared to accelerate bone healing and reduce lameness (compared with control treatment) and apparently augmented bone healing more than rhBMP-2/ACS (0.4 mg/mL). Compared with control dogs, rhBMP-2/ACS-treated dogs required antimicrobial treatments less frequently.


C-7 analogues of progesterone as potent inhibitors of the P-glycoprotein efflux pump.
The P-glycoprotein product (Pgp) of the MDR1 gene has been implicated in the multiple drug resistance phenotype expressed by many cancers. Functioning as an efflux pump, P-glycoprotein prevents the accumulation of high intracellular concentrations of substrates. We have taken a rational approach to designing inhibitors of P-glycoprotein function, selecting a natural substrate (progesterone) as our lead compound. We hypothesized that progesterone, substituted at C-7 with an aromatic moiety(s), would exhibit reduced Pgp affinity, significantly increased antiPgp activity, and reduced affinity for progesterone receptors (PGR). We synthesized 7 alpha-[4'-(aminophenyl)thio]pregna-4-ene-3,20-dione (2), which comprises a C-7 alpha thiol bridge linking an aminophenyl moiety to progesterone, from pregna-4,6-diene-3,20-dione (1). The subsequent addition reaction of 2 with the appropriate isocyanate produced an initial series of compounds (3-6). Compounds 3-5 (respectively, -CH(2)CH(2)Cl; -CH(2)CH(3); and -CH(CH(3))C(6)H(5)) exhibit a significantly increased ability to inhibit P-glycoprotein. Potency for restoring doxorubicin accumulation in MDR1-transduced human breast cancer cells is increased up to 60-fold as compared with progesterone. Compound 5 has greater potency than verapamil and is equipotent with cyclosporin A, for inhibiting P-glycoprotein function. Furthermore, 5 does not bind to PGR, implying a potential reduction in in vivo toxicity. These data identify C-7-substituted progesterone analogues and 5, in particular, as rationally designed antiPgp compounds worthy of further evaluation/development.


Frequency of Acentric Fragments Are Associated with Cancer Risk in Subjects Exposed to Ionizing Radiation.
Biomonitoring is currently applied in the estimation of health risks after overexposure to ionizing radiation (IR). The aim of this study was to compare the association of dicentric chromosomes and acentric fragments (AF) with cancer risk in subjects exposed to IR, as well as in control subjects. The study was performed on 3,574 subjects (2,030 subjects exposed to IR and 1,544 control subjects). The mean follow-up period was 8 years. In subjects reporting exposure to IR, the presence of AFs and dicentric chromosomes was associated with a significant increase in cancer risk, hazard ratio (HR)=1.78 (95% confidence interval (CI)=1.01-3.13) and HR=1.73 (95% CI=1.03-2.90), respectively. AFs are associated with cancer risk and have a similar sensitivity to dicentric chromosomes in subjects exposed to IR. Because automated AF scoring can be easily introduced using fast flow cytometry combined with the pan-centromere staining, this biomarker may hold promise as a potential sensitive biomarker of exposure to IR and cancer risk.


Nodular regenerative hyperplasia in partial hepatectomy specimens.
To investigate possible associations of nodular regenerative hyperplasia (NRH) with antitumor chemotherapy, we reviewed 72 partial hepatic resections (55 with metastatic tumor, 12 hepatocellular carcinomas, and five benign neoplasms). Thirty autopsy livers from adults without malignancies served as controls. Studies included hematoxylin and eosin, reticulin, and trichrome stains and immunostaining for proliferating cell nuclear antigen (PCNA). Five of 72 livers (7%) had NRH. All five patients had received chemotherapy, one by intrahepatic artery infusion. Four had received chemotherapy 2 months or less before undergoing partial hepatectomy. These five cases represented 15% of the 33 patients who received chemotherapy. No NRH was seen in autopsy control livers. In contrast to NRH, multiple hyperplastic foci were seen in 28 of 72 livers (39%). This finding did not correlate with chemotherapy. Two of 30 control livers (7%) showed similar mild regenerative changes. In only one case of NRH was PCNA staining increased over controls. A band of PCNA-positive hepatocytes was seen adjacent to the tumor in 21 cases, suggesting that the presence of tumor may cause a local increase in PCNA expression. Mitoses in hepatocytes and assessment of the thickness of liver cell plates were more sensitive indicators of regeneration than PCNA. Vascular changes, such as sinusoidal fibrosis (11 of 72 cases), thickened hepatic arterioles (13 of 72 cases), and mild thickening of central veins (10 of 72 cases), did not correlate with NRH, hyperplastic foci, or chemotherapy. No cases of hepatoportal sclerosis were identified.


Recent changes in mountain grasslands: a vegetation resampling study.
Understanding how land-use changes affect different facets of plant biodiversity in seminatural European grasslands is of particular importance for biodiversity conservation. As conclusions of previous experimental or synchronic observational studies did not converge toward a general agreement, assessing the recent trends in vegetation change in various grassland systems using a diachronic approach is needed. In this resurvey study, we investigated the recent changes in grassland vegetation of the French Jura Mountains, a region with a long tradition of pastoralism. We compared the floristic composition of 150 grassland plots recorded between 1990 and 2000 with new relevés made in 2012 on the same plots. We considered taxonomic, phylogenetic and functional diversity as well as ecological characteristics of the plant communities derived from ecological indicator values and life strategies of the species. PCA of the floristic composition revealed a significant general trend linked to the sampling year. Wilcoxon paired tests showed that contemporary communities were generally more dominated by grass species and presented a higher tolerance to defoliation, a higher pastoral value, and a higher nutrient indicator value. Comparisons revealed a decrease in phylogenetic and functional diversity. By contrast, local species richness has slightly increased. The intensity of change in species composition, measured by Hellinger distance between pairs of relevés, was dependent on neither the time lag between the two surveys, the author of the first relevé nor its location or elevation. The most important changes were observed in grasslands that previously presented low pastoral value, low grass cover, low tolerance to defoliation, and high proportion of stress-tolerant species. This trend was likely linked to the intensification of grassland management reported in the region, with a parallel increase in mowing frequency, grazing pressure, and fertilization level. More restrictive specifications should be applied to agricultural practices to avoid overexploitation of mountain species-rich grasslands and its negative consequences on their biodiversity and resilience.


An "off-the-shelf" capillary microfluidic device that enables tuning of the droplet breakup regime at constant flow rates.
The fabrication of glass capillary microfluidic devices is technically challenging, often hampering use of the design. We describe a new technique, based on commercially available components, for assembling flow focusing capillary devices that can readily be taken apart and cleaned between uses. This design strategy allows for generation of both water-in-oil and oil-in-water emulsions in the same device after an ethanol rinse. The modularity of the device enables the adjustment of the tip separation between the two inner capillaries during droplet generation, which enables tuning of the age of the interface. Time-dependent surfactant diffusion to the interface changes the interfacial tension, thus providing an approach for adjusting the capillary number in addition to the usual method of changing flow rates. This design enables the tuning of the mode of breakup and the droplet size.


Infection of mast cells with live streptococci causes a toll-like receptor 2- and cell-cell contact-dependent cytokine and chemokine response.
Mast cells (MCs) are strongly implicated in immunity toward bacterial infection, but the molecular mechanisms by which MCs contribute to the host response are only partially understood. We addressed this issue by examining the direct effects of a Gram-positive pathogen, Streptococcus equi, on bone marrow-derived MCs (BMMCs). Ultrastructural analysis revealed extensive formation of dilated rough endoplasmic reticulum in response to bacterial infection, indicating strong induction of protein synthesis. However, the BMMCs did not show signs of extensive degranulation, and this was supported by only slow release of histamine in response to infection. Coculture of live bacteria with BMMCs caused a profound secretion of CCL2/MCP-1, CCL7/MCP-3, CXCL2/MIP-2, CCL5/RANTES, interleukin-4 (IL-4), IL-6, IL-12, IL-13, and tumor necrosis factor alpha, as shown by antibody-based cytokine/chemokine arrays and/or enzyme-linked immunosorbent assay. In contrast, heat-inactivated bacteria caused only minimal cytokine/chemokine release. The cytokine/chemokine responses were substantially attenuated in Toll-like receptor 2-deficient BMMCs and were strongly dependent on cell-cell contacts between bacteria and BMMCs. Gene chip microarray analysis confirmed a massively upregulated expression of the genes coding for the secreted cytokines and chemokines and also identified a pronounced upregulation of numerous additional genes, including transcription factors, signaling molecules, and proteases. Together, the present study outlines MC-dependent molecular events associated with Gram-positive infection and thus provides an advancement in our understanding of how MCs may contribute to host defense toward bacterial insults.


Whole genome sequencing of Mycobacterium tuberculosis SB24 isolated from Sabah, Malaysia.
Mycobacterium tuberculosis (M. tuberculosis) is the causative agent of tuberculosis (TB) that causes millions of death every year. We have sequenced the genome of M. tuberculosis isolated from cerebrospinal fluid (CSF) of a patient diagnosed with tuberculous meningitis (TBM). The isolated strain was referred as M. tuberculosis SB24. Genomic DNA of the M. tuberculosis SB24 was extracted and subjected to whole genome sequencing using PacBio platform. The draft genome size of M. tuberculosis SB24 was determined to be 4,452,489 bp with a G + C content of 65.6%. The whole genome shotgun project has been deposited in NCBI SRA under the accession number SRP076503.


Reaction-diffusion waves of advance in the transition to agricultural economics.
In a previous paper [J. Fort and V. Méndez, Phys. Rev. Lett. 82, 867 (1999)], the possible importance of higher-order terms in a human population wave of advance has been studied. However, only a few such terms were considered. Here we develop a theory including all higher-order terms. Results are in good agreement with the experimental evidence involving the expansion of agriculture in Europe.


The Impact of Progressive Simulation-Based Training on Tourniquet Application.
The Advanced Combat Medical Experience (ACME) is a progressive simulation-based training held for second-year medical students at the Uniformed Services University (USU). This study explored the impact of participating in ACME on students' tourniquet application skills. A panel of emergency medicine physician experts developed an assessment to evaluate the participants' performance. Trained raters then scored students' tourniquet application performance before and after participating in ACME. We conducted a Wilcoxon signed-rank test to detect any significant difference in the participants' pretest and posttest ratings as well as time it took them to apply the tourniquet. Our results indicated a significant difference in the pre- and posttest ratings of students as well as the time it took them to apply the tourniquet. This study confirms the effectiveness of progressive simulation-based education for teaching TCCC skills to military medical trainees.


Quaternary-transformation-induced changes at the heme in deoxyhemoglobins.
Quaternary-structure-induced differences in both the high- and low-frequency regions of the resonance Raman spectrum of the heme have been detected in a variety of hemoglobins. These differences may be the result of (1) changes in the amino acid sequence, induced by genetic and chemical modifications, and (2) alterations in the quaternary structure. For samples in solution in low ionic strength buffers, differences in the 1357-cm-1 line (an electron-density-sensitive vibrational mode) correlate with differences in the 216-cm-1 line (the iron-histidine stretching mode). Thus, changes in the iron-histidine bond and changes in the pi-electron density of the porphyrin depend upon a common heme-globin interaction. The quaternary-structure-induced changes in the vibrational modes associated with the heme demonstrate that there is extensive communication between the heme and the globin and impact on models for the energetics of cooperativity. The local interactions of the iron-histidine mode are energetically small and destabilize the deoxy heme in the T structure with respect to the R structure. Therefore, these interactions must be larger in the ligated protein than in the deoxy protein to obtain a negative free energy of cooperativity. Additionally, our data imply that the deprotonation of the proximal histidine does not play a major role in the energetics of cooperativity. On the other hand, models for cooperativity that require conformational changes in the iron-histidine bond or direct interaction between the porphyrin and the protein are qualitatively consistent with the observed variation of heme electronic structure in concert with protein quaternary structure.


Characterization of human immunodeficiency virus type 1 (HIV-1) diversity and tropism in 145 patients with primary HIV-1 infection.
In the context of sexual transmission of human immunodeficiency virus type 1 (HIV-1), current findings suggest that the mucosal barrier is the major site of viral selection, transforming the complex inoculum to a small, homogeneous founder virus population. We analyzed HIV-1 transmission in relation to viral and host characteristics within the Zurich primary HIV-1 infection study. Clonal HIV-1 envelope sequences (on average 16 clones/patient) were isolated from the first available plasma samples during the early phase of infection from 145 patients with primary HIV-1 infection. Phylogenetic and tropism analyses were performed. Differences of viral diversities were investigated in association with several parameters potentially influencing HIV-1 transmission, eg, concomitant sexually transmitted infections (STIs) and mode of transmission. Median viral diversity within env C2-V3-C3 region was 0.39% (range 0.04%-3.23%). Viral diversity did not correlate with viral load, but it was slightly correlated with the duration of infection. Neither transmission mode, gender, nor STI predicted transmission of more heterogeneous founder virus populations that were found in 16 of 145 patients (11%; diversity >1%). Only 2 patients (1.4%) were assuredly infected with CXCR4-tropic HIV-1 within a R5/X4-tropic--mixed population, as revealed and confirmed using several genotypic prediction algorithms and phenotypic assays. Our findings suggest that transmission of multiple HIV-1 variants might be a complex process that is not dependent on mucosal factors alone. CXCR4-tropic viruses can be sexually transmitted in rare instances, but their clinical relevance remains to be determined.


Somatic-cell mutation induced by UVA and monochromatic UV radiation in repair-proficient and -deficient Drosophila melanogaster.
Near-ultraviolet light (UVA: 320-400 nm) constitutes a major part of sunlight UV. It is important to know the effect of UVA on the biological activities of organisms on the earth. We have previously reported that black light induces somatic-cell mutation in Drosophila larvae. To investigate which wavelength of the UVA is responsible for the mutation we have now carried out a series of monochromatic irradiations (310, 320, 330, 340, 360, 380 and 400 nm) on Drosophila larvae, using the large spectrograph of the National Institute for Basic Biology (Okazaki National Research Institutes, Okazaki, Japan). Mutagenic activity was examined by the Drosophila wing-spot test in which we observe mutant wing hair colonies (spots) on the wings of adult flies obtained from the treated larvae. The induction of mutation was highest by irradiation at 310 nm and decreased as the wavelength became longer. Neither the 380 nor the 400 nm light was mutagenic. Excision repair is known to protect cells from UV damage. In the excision-repair-deficient Drosophila, the mutagenic response induced by 310 nm irradiation was 24-fold higher than that of the wild-type (7.2 +/- 1.5 spots/wing/kJ vs 0.3 +/- 0.08 spots/wing/kJ), and at 320 nm the difference of the response was 14-fold (0.21 vs 0.015 +/- 0.005). In the case of irradiation at 330 and 340 nm the difference of the response was only two-fold (at 330 nm, 6.9 +/- 2.9 x 10(-3) vs 3.1 +/- 1.1 x 10(-3) spots/wing/kJ; at 340 nm, 3.5 +/- 0.9 x 10(-3) vs 2.0 +/- 0.7 x 10(-3). These results suggest that the lesion caused in the larvae by 320 nm irradiation may be similar to the damage induced by 310 nm and that the lights of 330 and 340 nm may induce damage different from the lesions induced by shorter-wavelength lights.


L(+)- and D(-)-lactate modulate rat renal tubular accumulation of amantadine in the presence and absence of bicarbonate.
The effect of L(+)-, D(-)- and racemic (DL)-lactate on the energy-dependent renal uptake of the achiral organic cation amantadine was determined with purified proximal and distal cortical tubule fragments isolated from rat kidneys. Kinetic parameters for uptake of amantadine were measured, under constant pH, in bicarbonate buffer (Krebs-Henseleit [KHS]), and in lactate buffers (5 mM) with different proportions of the enantiomers. Km for amantadine uptake increased in all lactate buffers compared with KHS for both proximal and distal tubules. Km for uptake in DL-lactate was similar to that in D(-)-lactate for proximal tubules and to L(+)-lactate in distal tubules, but Km in L(+)-lactate was higher than in D(-)-lactate for both tubules. Maximal transport capacity (Vmax) in DL-lactate and mixtures of enantiomers were similar to KHS but higher than in pure L(+)- and D(-)-lactate. In KHS, lactate inhibited energy-dependent amantadine uptake in a biphasic manner. Graded competitive inhibition of amantadine uptake was observed between 1 and 15 mM lactate for both proximal and distal tubules. This first phase (1-15 mM) inhibited 60% of amantadine uptake. The second phase (15-20 mM lactate) showed a much steeper slope and inhibited the remaining amantadine uptake. There were no differences in inhibitory potencies of the lactate enantiomers for either proximal tubules or distal tubules amantadine tubule uptake. Our present studies suggest that L(+)- and D(-)-lactate modulate amantadine transport by interacting directly with the bicarbonate-dependent transport mechanism(s).


Midazolam-induced amnesia reduces memory for details and affects the ERP correlates of recollection and familiarity.
Dual process models suggest that recognition memory is supported by familiarity and recollection processes. Previous research administering amnesic drugs and measuring ERPs during recognition memory have provided evidence for separable neural correlates of familiarity and recollection. This study examined the effect of midazolam-induced amnesia on memory for details and the proposed ERP correlates of recognition. Midazolam or saline was administered while subjects studied oriented pictures of common objects. ERPs were recorded during a recognition test 1 day later. Subjects' discrimination of old and new pictures as well as orientation discrimination was worse when they were given midazolam instead of saline. As predicted, the parietal old/new effect was decreased with the administration of midazolam. However, weaker effects on FN400 old/new effects were also observed. These results provide converging pharmacological and electrophysiological evidence that midazolam primarily affects recollection as indexed by parietal ERP old/new effects and memory for orientation, while also exerting some weaker effects on familiarity as indexed by FN400 old/new effects.


Tuning the drug efflux activity of an ABC transporter in vivo by in vitro selected DARPin binders.
ABC transporters use the energy from binding and hydrolysis of ATP to import or extrude substrates across the membrane. Using ribosome display, we raised designed ankyrin repeat proteins (DARPins) against detergent solubilized LmrCD, a heterodimeric multidrug ABC exporter from Lactococcus lactis. Several target-specific DARPin binders were identified that bind to at least three distinct, partially overlapping epitopes on LmrD in detergent solution as well as in native membranes. Remarkably, functional screening of the LmrCD-specific DARPin pools in L. lactis revealed three homologous DARPins which, when generated in LmrCD-expressing cells, strongly activated LmrCD-mediated drug transport. As LmrCD expression in the cell membrane was unaltered upon the co-expression of activator DARPins, the activation is suggested to occur at the level of LmrCD activity. Consistent with this, purified activator DARPins were found to stimulate the ATPase activity of LmrCD in vitro when reconstituted in proteoliposomes. This study suggests that membrane transporters are tunable in vivo by in vitro selected binding proteins. Our approach could be of biopharmaceutical importance and might facilitate studies on molecular mechanisms of ABC transporters.


Capsaicin Supplementation Reduces Physical Fatigue and Improves Exercise Performance in Mice.
Chili pepper is used as a food, seasoning and has been revered for its medicinal and health claims. It is very popular and is the most common spice worldwide. Capsaicin (CAP) is a major pungent and bioactive phytochemical in chili peppers. CAP has been shown to improve mitochondrial biogenesis and adenosine triphosphate (ATP) production. However, there is limited evidence around the effects of CAP on physical fatigue and exercise performance. The purpose of this study was to evaluate the potential beneficial effects of CAP on anti-fatigue and ergogenic functions following physiological challenge. Female Institute of Cancer Research (ICR) mice from four groups (n = 8 per group) were orally administered CAP for 4 weeks at 0, 205, 410, and 1025 mg/kg/day, which were respectively designated the vehicle, CAP-1X, CAP-2X, and CAP-5X groups. The anti-fatigue activity and exercise performance was evaluated using forelimb grip strength, exhaustive swimming time, and levels of serum lactate, ammonia, glucose, BUN (blood urea nitrogen) and creatine kinase (CK) after a 15-min swimming exercise. The grip strength and exhaustive swimming time of the CAP-5X group were significantly higher than other groups. CAP supplementation dose-dependently reduced serum lactate, ammonia, BUN and CK levels, and increased glucose concentration after the 15-min swimming test. In addition, CAP also increased hepatic glycogen content, an important energy source for exercise. The possible mechanism was relevant to energy homeostasis and the physiological modulations by CAP supplementation. Therefore, our results suggest that CAP supplementation may have a wide spectrum of bioactivities for promoting health, performance improvement and fatigue amelioration.


Enlargement of cerebral ventricles as an early indicator of encephalomyelitis.
Inflammatory disorders of the central nervous system such as multiple sclerosis and acute disseminated encephalomyelitis involve an invasion of immune cells that ultimately leads to white matter demyelination, neurodegeneration and development of neurological symptoms. A clinical diagnosis is often made when neurodegenerative processes are already ongoing. In an attempt to seek early indicators of disease, we studied the temporal and spatial distribution of brain modifications in experimental autoimmune encephalomyelitis (EAE). In a thorough magnetic resonance imaging study performed with EAE mice, we observed significant enlargement of the ventricles prior to disease clinical manifestation and an increase in free water content within the cerebrospinal fluid as demonstrated by changes in T2 relaxation times. The increase in ventricle size was seen in the lateral, third and fourth ventricles. In some EAE mice the ventricle size started returning to normal values during disease remission. In parallel to this macroscopic phenomenon, we studied the temporal evolution of microscopic lesions commonly observed in the cerebellum also starting prior to disease onset. Our data suggest that changes in ventricle size during the early stages of brain inflammation could be an early indicator of the events preceding neurological disease and warrant further exploration in preclinical and clinical studies.


Downstream of mutant KRAS, the transcription regulator YAP is essential for neoplastic progression to pancreatic ductal adenocarcinoma.
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor survival rates and frequently carries oncogenic KRAS mutation. However, KRAS has thus far not been a viable therapeutic target. We found that the abundance of YAP mRNA, which encodes Yes-associated protein (YAP), a protein regulated by the Hippo pathway during tissue development and homeostasis, was increased in human PDAC tissue compared with that in normal pancreatic epithelia. In genetically engineered Kras(G12D) and Kras(G12D):Trp53(R172H) mouse models, pancreas-specific deletion of Yap halted the progression of early neoplastic lesions to PDAC without affecting normal pancreatic development and endocrine function. Although Yap was dispensable for acinar to ductal metaplasia (ADM), an initial step in the progression to PDAC, Yap was critically required for the proliferation of mutant Kras or Kras:Trp53 neoplastic pancreatic ductal cells in culture and for their growth and progression to invasive PDAC in mice. Yap functioned as a critical transcriptional switch downstream of the oncogenic KRAS-mitogen-activated protein kinase (MAPK) pathway, promoting the expression of genes encoding secretory factors that cumulatively sustained neoplastic proliferation, a tumorigenic stromal response in the tumor microenvironment, and PDAC progression in Kras and Kras:Trp53 mutant pancreas tissue. Together, our findings identified Yap as a critical oncogenic KRAS effector and a promising therapeutic target for PDAC and possibly other types of KRAS-mutant cancers.


Clinical implications of Mycobacterium chimaera detection in thermoregulatory devices used for extracorporeal membrane oxygenation (ECMO), Germany, 2015 to 2016.
Mycobacterium chimaera, a non-tuberculous mycobacterium, was recently identified as causative agent of deep-seated infections in patients who had previously undergone open-chest cardiac surgery. Outbreak investigations suggested an aerosol-borne pathogen transmission originating from water contained in heater-cooler units (HCUs) used during cardiac surgery. Similar thermoregulatory devices are used for extracorporeal membrane oxygenation (ECMO) and M. chimaera might also be detectable in ECMO treatment settings. We performed a prospective microbiological study investigating the occurrence of M. chimaera in water from ECMO systems and in environmental samples, and a retrospective clinical review of possible ECMO-related mycobacterial infections among patients in a pneumological intensive care unit. We detected M. chimaera in 9 of 18 water samples from 10 different thermoregulatory ECMO devices; no mycobacteria were found in the nine room air samples and other environmental samples. Among 118 ECMO patients, 76 had bronchial specimens analysed for mycobacteria and M. chimaera was found in three individuals without signs of mycobacterial infection at the time of sampling. We conclude that M. chimaera can be detected in water samples from ECMO-associated thermoregulatory devices and might potentially pose patients at risk of infection. Further research is warranted to elucidate the clinical significance of M. chimaera in ECMO treatment settings.


Regulatory functions of hapten-reactive helper and suppressor T lymphocytes. III. Amplification of a generation of tumor-specific killer T-lymphocyte activities by suppressor T-cell-depleted hapten-reactive T lymphocytes.
2,4.6-trinitrophenyl (TNP)-reactive T-cell activities were raised in mice by immunization with TNP-isologous mouse gamma globulin. After establishing that TNP-reactive T lymphocytes can serve as amplifier cells for induction of killer T lymphocytes in allogeneic system, we explored the possibility of this hapten-reactive T-cell system to amplify tumor-specific killer T-lymphocyte activity in the syngeneic system. We utlized relatively weak immunogenic syngeneic plasmacytoma X5563 in C3H/He mice. Analysis of the TNP-reactive T-cell activities revealed that such T lymphocytes express the biological functions of both major subtypes of regulatory T cells, namely suppressors and helpers, and that TNP-reactive suppressor and helper T lymphocytes, respectively, differ in their relative susceptibility to specific inactivation by TNP conjugates of the nonimmunogenic D-amino acid copolymer, D-glutamic acid, and D-lysine (D-GL). By taking advantage of the relative susceptibility-difference to TNP-D-GL, selective inactivation of TNP-reactive suppressor T cells was induced by appropriate treatment with TNP-D-GL, and the generation of TNP-reactive helper T-cell activity was amplified. The supplement of augmented TNP-reactive helper T-cell activity to the system at the immunization with syngeneic X5563 with TNP-haptenation, resulted in a striking augmentation of induction of tumor-specific killer T-lymphocyte activity, and a considerable number of hosts survived after the challenge with lethal dose of viable tumor cells. Thus, appropriate manipulations designed to induce potent hapten-reactive helper T-lymphocytes provided the potential for a very effective mode of immunoprophylaxis against tumor.


Surgery with molecular fluorescence imaging using activatable cell-penetrating peptides decreases residual cancer and improves survival.
The completeness of tumor removal during surgery is dependent on the surgeon's ability to differentiate tumor from normal tissue using subjective criteria that are not easily quantifiable. A way to objectively assess tumor margins during surgery in patients would be of great value. We have developed a method to visualize tumors during surgery using activatable cell-penetrating peptides (ACPPs), in which the fluorescently labeled, polycationic cell-penetrating peptide (CPP) is coupled via a cleavable linker to a neutralizing peptide. Upon exposure to proteases characteristic of tumor tissue, the linker is cleaved, dissociating the inhibitory peptide and allowing the CPP to bind to and enter tumor cells. In mice, xenografts stably transfected with green fluorescent protein show colocalization with the Cy5-labeled ACPPs. In the same mouse models, Cy5-labeled free ACPPs and ACPPs conjugated to dendrimers (ACPPDs) delineate the margin between tumor and adjacent tissue, resulting in improved precision of tumor resection. Surgery guided by ACPPD resulted in fewer residual cancer cells left in the animal after surgery as measured by Alu PCR. A single injection of ACPPD dually labeled with Cy5 and gadolinium chelates enabled preoperative whole-body tumor detection by MRI, intraoperative guidance by real-time fluorescence, intraoperative histological analysis of margin status by fluorescence, and postoperative MRI tumor quantification. Animals whose tumors were resected with ACPPD guidance had better long-term tumor-free survival and overall survival than animals whose tumors were resected with traditional bright-field illumination only.


Longitudinal prevalence and faecal shedding of Chlamydia pecorum in sheep.
The prevalence and faecal shedding of Chlamydia spp. in sheep in Australia has not been well described. Two species-specific quantitative PCRs (qPCRs) targeting the chlamydial outer membrane protein cell surface antigen gene (ompA) were validated and used to determine the prevalence and faecal shedding of C. abortus and C. pecorum from faecal samples of lambs at three sampling times (weaning, post-weaning and pre-slaughter) from eight farms in South Australia, New South Wales, Victoria and Western Australia. A total of 3412 faecal samples were collected and screened from approximately 1189 lambs across the four states. C. abortus was not detected in any of the samples screened. The overall prevalence of C. pecorum was 1027/3412 (30.1%) and median bacterial concentrations at weaning, post-weaning and pre-slaughter were 1.8 × 10(7), 1.2 × 10(7) and 9.6 × 10(5)/g faeces, respectively. A subset of C. pecorum positive samples from each farm, (n = 48) was sequenced to confirm their identity. The present study demonstrates that C. pecorum is prevalent in Australian sheep, highlighting a need for further research on the impact of this bacterium on production.


A Systematic Review Evaluating Psychometric Properties of Parent or Caregiver Report Instruments on Child Maltreatment: Part 2: Internal Consistency, Reliability, Measurement Error, Structural Validity, Hypothesis Testing, Cross-Cultural Validity, and Criterion Validity.
Child maltreatment (CM) is global public health issue with devastating lifelong consequences. Global organizations have endeavored to eliminate CM; however, there is lack of consensus on what instruments are most suitable for the investigation and prevention of CM. This systematic review aimed to appraise the psychometric properties (other than content validity) of all current parent- or caregiver-reported CM instruments and recommend the most suitable for use. A systematic search of the CINAHL, Embase, ERIC, PsycINFO, PubMed, and Sociological Abstracts databases was performed. The evaluation of psychometric properties was conducted according to the COnsensus-based Standards for the selection of health Measurement INstruments (COSMIN) guidelines for systematic reviews of patient-report outcome measures. Responsiveness was beyond the scope of this systematic review, and content validity has been reported on in a companion paper (Part 1). Only instruments developed and published in English were included. Twenty-five studies reported on selected psychometric properties of 15 identified instruments. The methodological quality of the studies was overall adequate. The psychometric properties of the instruments were generally indeterminate or not reported due to incomplete or missing psychometric data; high-quality evidence on the psychometric properties was limited. No instruments could be recommended as most suitable for use in clinic and research. Nine instruments were identified as promising based on current psychometric data but would need further psychometric evidence for them to be recommended.


The intervertebral disc contains intrinsic circadian clocks that are regulated by age and cytokines and linked to degeneration.
The circadian clocks are internal timing mechanisms that drive ∼24-hour rhythms in a tissue-specific manner. Many aspects of the physiology of the intervertebral disc (IVD) show clear diurnal rhythms. However, it is unknown whether IVD tissue contains functional circadian clocks and if so, how their dysregulation is implicated in IVD degeneration. Clock gene dynamics in ex vivo IVD explants (from PER2:: luciferase (LUC) reporter mice) and human disc cells (transduced with lentivirus containing Per2::luc reporters) were monitored in real time by bioluminescence photon counting and imaging. Temporal gene expression changes were studied by RNAseq and quantitative reverse transcription (qRT)-PCR. IVD pathology was evaluated by histology in a mouse model with tissue-specific deletion of the core clock gene Bmal1. Here we show the existence of the circadian rhythm in mouse IVD tissue and human disc cells. This rhythm is dampened with ageing in mice and can be abolished by treatment with interleukin-1β but not tumour necrosis factor α. Time-series RNAseq revealed 607 genes with 24-hour patterns of expression representing several essential pathways in IVD physiology. Mice with conditional knockout of Bmal1 in their disc cells demonstrated age-related degeneration of IVDs. We have established autonomous circadian clocks in mouse and human IVD cells which respond to age and cytokines, and control key pathways involved in the homeostasis of IVDs. Genetic disruption to the mouse IVD molecular clock predisposes to IVD degeneration. These results support the concept that disruptions to circadian rhythms may be a risk factor for degenerative IVD disease and low back pain.


ONS Guidelines™ for Cancer Treatment-Related Skin Toxicity.
Management of cancer treatment-related skin toxicities can minimize treatment disruptions and improve patient well-being. This guideline aims to support patients and clinicians in decisions regarding management of cancer treatment-related skin toxicities. A panel developed a guideline for management of cancer treatment-related skin toxicities using GRADE (Grading of Recommendations, Assessment, Development, and Evaluation) for certainty of evidence and the National Academies of Sciences, Engineering, and Medicine criteria for trustworthy guidelines. The Cochrane risk-of-bias tool assessed risk of bias. A quantitative or narrative synthesis of the evidence was completed. The panel issued seven conditional recommendations for epidermal growth factor receptor inhibitor rash, hand-foot skin reaction, hand-foot syndrome, and chemotherapy-induced alopecia. The panel suggested strategies for prevention and treatment for all toxicities except hand-foot syndrome, which only has a prevention recommendation. Cancer treatment-related skin toxicities can significantly affect quality of life. Incorporation of these interventions into clinical care can improve patient outcomes. //onf.ons.org/supplementary-material-ons-guidelines-cancer-treatment-related-skin-toxicity.


Halide-stabilizing residues of haloalkane dehalogenases studied by quantum mechanic calculations and site-directed mutagenesis.
Haloalkane dehalogenases catalyze cleavage of the carbon-halogen bond in halogenated aliphatic compounds, resulting in the formation of an alcohol, a halide, and a proton as the reaction products. Three structural features of haloalkane dehalogenases are essential for their catalytic performance: (i) a catalytic triad, (ii) an oxyanion hole, and (iii) the halide-stabilizing residues. Halide-stabilizing residues are not structurally conserved among different haloalkane dehalogenases. The level of stabilization of the transition state structure of S(N)2 reaction and halide ion provided by each of the active site residues in the enzymes DhlA, LinB, and DhaA was quantified by quantum mechanic calculations. The residues that significantly stabilize the halide ion were assigned as the primary (essential) or the secondary (less important) halide-stabilizing residues. Site-directed mutagenesis was conducted with LinB enzyme to confirm location of its primary halide-stabilizing residues. Asn38Asp, Asn38Glu, Asn38Phe, Asn38Gln, Trp109Leu, Phe151Leu, Phe151Trp, Phe151Tyr, and Phe169Leu mutants of LinB were constructed, purified, and kinetically characterized. The following active site residues were classified as the primary halide-stabilizing residues: Trp125 and Trp175 of DhlA; Asn38 and Trp109 of LinB; and Asn41 and Trp107 of DhaA. All these residues make a hydrogen bond with the halide ion released from the substrate molecule, and their substitution results in enzymes with significantly modified catalytic properties. The following active site residues were classified as the secondary halide-stabilizing residues: Phe172, Pro223, and Val226 of DhlA; Trp207, Pro208, and Ile211 of LinB; and Phe205, Pro206, and Ile209 of DhaA. The differences in the halide stabilizing residues of three haloalkane dehalogenases are discussed in the light of molecular adaptation of these enzymes to their substrates.


Improving pediatric cardiac surgical care in developing countries: matching resources to needs.
This article reviews a systematic approach to the design and support of pediatric cardiac surgery programs in the developing world with the guidance and strategies of Children's HeartLink, an experienced non-government organization for more than 40 years. An algorithm with criteria for the selection of a partner site is outlined. A comprehensive education strategy from the physician to the allied health care provider is the mainstay for successful program development. In a partner program, the road to successful advancement and change depends on many factors, such as government support, hospital administration support, medical staff leadership, and a committed and motivated faculty with requisite skills, incentives, and resources. In addition to these factors, it is essential that the development effort includes considerations of environment (eg, governmental support, regulatory environment, and social structure) and health system (elements related to affordability, access, and awareness of care) that impact success. Partner programs should be willing to initiate a clinical database with the intent to analyze and critique their results to optimize quality assurance and improve outcomes.


A Longitudinal Study of Eating Rituals in Patients With Anorexia Nervosa.
Background: Eating rituals are any problematic behaviors involving food. They are usually observed in patients with anorexia nervosa, but research into these behaviors and their role in treatment outcomes is lacking. Objective: We set out to assess the presence of eating rituals in patients with anorexia nervosa treated by means of intensive enhanced cognitive behavioral therapy (ICBT-E), in addition to their change over time and role as potential predictors of treatment outcome. Materials and Methods: Ninety adult female inpatients with anorexia nervosa were recruited. The Participants' body mass index (BMI), and scores for Starvation Symptoms Inventory (SSI), Eating Disorder Examination (EDE), and Brief Symptom Inventory (BSI) were recorded, and a purpose-designed 9-item checklist of eating rituals was completed by trained dieticians during assisted eating - an integral part of the ICBT-E. The Structured Clinical Interview for DSM-IV was used at admission to identify the presence of coexisting axis I psychiatric disorders. All other tests were administered at baseline (admission), the end of treatment and 6-month follow-up. BMI, EDE, and BSI were also re-administered after 4 weeks of treatment in order to examine how refeeding affects these variables. Results: We found a correlation at baseline between eating rituals and both general and eating-disorder psychopathology scores. Eating rituals were also associated with the presence of at least one comorbid anxiety disorder. ICBT-E treatment was associated with a significant reduction in eating rituals, as well as a significant increase in BMI and improved eating-disorder and general psychopathology. However, our most relevant finding was that neither baseline eating ritual scores nor their change during treatment was associated with either BMI or general or eating-disorder psychopathology scores taken at either the end of therapy or at 6-month follow-up. Conclusion: Neither the presence of nor change in eating rituals influence treatment outcomes in patients with anorexia nervosa.


Clinical Updates in Women's Health Care Summary: Ischemic Heart Disease: Primary and Preventive Care Review.
Cardiovascular disease is the leading cause of death among women in the United States. Obstetrician-gynecologists often are the sole health care providers for otherwise healthy women. Therefore, they must be aware of risk factors, signs, and symptoms of cardiovascular disease and be prepared to recognize and treat this condition in patients as well as provide referrals when specialized care is indicated. Women with cardiac problems typically present with chest discomfort; however, they also are more likely than healthy women to present with other common symptoms, such as shortness of breath, back pain, jaw pain, or nausea and vomiting. This monograph, with a primary focus on ischemic heart disease (IHD), discusses the basic anatomy of the heart and coronary arteries; vascular biology; pathogenesis of atherosclerosis; and the screening, prevention, diagnosis, and treatment of IHD as well as the multiple risk factors associated with the development of IHD that contribute to both increased morbidity and mortality.


3D Anisotropic Thermal Conductivity of Exfoliated Rhenium Disulfide.
ReS2 represents a different class of 2D materials, which is characterized by low symmetry having 1D metallic chains within the planes and extremely weak interlayer bonding. Here, the thermal conductivity of single-crystalline ReS2 in a distorted 1T phase is determined at room temperature for the in-plane directions parallel and perpendicular to the Re-chains, and the through-plane direction using time-domain thermoreflectance. ReS2 is prepared in the form of flakes having thicknesses of 60-450 nm by micromechanical exfoliation, and their crystalline orientations are identified by polarized Raman spectroscopy. The in-plane thermal conductivity is higher along the Re-chains, (70 ± 18) W m-1 K-1 , as compared to transverse to the chains, (50 ± 13) W m-1 K-1 . As expected from the weak interlayer bonding, the through-plane thermal conductivity is the lowest observed to date for 2D materials, (0.55 ± 0.07) W m-1 K-1 , resulting in a remarkably high anisotropy of (130 ± 40) and (90 ± 30) for the two in-plane directions. The thermal conductivity and interface thermal conductance of ReS2 are discussed relative to the other 2D materials.


Route of Delivery Modulates the Efficacy of Mesenchymal Stem Cell Therapy for Myocardial Infarction: A Meta-Analysis of Preclinical Studies and Clinical Trials.
Accumulating data support a therapeutic role for mesenchymal stem cell (MSC) therapy; however, there is no consensus on the optimal route of delivery. We tested the hypothesis that the route of MSC delivery influences the reduction in infarct size and improvement in left ventricular ejection fraction (LVEF). We performed a meta-analysis investigating the effect of MSC therapy in acute myocardial infarction (AMI) and chronic ischemic cardiomyopathy preclinical studies (58 studies; n=1165 mouse, rat, swine) which revealed a reduction in infarct size and improvement of LVEF in all animal models. Route of delivery was analyzed in AMI swine studies and clinical trials (6 clinical trials; n=334 patients). In AMI swine studies, transendocardial stem cell injection reduced infarct size (n=49, 9.4% reduction; 95% confidence interval, -15.9 to -3.0), whereas direct intramyocardial injection, intravenous infusion, and intracoronary infusion indicated no improvement. Similarly, transendocardial stem cell injection improved LVEF (n=65, 9.1% increase; 95% confidence interval, 3.7 to 14.5), as did direct intramyocardial injection and intravenous infusion, whereas intracoronary infusion demonstrated no improvement. In humans, changes of LVEF paralleled these results, with transendocardial stem cell injection improving LVEF (n=46, 7.0% increase; 95% confidence interval, 2.7 to 11.3), as did intravenous infusion, but again intracoronary infusion demonstrating no improvement. MSC therapy improves cardiac function in animal models of both AMI and chronic ischemic cardiomyopathy. The route of delivery seems to play a role in modulating the efficacy of MSC therapy in AMI swine studies and clinical trials, suggesting the superiority of transendocardial stem cell injection because of its reduction in infarct size and improvement of LVEF, which has important implications for the design of future studies.


Protective and therapeutic effects of Curcuma xanthorrhiza on hepatotoxin-induced liver damage.
Curcuma xanthorrhiza Roxb. (Zingiberaceae family, commonly known as temu lawak or Javanese turmeric in Indonesia), which is found both wild and cultivated in Indonesia, has been traditionally used for medicinal purposes. C. xanthorrhiza is also used as a tonic in Indonesia. The aim of the present study is to clarify whether C. xanthorrhiza treatment may prevent acute liver damage induced by acetaminophen and carbon tetrachloride in mice. The results clearly indicated that extract of C. xanthorrhiza could reduce significantly the acute elevation of serum transaminases levels induced by the two kinds of hepatotoxins, and alleviated the degree of liver damage at 24 hours after the intraperitoneal administration of two hepatotoxins. It may be concluded that C. xanthorrhiza can protect the liver from various hepatotoxins, hence C. xanthorrhiza could be useful in the treatment of liver injuries and has promise as a kind of broad spectrum hepatoprotective agent.


Reconstitution Of β-catenin degradation in Xenopus egg extract.
Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways(1-3). Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, β-catenin. Two different methods are described to assess β-catenin protein degradation in Xenopus egg extract. One method is visually informative ([(35)S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess β-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of β-catenin degradation.


The distribution of 13 GABAA receptor subunit mRNAs in the rat brain. II. Olfactory bulb and cerebellum.
In an effort to determine subunit compositions of in vivo GABAA receptors, the cellular localization of 13 subunit encoding mRNAs (alpha 1-alpha 6, beta 1-beta 3, gamma 2-gamma 3, delta) was determined in the rat olfactory bulb and cerebellum. Cerebellar granule cells expressed significant quantities of alpha 1, alpha 6, beta 2, beta 3, gamma 2, and delta mRNAs. They contained very much lower levels of alpha 4, beta 1, and gamma 3 mRNAs, and the alpha 2, alpha 3, alpha 5, and gamma 1 genes appeared to be silent. Purkinje cells contained only the alpha 1, beta 2, beta 3, and gamma 2 mRNAs. Putative Bergmann glial cells were found to contain the gamma 1 mRNA and possibly the alpha 2 mRNA. In the molecular layer, only the alpha 1, beta 2 and gamma 2 mRNAs were expressed in stellate/basket cells. The alpha 3 probe hybridized weakly to targets in the molecular layer. The inferior olivary nucleus contained significant quantities of alpha 2, alpha 4, and gamma 1 transcripts, with the alpha 1, alpha 3, beta 2, beta 3, and gamma 2 mRNAs also present. In the olfactory bulb, mitral cells were found to express the alpha 1, beta 1, beta 2, beta 3, and gamma 2 mRNAs strongly and the alpha 3 mRNA weakly. Tufted cells contained alpha 1, alpha 3, beta 2, beta 3, and gamma 2 mRNAs and, occasionally, the alpha 2 mRNA. In the internal granule cells the alpha 2, alpha 4, alpha 5, beta 3, and delta mRNAs were all present. Low levels of alpha 3, gamma 1, gamma 2, and gamma 3 mRNAs were also noted in these cells. Periglomerular cells expressed low levels of alpha 2, alpha 3, alpha 4, beta 2, beta 3, gamma 1, gamma 2, and gamma 3 mRNAs. No alpha 6 mRNA was present in the olfactory bulb. Correlations that are general ones from other brain regions are the colocalizations of alpha 1 beta 2, alpha 2 beta 3, and alpha 4 delta mRNAs. In both the olfactory bulb and cerebellum, alpha 1 beta 2 gamma 2 receptor cores are probably employed. The delta-subunit mRNA appears to codistribute with alpha-subunit mRNAs (alpha 4 and alpha 6) associated with GABAA subunits that fail to bind benzodiazepine agonists.


Designing sequential transcription logic: a simple genetic circuit for conditional memory.
The ability to learn and respond to recurrent events depends on the capacity to remember transient biological signals received in the past. Moreover, it may be desirable to remember or ignore these transient signals conditioned upon other signals that are active at specific points in time or in unique environments. Here, we propose a simple genetic circuit in bacteria that is capable of conditionally memorizing a signal in the form of a transcription factor concentration. The circuit behaves similarly to a "data latch" in an electronic circuit, i.e. it reads and stores an input signal only when conditioned to do so by a "read command." Our circuit is of the same size as the well-known genetic toggle switch (an unconditional latch) which consists of two mutually repressing genes, but is complemented with a "regulatory front end" involving protein heterodimerization as a simple way to implement conditional control. Deterministic and stochastic analysis of the circuit dynamics indicate that an experimental implementation is feasible based on well-characterized genes and proteins. It is not known, to which extent molecular networks are able to conditionally store information in natural contexts for bacteria. However, our results suggest that such sequential logic elements may be readily implemented by cells through the combination of existing protein-protein interactions and simple transcriptional regulation.


Temporal trend (1988-2008) of hexabromocyclododecane enantiomers in herring gull eggs from the German coastal region.
Levels of α-, β-, and γ-hexabromocyclododecane (HBCD) were determined in pooled eggs from herring gulls (Larus argentatus) sampled on three bird sanctuaries near the German North Sea coast between 1988 and 2008 (Mellum and Trischen) and the German Baltic Sea coast between 1998 and 2008 (Heuwiese) and archived by the German Environmental Specimen Bank. Pressurized fluid extraction, gel permeation chromatography, and LC-MS/MS using (13)C(12)-labelled isotope standards and a chiral column were applied. α-HBCD was the dominating diastereomer and ranged between 3.7 and 107 ng g(-1)lw while β- and γ-HBCD were throughout close to LOQ. The highest α-HBCD concentration was found in eggs from Mellum sampled in the year 2000. Interestingly, HBCD in eggs from the three islands displayed similar time courses with levels increasing to a peak contamination around 2000 and decreasing levels ever since. Chiral signatures of α-HBCD in eggs differed among the islands but indicated a preferential enrichment of the first eluting enantiomer (-)-α-HBCD.


Molecular and genetic regulation of pig pancreatic islet cell development.
Reliance on rodents for understanding pancreatic genetics, development and islet function could limit progress in developing interventions for human diseases such as diabetes mellitus. Similarities of pancreas morphology and function suggest that porcine and human pancreas developmental biology may have useful homologies. However, little is known about pig pancreas development. To fill this knowledge gap, we investigated fetal and neonatal pig pancreas at multiple, crucial developmental stages using modern experimental approaches. Purification of islet β-, α- and δ-cells followed by transcriptome analysis (RNA-seq) and immunohistology identified cell- and stage-specific regulation, and revealed that pig and human islet cells share characteristic features that are not observed in mice. Morphometric analysis also revealed endocrine cell allocation and architectural similarities between pig and human islets. Our analysis unveiled scores of signaling pathways linked to native islet β-cell functional maturation, including evidence of fetal α-cell GLP-1 production and signaling to β-cells. Thus, the findings and resources detailed here show how pig pancreatic islet studies complement other systems for understanding the developmental programs that generate functional islet cells, and that are relevant to human pancreatic diseases.