Relevant baseline characteristics for describing patients with knee osteoarthritis: results from a Delphi survey.
Inclusion/exclusion criteria and baseline characteristics are essential for assessing the applicability of trial results to a given patient and the comparability of study populations for meta-analyses. This Delphi survey aimed to generate a set of baseline characteristics for describing patients with knee osteoarthritis enrolled in clinical studies. Survey participants comprised clinical experts (n = 23; mean age 54 y; from 4 continents) that had authored at least two randomized trials on knee osteoarthritis. First, given a prepared list of baseline patient characteristics, the experts were asked to add characteristics they considered important for assessing comparability of patient populations in different trials that evaluated the efficacy of non-surgical interventions for treating knee osteoarthritis. Next, they were asked to rate the importance of each characteristic, on a scale of 0 (not important) to 10 (highly important), according to three outcome categories: pain, function, and structure. Participants identified 121 baseline characteristics. A rating ≥7 points was assigned to 39 characteristics (e.g., age, depression, global knee pain, daily dose of pain killers, Kellgren-Lawrence grading); of these, 20 were related to pain, 15 to function, and 23 to structural outcomes. Global knee pain was the only baseline characteristic that fulfilled among experts the predefined consensus criteria. Experts identified a large number of characteristics for describing patients with knee osteoarthritis. Disagreement and uncertainty prevailed over the relevance of these characteristics. Our findings justified further efforts to define appropriate, broadly acceptable sets of baseline characteristics for describing patients with knee osteoarthritis.


Defining the epigenetic mechanism of asymmetric cell division of Schizosaccharomyces japonicus yeast.
A key question in developmental biology addresses the mechanism of asymmetric cell division. Asymmetry is crucial for generating cellular diversity required for development in multicellular organisms. As one of the potential mechanisms, chromosomally borne epigenetic difference between sister cells that changes mating/cell type has been demonstrated only in the Schizosaccharomyces pombe fission yeast. For technical reasons, it is nearly impossible to determine the existence of such a mechanism operating during embryonic development of multicellular organisms. Our work addresses whether such an epigenetic mechanism causes asymmetric cell division in the recently sequenced fission yeast, S. japonicus (with 36% GC content), which is highly diverged from the well-studied S. pombe species (with 44% GC content). We find that the genomic location and DNA sequences of the mating-type loci of S. japonicus differ vastly from those of the S. pombe species. Remarkably however, similar to S. pombe, the S. japonicus cells switch cell/mating type after undergoing two consecutive cycles of asymmetric cell divisions: only one among four "granddaughter" cells switches. The DNA-strand-specific epigenetic imprint at the mating-type locus1 initiates the recombination event, which is required for cellular differentiation. Therefore the S. pombe and S. japonicus mating systems provide the first two examples in which the intrinsic chirality of double helical structure of DNA forms the primary determinant of asymmetric cell division. Our results show that this unique strand-specific imprinting/segregation epigenetic mechanism for asymmetric cell division is evolutionary conserved. Motivated by these findings, we speculate that DNA-strand-specific epigenetic mechanisms might have evolved to dictate asymmetric cell division in diploid, higher eukaryotes as well.


Therapeutic alliance in schizophrenia: the role of recovery orientation, self-stigma, and insight.
The present study examined variables related to the quality of the therapeutic alliance in out-patients with schizophrenia. We expected recovery orientation and insight to be positively, and self-stigma to be negatively associated with a good therapeutic alliance. We expected these associations to be independent from age, clinical symptoms (i.e. positive and negative symptoms, depression), and more general aspects of relationship building like avoidant attachment style and the duration of treatment by the current therapist. The study included 156 participants with DSM-IV diagnoses of schizophrenia or schizoaffective disorder in the maintenance phase of treatment. Therapeutic alliance, recovery orientation, self-stigma, insight, adult attachment style, and depression were assessed by self-report. Symptoms were rated by interviewers. Hierarchical multiple regressions revealed that more recovery orientation, less self-stigma, and more insight independently were associated with a better quality of the therapeutic alliance. Clinical symptoms, adult attachment style, age, and the duration of treatment by current therapist were unrelated to the quality of the therapeutic alliance. Low recovery orientation and increased self-stigma might undermine the therapeutic alliance in schizophrenia beyond the detrimental effect of poor insight. Therefore in clinical settings, besides enhancing insight, recovery orientation, and self-stigma should be addressed.


Cell-matrix interactions in mammary gland development and breast cancer.
The mammary gland is an organ that at once gives life to the young, but at the same time poses one of the greatest threats to the mother. Understanding how the tissue develops and functions is of pressing importance in determining how its control mechanisms break down in breast cancer. Here we argue that the interactions between mammary epithelial cells and their extracellular matrix (ECM) are crucial in the development and function of the tissue. Current strategies for treating breast cancer take advantage of our knowledge of the endocrine regulation of breast development, and the emerging role of stromal-epithelial interactions (Fig. 1). Focusing, in addition, on the microenvironmental influences that arise from cell-matrix interactions will open new opportunities for therapeutic intervention. We suggest that ultimately a three-pronged approach targeting endocrine, growth factor, and cell-matrix interactions will provide the best chance of curing the disease.


Recanalization and rupture of a giant vertebral artery aneurysm after hunterian ligation: case report.
Recanalization and subsequent rupture of giant aneurysms of the posterior circulation after Hunterian ligation is an extremely rare event that has been noted to occur with basilar apex, basilar trunk, and vertebrobasilar junction aneurysms. We report the case of a giant, previously unruptured right vertebral artery aneurysm, which recanalized from the contralateral vertebral artery and subsequently ruptured after previously performed angiography showed complete thrombosis of the aneurysm. A 72-year-old woman presented with headaches, ataxia, and lower extremity weakness. A giant 3-cm right vertebral artery aneurysm was found during the patient evaluation. Because of the size of the aneurysm and the absence of a discrete neck, Hunterian ligation was performed. After treatment, angiograms showed no filling of the aneurysm from either the right or left vertebral artery. Nine days later, after the patient developed lethargy and nausea, repeat angiography showed that a small portion of the aneurysmal base had recanalized. The next day, the patient had a massive subarachnoid hemorrhage and subsequently died. We think that this is a previously undescribed complication associated with direct arterial ligation of giant vertebral artery aneurysms. Patients with aneurysms treated using Hunterian ligation need to be followed up closely. Even aneurysms that have minimal recanalization are at risk for subarachnoid hemorrhage.


Parental burnout during the COVID-19 pandemic.
Increased and long-term parental stress related to one's parental role can lead to parental burnout. In the early phase of the COVID-19 pandemic, families experienced intensified pressure due to the government-initiated contact restrictions applied to prevent the spread of the virus in the population. This study investigates the risk factors and predictors of parental burnout in a large sample of parents (N = 1488) during the COVID-19 pandemic in Norway. Demographic and psychosocial factors were assessed at two timepoints: at the beginning of the pandemic outbreak in March 2020 (T1) and at 3 months follow-up (T2). A hierarchical regression analysis was applied to identify the factors that contribute to parental burnout at T2. Parental burnout was additionally explored across subgroups. Findings revealed that younger age was associated with more parental burnout. Concurrent (T2) use of unhelpful coping strategies, insomnia symptoms, parental stress, and less parental satisfaction was significantly associated with the presence of greater parental burnout (T2). Additionally, parental stress and satisfaction measured in the earliest phase of the pandemic (T1) were associated with parental burnout 3 months later (T2) over and above concurrent parental stress/satisfaction. Unemployed parents and individuals with a mental health condition were identified as subgroups with substantially heightened levels of parental burnout.


Motility-indole-lysine medium for presumptive identification of enteric pathogens of Enterobacteriaceae.
Detection of lysine decarboxylase activity is a useful supplement to reactions on triple sugar-iron (TSI) and urea agars in the initial examination of suspected pathogenic isolates from fecal cultures. Owing to the added value of motility and indole production in the differentiation of enteric pathogens, we prepared and evaluated a motility-indole-lysine (MIL) medium. The following 890 organisms were tested: 264 Shigella, 2 Edwardsiella, 182 Salmonella enteritidis, 235 S. typhi, 3 Arizona, 32 Yersinia enterocolitica, and 172 other members of the family Enterobacteriaceae. With few exceptions the MIL medium gave the same results as the standard motility, indole, and lysine decarboxylase (Moeller) test media. All discrepancies were with the indole reaction, which was weak in 2 of 67 strains of Escherichia coli and falsely negative in 6 of 32 strains of Y. enterocolitica. When both TSI agar and lysine-iron agar (LIA) slants are used in the evaluation isolates from fecal cultures, detection of H2S is duplicated. Both LIA and MIL medium detect lysine decarboxylase and deaminase activity equally well. Because of its ability to detect motility and indole production, the MIL medium is more useful than LIA when used with TSI agar. The combination of TSI agar, MIL medium, and urea agar enables reliable initial recognition of enteric pathogens of the Enterobacteriaceae.


Effects of asynchrony on rabbit blastocyst development.
The development of synchronously and asynchronously transferred rabbit morulae (recovered at Day 3 p.c.) and blastocysts (recovered at Day 4 p.c.) was investigated before the anticipated time of implantation. The results obtained with various techniques (evaluation of gross morphology, measurement of diameter, thymidine incorporation, light and electron microscopy) led basically to the same conclusions. Embryos being asynchronously transferred to the uterus of recipient rabbits survived, at least in terms of certain cellular functions like cell proliferation, for more than 2 days. Development, however, was clearly retarded and ultrastructural examination revealed substantial cell damage. Some blastocysts showed, even after 3 days, normal growth and cell proliferation indicating considerable differences between individuals in the ability to compensate for suboptimal developmental conditions before implantation. In general, this ability was greater in the transferred Day-3 morulae than in the Day-4 blastocysts. Embryonic growth and the ability to dissolve the zona pellucida, to synthesize crystalloid bodies and to differentiate extraembryonic endoderm indicated the maintenance of some developmental functions under asynchronous conditions. Blastocyst development was influenced by the progestational stage of the recipient. At 1 day after transfer into asynchronous older uteri, blastocyst diameters were larger and cell proliferation was increased compared with all other groups, suggesting an attempt of the blastocyst to adjust to the more advanced maternal milieu. Development in asynchronous younger uteri was delayed. No comparable differences in development were found in cultured embryos for which the media had been supplemented with flushings from the same progestational uterine stages as used for transfer. Thymidine incorporation in cultured embryos did not differ between the various supplements (P greater than 0.05) and was generally lower than in chronologically aged asynchronously transferred embryos (P less than 0.05 for Day-3 and P less than 0.001 or P greater than 0.05 for Day-4 embryos).


The Lsm1-7/Pat1 complex binds to stress-activated mRNAs and modulates the response to hyperosmotic shock.
RNA-binding proteins (RBPs) establish the cellular fate of a transcript, but an understanding of these processes has been limited by a lack of identified specific interactions between RNA and protein molecules. Using MS2 RNA tagging, we have purified proteins associated with individual mRNA species induced by osmotic stress, STL1 and GPD1. We found members of the Lsm1-7/Pat1 RBP complex to preferentially bind these mRNAs, relative to the non-stress induced mRNAs, HYP2 and ASH1. To assess the functional importance, we mutated components of the Lsm1-7/Pat1 RBP complex and analyzed the impact on expression of osmostress gene products. We observed a defect in global translation inhibition under osmotic stress in pat1 and lsm1 mutants, which correlated with an abnormally high association of both non-stress and stress-induced mRNAs to translationally active polysomes. Additionally, for stress-induced proteins normally triggered only by moderate or high osmostress, in the mutants the protein levels rose high already at weak hyperosmosis. Analysis of ribosome passage on mRNAs through co-translational decay from the 5' end (5P-Seq) showed increased ribosome accumulation in lsm1 and pat1 mutants upstream of the start codon. This effect was particularly strong for mRNAs induced under osmostress. Thus, our results indicate that, in addition to its role in degradation, the Lsm1-7/Pat1 complex acts as a selective translational repressor, having stronger effect over the translation initiation of heavily expressed mRNAs. Binding of the Lsm1-7/Pat1p complex to osmostress-induced mRNAs mitigates their translation, suppressing it in conditions of weak or no stress, and avoiding a hyperresponse when triggered.


Genome-wide Association Studies of Posttraumatic Stress Disorder in 2 Cohorts of US Army Soldiers.
Posttraumatic stress disorder (PTSD) is a prevalent, serious public health concern, particularly in the military. The identification of genetic risk factors for PTSD may provide important insights into the biological foundation of vulnerability and comorbidity. To discover genetic loci associated with the lifetime risk for PTSD in 2 cohorts from the Army Study to Assess Risk and Resilience in Servicemembers (Army STARRS). Two coordinated genome-wide association studies of mental health in the US military contributed participants. The New Soldier Study (NSS) included 3167 unique participants with PTSD and 4607 trauma-exposed control individuals; the Pre/Post Deployment Study (PPDS) included 947 unique participants with PTSD and 4969 trauma-exposed controls. The NSS data were collected from February 1, 2011, to November 30, 2012; the PDDS data, from January 9 to April 30, 2012. The primary analysis compared lifetime DSM-IV PTSD cases with trauma-exposed controls without lifetime PTSD. Data were analyzed from March 18 to December 27, 2015. Association analyses for PTSD used logistic regression models within each of 3 ancestral groups (European, African, and Latino American) by study, followed by meta-analysis. Heritability and genetic correlation and pleiotropy with other psychiatric and immune-related disorders were estimated. The NSS population was 80.7% male (6277 of 7774 participants; mean [SD] age, 20.9 [3.3] years); the PPDS population, 94.4% male (5583 of 5916 participants; mean [SD] age, 26.5 [6.0] years). A genome-wide significant locus was found in ANKRD55 on chromosome 5 (rs159572; odds ratio [OR], 1.62; 95% CI, 1.37-1.92; P = 2.34 × 10-8) and persisted after adjustment for cumulative trauma exposure (adjusted OR, 1.64; 95% CI, 1.39-1.95; P = 1.18 × 10-8) in the African American samples from the NSS. A genome-wide significant locus was also found in or near ZNF626 on chromosome 19 (rs11085374; OR, 0.77; 95% CI, 0.70-0.85; P = 4.59 × 10-8) in the European American samples from the NSS. Similar results were not found for either single-nucleotide polymorphism in the corresponding ancestry group from the PPDS sample, in other ancestral groups, or in transancestral meta-analyses. Single-nucleotide polymorphism-based heritability was nonsignificant, and no significant genetic correlations were observed between PTSD and 6 mental disorders or 9 immune-related disorders. Significant evidence of pleiotropy was observed between PTSD and rheumatoid arthritis and, to a lesser extent, psoriasis. In the largest genome-wide association study of PTSD to date, involving a US military sample, limited evidence of association for specific loci was found. Further efforts are needed to replicate the genome-wide significant association with ANKRD55-associated in prior research with several autoimmune and inflammatory disorders-and to clarify the nature of the genetic overlap observed between PTSD and rheumatoid arthritis and psoriasis.


Diabetes knowledge in nursing homes and home-based care services: a validation study of the Michigan Diabetes Knowledge Test adapted for use among nursing personnel.
Providing high-quality diabetes care in nursing homes and home-based care facilities requires suitable instruments to evaluate the level of diabetes knowledge among the health-care providers. Thus, the aim of this study was to examine the psychometric properties of the Michigan Diabetes Knowledge Test adapted for use among nursing personnel. The study included 127 nursing personnel (32 registered nurses, 69 nursing aides and 26 nursing assistants) at three nursing homes and one home-based care facility in Norway. We examined the reliability and content and construct validity of the Michigan Diabetes Knowledge Test. The items in both the general diabetes subscale and the insulin-use subscale were considered relevant and appropriate. The instrument showed satisfactory properties for distinguishing between groups. Item response theory-based measurements and item information curves indicate maximum information at average or lower knowledge scores. Internal consistency and the item-total correlations were quite weak, indicating that the Michigan Diabetes Knowledge Test measures a set of items related to various relevant knowledge topics but not necessarily related to each other. The Michigan Diabetes Knowledge Test measures a broad range of topics relevant to diabetes care. It is an appropriate instrument for identifying individual and distinct needs for diabetes education among nursing personnel. The knowledge gaps identified by the Michigan Diabetes Knowledge Test could also provide useful input for the content of educational activities. However, some revision of the test should be considered.


Taking risks with translational research.
The recent bankruptcy filing by deCODE, a company with an exceptional pedigree in associating genetic variance with disease onset, highlights the commercial risks of translational research. Indeed, deCODE's approach was similar to that adapted by academic researchers who seek to connect genetics and disease. We argue here that neither a purely corporate nor purely academic model is entirely appropriate for such research. Instead, we suggest that the private sector undertake the high-throughput elements of translational research, while the public sector and governments assume the role of providing long-term funding to develop gifted scientists with the confidence to attempt to use genetic data as a stepping stone to a truly mechanistic understanding of complex disease.


Crystal structure of the RNA-guided immune surveillance Cascade complex in Escherichia coli.
Clustered regularly interspaced short palindromic repeats (CRISPR) together with CRISPR-associated (Cas) proteins form the CRISPR/Cas system to defend against foreign nucleic acids of bacterial and archaeal origin. In the I-E subtype CRISPR/Cas system, eleven subunits from five Cas proteins (CasA1B2C6D1E1) assemble along a CRISPR RNA (crRNA) to form the Cascade complex. Here we report on the 3.05 Å crystal structure of the 405-kilodalton Escherichia coli Cascade complex that provides molecular details beyond those available from earlier lower-resolution cryo-electron microscopy structures. The bound 61-nucleotide crRNA spans the entire 11-protein subunit-containing complex, where it interacts with all six CasC subunits (named CasC1-6), with its 5' and 3' terminal repeats anchored by CasD and CasE, respectively. The crRNA spacer region is positioned along a continuous groove on the concave surface generated by the aligned CasC1-6 subunits. The five long β-hairpins that project from individual CasC2-6 subunits extend across the crRNA, with each β-hairpin inserting into the gap between the last stacked base and its adjacent splayed counterpart, and positioned within the groove of the preceding CasC subunit. Therefore, instead of continuously stacking, the crRNA spacer region is divided into five equal fragments, with each fragment containing five stacked bases flanked by one flipped-out base. Each of those crRNA spacer fragments interacts with CasC in a similar fashion. Furthermore, our structure explains why the seed sequence, with its outward-directed bases, has a critical role in target DNA recognition. In conclusion, our structure of the Cascade complex provides novel molecular details of protein-protein and protein-RNA alignments and interactions required for generation of a complex mediating RNA-guided immune surveillance.


Polymorphisms of the XRCC1, XRCC3, & XPD genes, and colorectal cancer risk: a case-control study in Taiwan.
Recent studies relating to the association between DNA repair-gene polymorphisms and colorectal cancer risk would, to the best of our knowledge, appear to be very limited. This study was designed to examine the polymorphisms associated with three DNA repair genes, namely: XRCC1 Arg399Gln, XRCC3 Thr241Met and XPD Lys751Gln, and investigate their role as susceptibility markers for colorectal cancer. We conducted a case-control study including 727 cases of cancer and 736 hospital-based age- and sex-matched healthy controls to examine the role of genetic polymorphisms of three DNA-repair genes (XRCC1, XRCC3 and XPD) in the context of colorectal cancer risk for the Taiwanese population. Genomic DNA isolated from 10 ml whole blood was used to genotype XRCC1 Arg399Gln, XRCC3 Thr241Met and XPD Lys751Gln by means of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. The risk for colorectal cancer did not appear to differ significantly amongst individuals featuring the XRCC1 399Arg/Arg genotype (OR = 1.18; 95% CI, 0.96-1.45), the XRCC3 241Thr/Thr genotype (OR = 1.25; 95% CI, 0.88-1.79) or the XPD 751Gln allele (OR = 1.20; 95% CI, 0.90-1.61), although individuals featuring a greater number of risk genotypes (genotype with OR greater than 1) did experience a higher risk for colorectal cancer when compared to those who didn't feature any risk genotypes (Trend test P = 0.03). Compared with those individuals who didn't express any putative risk genotypes, individuals featuring all of the putative risk genotypes did experience a significantly greater cancer risk (OR = 2.43, 95% CI = 1.21-4.90), particularly for individuals suffering tumors located in the rectum (OR = 3.18, 95% CI = 1.29-7.82) and diagnosed prior to the age of 60 years (OR = 4.90, 95% CI = 1.72-14.0). Our results suggest that DNA-repair pathways may simultaneously modulate the risk of colorectal cancer for the Taiwanese population, and, particularly for rectal cancer and younger patients.


Investigation of the relocation of cytosolic phospholipase A2 and annexin V in activated platelets.
Cytosolic phospholipase A(2) is a Ca(2+)-dependent enzyme that acts on membrane phospholipids to release arachidonic acid, which in platelets is converted to thromboxane A(2). Annexin V is a Ca(2+)-dependent, phospholipid-binding protein, which is proposed to regulate inflammation by inhibiting cytosolic phospholipase A(2). Here, we have studied the association of cytosolic phospholipase A(2) and annexin V with platelet membranes after thrombin stimulation. In a time-dependent manner, an exact correlation was found between the membrane association of cytosolic phospholipase A(2) and annexin V. Calcium from the intracellular stores was sufficient for the relocation of intracellular annexin V and cytosolic phospholipase A(2) to platelet membranes. Activation in the presence of arginyl-glycyl-aspartyl-serine (RGDS), which inhibits binding of fibrinogen to its adhesive ligand, does not alter the amount of cytosolic phospholipase A(2) or annexin V that binds to membranes. When activation-induced actin polymerisation was prevented by cytochalasin E, the recovery of both annexin V and cytosolic phospholipase A(2) remained unchanged. However, complete depolymerisation of the cytoskeleton with DNase I almost abolished the association of cytosolic phospholipase A(2) with the membranes, and it completely abolished the relocation of annexin V to platelet membranes. Finally, we show that cytosolic phospholipase A(2) can be specifically purified from platelet membranes by affinity chromatography on GST-annexin V and that immunoprecipitation using antibodies against cytosolic phospholipase A(2) copurify annexin V and cytosolic phospholipase A(2) from activated platelets. These findings suggest that following platelet activation with thrombin, both cytosolic phospholipase A(2) and annexin V, relocate to platelet membranes where they interact. An intact cytoskeleton seems to be a prerequisite for the interaction of cytosolic phospholipase A(2) and annexin V with platelet membranes. The incorporation of cytosolic phospholipase A(2) into the membrane fraction of thrombin-activated platelets parallels that of annexin V, which suggests an interaction between the two proteins.


Internal and external dynamics in language: evidence from verb regularity in a historical corpus of English.
Human languages are rule governed, but almost invariably these rules have exceptions in the form of irregularities. Since rules in language are efficient and productive, the persistence of irregularity is an anomaly. How does irregularity linger in the face of internal (endogenous) and external (exogenous) pressures to conform to a rule? Here we address this problem by taking a detailed look at simple past tense verbs in the Corpus of Historical American English. The data show that the language is open, with many new verbs entering. At the same time, existing verbs might tend to regularize or irregularize as a consequence of internal dynamics, but overall, the amount of irregularity sustained by the language stays roughly constant over time. Despite continuous vocabulary growth, and presumably, an attendant increase in expressive power, there is no corresponding growth in irregularity. We analyze the set of irregulars, showing they may adhere to a set of minority rules, allowing for increased stability of irregularity over time. These findings contribute to the debate on how language systems become rule governed, and how and why they sustain exceptions to rules, providing insight into the interplay between the emergence and maintenance of rules and exceptions in language.


Optimized SNR simultaneous multispectral photoacoustic imaging with laser diodes.
Multispectral photoacoustic laser diode systems have multiple wavelengths available simultaneously. In addition to multispectral imaging, this can be exploited to increase the signal to noise ratio (SNR) by combining these wavelengths to form a combined image, but at the loss of spectral information. Here, a novel signal processing concept is introduced, which optimizes the SNR in the reconstructions of single wavelength data from combined acquisitions while simultaneously permitting to obtain a higher SNR fused image from the same data. The concept is derived for an arbitrary number of wavelengths; it is also applicable at low pulse repetition frequencies. The concept is applied in an experiment using two wavelengths, verifying the theoretical results.


Prospective study of the frequency and outcomes of patients with suspected pulmonary embolism administered heparin prior to confirmatory imaging.
The administration of empiric systemic anticoagulation (ESA) before confirmatory radiographic testing in patients with suspected pulmonary embolism (PE) may improve outcomes, but no data have been published regarding current practice. We describe the use of ESA in a large prospective cohort of emergency department (ED) patients and report the outcomes of those treated with ESA compared with patients not receiving ESA. 12-center, noninterventional study of ED patients who presented with symptoms concerning for PE. Clinical data including pretest probability and decision to start ESA were recorded at point of care by attending physicians. Patients were followed for adverse in-hospital outcomes and recurrence of venous thromboembolism. ESA was initiated 342/7932 (4.3%) of enrolled patients, including 142/618 (23%) patients with high pretest probability. Patients receiving ESA had more abnormal vital signs and were more likely to have a history of venous thromboembolism than those who did not receive ESA. Overall, 481/7,932 (6.1%) had PE diagnosed, 72/481 (15.0%) with PE had ESA, and 72/342 (21%) of ESA patients had PE. Three patients (0.9%, 95%CI: 0.2-2.5%) who received ESA suffered hemorrhagic complications compared with 38 patients (0.5%, 95%CI: 0.4-0.7%) who did not receive ESA. In this multicenter sample, ED physicians administered ESA to a small, generally more acutely ill subset of patients with high pretest probability of PE, and very few had hemorrhagic complications. ESA was not associated with any clear difference in outcomes. More study is needed to clarify the risk versus benefit of ESA.


Is the systemic microvascular endothelial glycocalyx in peritoneal dialysis patients related to peritoneal transport?
The capillary wall coated by the endothelial glycocalyx is the main transport barrier during peritoneal dialysis (PD). Here, we investigated the relationships between measurements of the systemic endothelial glycocalyx and peritoneal transport in PD patients. We performed sidestream darkfield (SDF) imaging of the sublingual microvasculature in 15 patients, measured the perfused boundary region (PBR), which includes the permeable part of the glycocalyx, and calculated the estimated blood vessel density (EBVD). All patients underwent a peritoneal permeability analysis. No relationships were present between the imaging and peritoneal transport parameters, neither in the group as a whole nor in fast transporters. In patients with nonfast peritoneal transport status, PBR had a negative relationship with EBVD and small solute transport, and a positive one with net ultrafiltration (NUF). The EBVD showed a positive correlation with glucose absorption and a negative one with NUF. We found no relationships with the peritoneal transport of albumin. No relationships are present between the systemic endothelial glycocalyx, which was assessed by SDF, and peritoneal transport. In nonfast transporters, a reduction in blood vessel density caused by endothelial glycocalyx alterations or a thicker permeable phase of the glycocalyx delaying the access of small solutes to the small pores may be important. .


Consensus guidelines for the detection of immunogenic cell death.
Apoptotic cells have long been considered as intrinsically tolerogenic or unable to elicit immune responses specific for dead cell-associated antigens. However, multiple stimuli can trigger a functionally peculiar type of apoptotic demise that does not go unnoticed by the adaptive arm of the immune system, which we named "immunogenic cell death" (ICD). ICD is preceded or accompanied by the emission of a series of immunostimulatory damage-associated molecular patterns (DAMPs) in a precise spatiotemporal configuration. Several anticancer agents that have been successfully employed in the clinic for decades, including various chemotherapeutics and radiotherapy, can elicit ICD. Moreover, defects in the components that underlie the capacity of the immune system to perceive cell death as immunogenic negatively influence disease outcome among cancer patients treated with ICD inducers. Thus, ICD has profound clinical and therapeutic implications. Unfortunately, the gold-standard approach to detect ICD relies on vaccination experiments involving immunocompetent murine models and syngeneic cancer cells, an approach that is incompatible with large screening campaigns. Here, we outline strategies conceived to detect surrogate markers of ICD in vitro and to screen large chemical libraries for putative ICD inducers, based on a high-content, high-throughput platform that we recently developed. Such a platform allows for the detection of multiple DAMPs, like cell surface-exposed calreticulin, extracellular ATP and high mobility group box 1 (HMGB1), and/or the processes that underlie their emission, such as endoplasmic reticulum stress, autophagy and necrotic plasma membrane permeabilization. We surmise that this technology will facilitate the development of next-generation anticancer regimens, which kill malignant cells and simultaneously convert them into a cancer-specific therapeutic vaccine.


Suggestive linkage of familial mesial temporal lobe epilepsy to chromosome 3q26.
To describe the clinical findings in a family with a benign form of mesial temporal lobe epilepsy and to identify the causative genetic factors. All participants were personally interviewed and underwent neurologic examination. The affected subjects underwent EEG and most of them neuroradiological examinations (MRI). All family members were genotyped with the HumanCytoSNP-12 v1.0 beadchip and linkage analysis was performed with Merlin and Simwalk2 programs. Exome sequencing was performed on HiSeq2000, after exome capture with SureSelect 50 Mb kit v2.0. The family had 6 members with temporal lobe epilepsy. Age at seizure onset ranged from 8 to 13 years. Five patients had epigastric auras often associated to oro-alimentary automatic activity, 3 patients presented loss of contact, and 2 experienced secondary generalizations. Febrile seizures occurred in 2 family members, 1 of whom also had temporal lobe epilepsy. EEG showed focal slow waves and epileptic abnormalities on temporal regions in 1 patient and was normal in the other affected individuals. MRI was normal in all temporal lobe epilepsy patients. We performed single nucleotide polymorphism-array linkage analysis of the family and found suggestive evidence of linkage (LOD score=2.106) to a region on chromosome 3q26. Haplotype reconstruction supported the linkage data and showed that the majority of unaffected family members carried the haplotype at risk. Whole exome sequencing failed to identify pathogenic mutations in genes of the candidate region. Our data suggest the existence of a novel locus for benign familial mesial temporal lobe epilepsy on chromosome 3q26. Our failure to identify pathogenic mutations in genes of this region may be due to limitations of the exome sequencing technology.


Functional characterisation of two ferric-ion coordination modes of TtFbpA, the periplasmic subunit of an ABC-type iron transporter from Thermus thermophilus HB8.
The ferric ion binding protein A of Thermus thermophilus HB8 (TtFbpA) is the periplasmic subunit of an ABC-type iron transporter. Two Fe3+-bound crystal structures at pH 5.5 and pH 7.5 and one apo structure have been reported for TtFbpA. In addition to three Tyr residues, TtFbpA coordinates with Fe3+ using two monodentate HCO3- and one H2O to form a six-coordinated mode at pH 5.5 or one bidentate CO32- to form a five-coordinated mode at pH 7.5. We investigated the biological significance of these Fe3+-bound forms of TtFbpA and the synergistic anions (HCO3- and CO32-). Quantum mechanical calculations in silico indicated that only these coordination modes were plausible out of six possibilities. Comparison of the crystal structures revealed a key motif, RZX1X2L(I/V), that would couple the Fe3+ coordination mode and the TtFbpA protein conformation. Both gel filtration chromatography and isothermal titration calorimetry showed that TtFbpA could bind Fe3+ at pH 7.5 but not at pH 5.5. Isothermal titration calorimetry also revealed that the binding at pH 7.5 was a three-step endothermic reaction that required NaHCO3. These results indicate that the holo structure at pH 5.5 is unstable in solution and may correspond to a transition state of TtFbpA-Fe3+ binding at pH 7.5 because HCO3- is much more abundant than CO32- at both pH values. Reorganisation of the synergistic ions and coupled protein conformational change will occur to form the stable TtFbpA-Fe3+ complex at pH 7.5, but not at pH 5.5. Identification of such a transition state will contribute to molecular design of novel FbpA inhibitors.


Evidence that a novel tetracycline resistance gene found on two Bacteroides transposons encodes an NADP-requiring oxidoreductase.
Two transposons, Tn4351 and Tn4400, which were originally isolated from the obligate anaerobe Bacteroides fragilis, carry a tetracycline resistance (Tcr) gene that confers resistance only on aerobically grown Escherichia coli. This aerobic Tcr gene, designated tetX, has been shown previously to act by chemically modifying tetracycline in a reaction that appears to require oxygen. We have now obtained the DNA sequence of tetX and 0.6 kb of its upstream region from Tn4400. Analysis of the DNA sequence of tetX revealed that this gene encoded a 43.7-kDa protein. The deduced amino acid sequence of the amino terminus of the protein had homology with a number of enzymes, all of which had in common a requirement for NAD(P). In an earlier study, we had observed that disrupted cells, unlike intact cells, could not carry out the alteration of tetracycline. We have now shown that if NADPH (1 mM) is added to the disrupted cell preparation, alteration of tetracycline occurs. Thus, TetX appears to be an NADP-requiring oxidoreductase. Tn4400 conferred a fivefold-lower level of tetracycline resistance than Tn4351. This finding appears to be due to a lower level of expression of the tetX on Tn4400, because the activity of a tetX-lacZ fusion from Tn4400 was 10-fold lower than that of the same fusion from Tn4351. A comparison of the sequence of the tetX region on Tn4351 with that on Tn4400 showed that the only difference between the upstream regions of the two transposons was a 4-base change 350 bp upstream of the start of the tetX coding region. The 4-base change difference creates a good consensus -35 region on Tn4351 that is not present on Tn4400 and could be creating an extra promoter.


Migration towards Bangladesh coastlines projected to increase with sea-level rise through 2100.
To date, projections of human migration induced by sea-level change (SLC) largely suggest large-scale displacement away from vulnerable coastlines. However, results from our model of Bangladesh suggest counterintuitively that people will continue to migrate toward the vulnerable coastline irrespective of the flooding amplified by future SLC under all emissions scenarios until the end of this century. We developed an empirically calibrated agent-based model of household migration decision-making that captures the multi-faceted push, pull and mooring influences on migration at a household scale. We then exposed ~4800 000 simulated migrants to 871 scenarios of projected 21st-century coastal flooding under future emissions pathways. Our model does not predict flooding impacts great enough to drive populations away from coastlines in any of the scenarios. One reason is that while flooding does accelerate a transition from agricultural to non-agricultural income opportunities, livelihood alternatives are most abundant in coastal cities. At the same time, some coastal populations are unable to migrate, as flood losses accumulate and reduce the set of livelihood alternatives (so-called 'trapped' populations). However, even when we increased access to credit, a commonly-proposed policy lever for incentivizing migration in the face of climate risk, we found that the number of immobile agents actually rose. These findings imply that instead of a straightforward relationship between displacement and migration, projections need to consider the multiple constraints on, and preferences for, mobility. Our model demonstrates that decision-makers seeking to affect migration outcomes around SLC would do well to consider individual-level adaptive behaviors and motivations that evolve through time, as well as the potential for unintended behavioral responses.


Whole Chromosome Instability induces senescence and promotes SASP.
Age-related accumulation of ploidy changes is associated with decreased expression of genes controlling chromosome segregation and cohesin functions. To determine the consequences of whole chromosome instability (W-CIN) we down-regulated the spindle assembly checkpoint component BUB1 and the mitotic cohesin SMC1A, and used four-color-interphase-FISH coupled with BrdU incorporation and analyses of senescence features to reveal the fate of W-CIN cells. We observed significant correlations between levels of not-diploid cells and senescence-associated features (SAFs). W-CIN induced DNA double strand breaks and elevated oxidative stress, but caused low apoptosis. SAFs of W-CIN cells were remarkably similar to those induced by replicative senescence but occurred in only 13 days versus 4 months. Cultures enriched with not-diploid cells acquired a senescence-associated secretory phenotype (SASP) characterized by IL1B, CXCL8, CCL2, TNF, CCL27 and other pro-inflammatory factors including a novel SASP component CLEC11A. These findings suggest that W-CIN triggers premature senescence, presumably to prevent the propagation of cells with an abnormal DNA content. Cells deviating from diploidy have the ability to communicate with their microenvironment by secretion of an array of signaling factors. Our results suggest that aneuploid cells that accumulate during aging in some mammalian tissues potentially contribute to age-related pathologies and inflammation through SASP secretion.


Associations between short/medium-term variations in black smoke air pollution and mortality in the Glasgow conurbation, UK.
To examine associations between short/medium-term variations in black smoke air pollution and mortality in the population of Glasgow and the adjacent towns of Renfrew and Paisley over a 25-year period at different time lags (0-30 days). Generalised linear (Poisson) models were used to investigate the relationship between lagged black smoke concentrations and daily mortality, with allowance for confounding by cold temperature, between 1974 and 1998. When a range of lag periods were investigated significant associations were noted between temperature-adjusted black smoke exposure and all-cause mortality at lag periods of 13-18 and 19-24 days, and respiratory mortality at lag periods of 1-6, 7-12, and 13-18 days. Significant associations between cardiovascular mortality and temperature-adjusted black smoke were not observed. After adjusting for the effects of temperature a 10 μg m(-3) increase in black smoke concentration on a given day was associated with a 0.9% [95% Confidence Interval (CI): 0.3-1.5%] increase in all cause mortality and a 3.1% [95% CI: 1.4-4.9%] increase in respiratory mortality over the ensuing 30-day period. In contrast for a 10 μg m(-3) increase in black smoke concentration over 0-3 day lag period, the temperature adjusted exposure mortality associations were substantially lower (0.2% [95% CI: − 0.0-0.4%] and 0.3% [95% CI: − 0.2-0.8%] increases for all-cause and respiratory mortality respectively). This study has provided evidence of association between black smoke exposure and mortality at longer lag periods than have been investigated in the majority of time series analyses.


The LIM protein Ajuba influences p130Cas localization and Rac1 activity during cell migration.
Cell migration requires extension of lamellipodia that are stabilized by formation of adhesive complexes at the leading edge. Both processes are regulated by signaling proteins recruited to nascent adhesive sites that lead to activation of Rho GTPases. The Ajuba/Zyxin family of LIM proteins are components of cellular adhesive complexes. We show that cells from Ajuba null mice are inhibited in their migration, without associated abnormality in adhesion to extracellular matrix proteins, cell spreading, or integrin activation. Lamellipodia production, or function, is defective and there is a selective reduction in the level and tyrosine phosphorylation of FAK, p130Cas, Crk, and Dock180 at nascent focal complexes. In response to migratory cues Rac activation is blunted in Ajuba null cells, as detected biochemically and by FRET analysis. Ajuba associates with the focal adhesion-targeting domain of p130Cas, and rescue experiments suggest that Ajuba acts upstream of p130Cas to localize p130Cas to nascent adhesive sites in migrating cells thereby leading to the activation of Rac.


Reductions in dietary energy density are associated with weight loss in overweight and obese participants in the PREMIER trial.
Dietary energy density (ED) reductions are associated with energy intake (EI) reductions. Little is known about influences on body weight (BW). We examined the effects of behavioral interventions on ED values and explored how 6-mo ED changes relate to BW. Prehypertensive and hypertensive persons were randomly assigned to 1 of 3 groups: the established group received an 18-session intervention implementing well-established hypertension recommendations (eg, weight loss, sodium reduction, and physical activity), the established+Dietary Approaches to Stop Hypertension (DASH) group received an 18-session intervention also implementing the DASH diet, and the advice group received 1 session on these topics. Two 24-h dietary recalls were collected (n=658). Each group had significant declines in EI, ED, and BW. The established and established+DASH groups had the greatest EI and BW reductions. The established+DASH group had the greatest ED reduction and the greatest increase in the weight of food consumed. When groups were combined and analyzed by ED change tertiles, participants in the highest tertile (ie, largest ED reduction) lost more weight (5.9 kg) than did those in the middle (4.0 kg) or lowest (2.4 kg) tertile. Participants in the highest and middle tertiles increased the weight of food they consumed (300 and 80 g/d, respectively) but decreased their EI (500 and 250 kcal/d). Conversely, those in the lowest tertile decreased the weight of food consumed (100 g/d), with little change in EI. The highest and middle tertiles had favorable changes in fruit, vegetable, vitamin, and mineral intakes. Both large and modest ED reductions were associated with weight loss and improved diet quality.


Control of vessel diameters mediated by flow-induced outward vascular remodeling in vitro.
Vascular networks consist of hierarchical structures of various diameters and are necessary for efficient blood distribution. Recent advances in vascular tissue engineering and bioprinting have allowed us to construct large vessels, such as arteries, small vessels, such as capillaries and microvessels, and intermediate-scale vessels, such as arterioles, individually. However, little is known about the control of vessel diameters between small vessels and intermediate-scale vessels. Here, we focus on vascular remodeling, which creates lasting structural changes in the vessel wall in response to hemodynamic stimuli, to regulate vessel diameters in vitro. The purpose of this study is to control the vessel diameter at an intermediate scale by inducing outward remodeling of microvessels in vitro. Human umbilical vein endothelial cells and mesenchymal stem cells were cocultured in a microfluidic device to construct microvessels, which were then perfused with a culture medium to induce outward vascular remodeling. We successfully constructed vessels with diameters of 40-150 μm in perfusion culture, whereas vessels with diameters of <20 μm were maintained in static culture. We also revealed that the in vitro vascular remodeling was mediated by NO pathways and MMP-9. These findings provide insight into the regulation of diameters of tissue-engineered blood vessels. This is an important step toward the construction of hierarchical vascular networks within biofabricated three-dimensional systems.


Antibiotic treatment failure in four common infections in UK primary care 1991-2012: longitudinal analysis.
To characterise failure of antibiotic treatment in primary care in the United Kingdom in four common infection classes from 1991 to 2012. Longitudinal analysis of failure rates for first line antibiotic monotherapies associated with diagnoses for upper and lower respiratory tract infections, skin and soft tissue infections, and acute otitis media. Routine primary care data from the UK Clinical Practice Research Datalink (CPRD). Adjusted rates of treatment failure defined by standardised criteria and indexed to year 1 (1991=100). From 58 million antibiotic prescriptions in CPRD, we analysed 10,967,607 monotherapy episodes for the four indications: 4,236,574 (38.6%) for upper respiratory tract infections; 3,148,947 (28.7%) for lower respiratory tract infections; 2,568,230 (23.4%) for skin and soft tissue infections; and 1,013,856 (9.2%) for acute otitis media. In 1991, the overall failure rate was 13.9% (12.0% for upper respiratory tract infections; 16.9% for lower respiratory tract infections; 12.8% for skin and soft tissue infections; and 13.9% for acute otitis media). By 2012, the overall failure rate was 15.4%, representing an increase of 12% compared with 1991 (adjusted value indexed to first year (1991) 112, 95% confidence interval 112 to 113). The highest rate was seen in lower respiratory tract infections (135, 134 to 136). While failure rates were below 20% for most commonly prescribed antibiotics (amoxicillin, phenoxymethylpenicillin (penicillin-V), and flucloxacillin), notable increases were seen for trimethoprim in the treatment of upper respiratory tract infections (from 29.2% in 1991-95 to 70.1% in 2008-12) and for ciprofloxacin (from 22.3% in 1991-95 to 30.8% in 2008-12) and cefalexin (from 22.0% in 1991-95 to 30.8% in 2008-12) in the treatment of lower respiratory tract infections. Failure rates for broad spectrum penicillins, macrolides, and flucloxacillin remained largely stable. From 1991 to 2012, more than one in 10 first line antibiotic monotherapies for the selected infections were associated with treatment failure. Overall failure rates increased by 12% over this period, with most of the increase occurring in more recent years, when antibiotic prescribing in primary care plateaued and then increased.


The genome of the basidiomycetous yeast and human pathogen Cryptococcus neoformans.
Cryptococcus neoformans is a basidiomycetous yeast ubiquitous in the environment, a model for fungal pathogenesis, and an opportunistic human pathogen of global importance. We have sequenced its approximately 20-megabase genome, which contains approximately 6500 intron-rich gene structures and encodes a transcriptome abundant in alternatively spliced and antisense messages. The genome is rich in transposons, many of which cluster at candidate centromeric regions. The presence of these transposons may drive karyotype instability and phenotypic variation. C. neoformans encodes unique genes that may contribute to its unusual virulence properties, and comparison of two phenotypically distinct strains reveals variation in gene content in addition to sequence polymorphisms between the genomes.


Sheehan's syndrome.
Sheehan's syndrome occurs as a result of ischemic pituitary necrosis due to severe postpartum hemorrhage. It may be rarely seen without massive bleeding or after normal delivery. Improvement in obstetric care and availability of rapid blood transfusion coincided with a remarkable reduction in the frequency of Sheehan's syndrome particularly in western society. But it has recently been reported more often from well-developed countries. It is one of the most common causes of hypopituitarism in underdeveloped or developing countries. Enlargement of pituitary gland, small sella size, disseminated intravascular coagulation and autoimmunity have been suggested to play a role in the pathogenesis of Sheehan's syndrome in women who suffer from severe postpartum hemorrhage. The patients may seek medical advice because of various presentations ranging from non-specific symptoms to coma and the clinical manifestation may change from one patient to another. Failure of postpartum lactation and failure to resume menses after delivery are the most common presenting symptoms. Although a small percentage of patients with Sheehan's syndrome may cause abrupt onset severe hypopituitarism immediately after delivery, most patients have a mild disease and go undiagnosed and untreated for a long time. It may result in partial or panhypopituitarism and GH is one of the hormones lost earliest. The great majority of the patients has empty sella on CT or MRI. Lymphocytic hypophysitis should be kept in mind in differential diagnosis. In this review, the old and recent data regarding Sheehan's syndrome are presented.


Biothiol-Activatable Bioluminescent Coelenterazine Derivative for Molecular Imaging in Vitro and in Vivo.
There is a high demand for sensitive biothiol probes targeting cysteine, glutathione, and homocysteine. These biothiols are known as playing essential roles to maintain homeostasis and work as indicators of many diseases. This work presents a bioluminescent probe (named AMCM) to detect biothiols in live mammalian cells and in vivo with a limit of detection of 0.11 μM for cysteine in solution and high selectivity for biothiols, making it suitable for real-time biothiol detection in biological systems. Upon application to live cells, AMCM showed low cytotoxicity and sensitively reported bioluminescence in response to changes of biothiol levels. Furthermore, a bioluminescence resonance energy transfer system consisting of AMCM combined with the near-infrared fluorescent protein iRFP713 was applied to in vivo imaging, with emitted tissue-permeable luminescence in living mice. In summary, this work demonstrates that AMCM is of high practical value for the detection of biothiols in living cells and for deep tissue imaging in living animals.


'South Asian cocktail'--the concurrent use of opioids, benzodiazepines and antihistamines among injecting drug users in Nepal and associations with HIV risk behaviour.
Data of the Central Bureau of Statistic of Nepal from 2008 show a total of more than 46,000 illegal drug users, out of which 61% are injecting drug users (IDU). An injecting mix of medicines like opioids, benzodiazepines and antihistamines (the so-called South Asian cocktail) was prevalent. Furthermore, it is estimated that about 70,000 people are living with human immunodeficiency virus (HIV). The government of Nepal has started realizing and recognizing drug use and HIV as significant health and social issues. Harm reduction programs such as needle syringe exchange and opioid substitution treatment are being implemented. The aim of this study is to obtain specific knowledge on the drug use behaviour and the health status of drug users with a focus on HIV in drug users with concurrent injection of opioids, benzodiazepines and antihistamines. After an initial mapping of Kathmandu Valley, 300 drug users in contact with different treatment and counselling centres were randomly chosen for the interviews. The research questionnaire was designed according to the European Addiction Severity Index (EuropASI) and Maudsley Addiction Profile standards. Ninety-one percent of the respondents are male and 9% female. Mean age is 28.7 years. Ninety-five percent are injecting drug users with a mean of 8.7 years of drug use history. Eighty-six percent are injecting different 'cocktails', usually made of buprenorphine, diazepam, promethazine and/or other substances (30-day prevalence). Similarly, 48% use heroin, whereas only 2% take cocaine/crack. Among those tested for HIV (N = 223), 33% are positive (25% of the sample population). Compared to the other drug users (mainly heroin), the cocktail users show a higher HIV infection rate and more co-infections. Furthermore, risk behaviour, as e.g. needle sharing, is much more common among the cocktail users. Currently, the mixture of medicines, opioids, benzodiazepines and antihistamines, is the predominant drug in Nepal; the pharmaceutical drugs needed to prepare the cocktail are less expensive than heroin and relatively easy to acquire. The cocktail users show a higher risk behaviour regarding the transmission of HIV than heroin drug users. It needs to be considered which HIV prevention measures are necessary to target the specific needs of drug users who inject a mixture of opioids, benzodiazepines and antihistamines, since the available services (such as needle syringe exchange) do not seem to cover their specific needs (high percentage of needle sharing).


Loss of phase-locking in non-weakly coupled inhibitory networks of type-I model neurons.
Synchronization of excitable cells coupled by reciprocal inhibition is a topic of significant interest due to the important role that inhibitory synaptic interaction plays in the generation and regulation of coherent rhythmic activity in a variety of neural systems. While recent work revealed the synchronizing influence of inhibitory coupling on the dynamics of many networks, it is known that strong coupling can destabilize phase-locked firing. Here we examine the loss of synchrony caused by an increase in inhibitory coupling in networks of type-I Morris-Lecar model oscillators, which is characterized by a period-doubling cascade and leads to mode-locked states with alternation in the firing order of the two cells, as reported recently by Maran and Canavier (J Comput Nerosci, 2008) for a network of Wang-Buzsáki model neurons. Although alternating-order firing has been previously reported as a near-synchronous state, we show that the stable phase difference between the spikes of the two Morris-Lecar cells can constitute as much as 70% of the unperturbed oscillation period. Further, we examine the generality of this phenomenon for a class of type-I oscillators that are close to their excitation thresholds, and provide an intuitive geometric description of such "leap-frog" dynamics. In the Morris-Lecar model network, the alternation in the firing order arises under the condition of fast closing of K( + ) channels at hyperpolarized potentials, which leads to slow dynamics of membrane potential upon synaptic inhibition, allowing the presynaptic cell to advance past the postsynaptic cell in each cycle of the oscillation. Further, we show that non-zero synaptic decay time is crucial for the existence of leap-frog firing in networks of phase oscillators. However, we demonstrate that leap-frog spiking can also be obtained in pulse-coupled inhibitory networks of one-dimensional oscillators with a multi-branched phase domain, for instance in a network of quadratic integrate-and-fire model cells. Finally, for the case of a homogeneous network, we establish quantitative conditions on the phase resetting properties of each cell necessary for stable alternating-order spiking, complementing the analysis of Goel and Ermentrout (Physica D 163:191-216, 2002) of the order-preserving phase transition map.


Injectable Coacervate Hydrogel for Delivery of Anticancer Drug-Loaded Nanoparticles in vivo.
In this study, bortezomib (BTZ, a cytotoxic water-insoluble anticancer drug) was encapsulated in micellar nanoparticles having a catechol-functionalized polycarbonate core through a pH-sensitive covalent bond between phenylboronic acid (PBA) in BTZ and catechol, and these drug-loaded micelles were incorporated into hydrogels to form micelle/hydrogel composites. A series of injectable, biodegradable hydrogels with readily tunable mechanical properties were formed and optimized for sustained delivery of the BTZ-loaded micelles through ionic coacervation between PBA-functionalized polycarbonate/poly(ethylene glycol) (PEG) "ABA" triblock copolymer and a cationic one having guanidinium- or thiouronium-functionalized polycarbonate as "A" block. An in vitro release study showed the pH dependence in BTZ release. At pH 7.4, the BTZ release from the micelle/hydrogel composite remained low at 7%, whereas in an acidic environment, ∼85% of BTZ was released gradually over 9 days. In vivo studies performed in a multiple myeloma MM.1S xenograft mouse model showed that the tumor progression of mice treated with BTZ-loaded micelle solution was similar to that of the control group, whereas those treated with the BTZ-loaded micelle/hydrogel composite resulted in significant delay in the tumor progression. The results demonstrate that this hydrogel has great potential for use in subcutaneous and sustained delivery of drug-loaded micelles with superior therapeutic efficacy.


Experimental and computational assessment of F-actin influence in regulating cellular stiffness and relaxation behaviour of fibroblasts.
In biomechanics, a complete understanding of the structures and mechanisms that regulate cellular stiffness at a molecular level remain elusive. In this paper, we have elucidated the role of filamentous actin (F-actin) in regulating elastic and viscous properties of the cytoplasm and the nucleus. Specifically, we performed colloidal-probe atomic force microscopy (AFM) on BjhTERT fibroblast cells incubated with Latrunculin B (LatB), which results in depolymerisation of F-actin, or DMSO control. We found that the treatment with LatB not only reduced cellular stiffness, but also greatly increased the relaxation rate for the cytoplasm in the peripheral region and in the vicinity of the nucleus. We thus conclude that F-actin is a major determinant in not only providing elastic stiffness to the cell, but also in regulating its viscous behaviour. To further investigate the interdependence of different cytoskeletal networks and cell shape, we provided a computational model in a finite element framework. The computational model is based on a split strain energy function of separate cellular constituents, here assumed to be cytoskeletal components, for which a composite strain energy function was defined. We found a significant influence of cell geometry on the predicted mechanical response. Importantly, the relaxation behaviour of the cell can be characterised by a material model with two time constants that have previously been found to predict mechanical behaviour of actin and intermediate filament networks. By merely tuning two effective stiffness parameters, the model predicts experimental results in cells with a partly depolymerised actin cytoskeleton as well as in untreated control. This indicates that actin and intermediate filament networks are instrumental in providing elastic stiffness in response to applied forces, as well as governing the relaxation behaviour over shorter and longer time-scales, respectively.


A dynamic strategy for wavelength sensing using the diffracted orders of a grating.
We propose a strategy for real time multicolour detection of light emitting particles under flow conditions, using the diffracted orders of a grating. The method relies on the fact that the spatial (angular/linear) separation between the orders of a diffraction grating is a wavelength dependent quantity. Thus, the difference in arrival times at a fixed detector for neighbouring orders produced by a moving, light-emitting particle can be used as a wavelength sensing mechanism. We demonstrate this functionality using a prototype PDMS microfluidic device that incorporates a focusing transmission grating. A key attribute of this approach is that it requires only a single sensitive detector, such as a photomultiplier tube, in contrast to many conventional approaches which require multiple sensitive detectors or a detector array.


Viruses in human cancers.
Viruses may contribute to the development of human tumors by different mechanisms: indirectly by inducing immunosuppression or by modifying the host cell genome without persistence of viral DNA; directly by inducing oncoproteins or by altering the expression of host cell proteins at the site of viral DNA integration. Human cancers associated with papillomavirus, hepatitis B virus, Epstein-Barr virus, and human T cell leukemia-lymphoma virus infections are responsible for approximately 15 percent of the worldwide cancer incidence. Cancer of the cervix and hepatocellular carcinoma account for about 80 percent of virus-linked cancers. Because experimental and epidemiologic data imply a causative role for viruses, particularly in cervical and liver cancer, viruses must be thought of as the second most important risk factor for cancer development in humans, exceeded only by tobacco consumption.


Population-scale sequencing reveals genetic differentiation due to local adaptation in Atlantic herring.
The Atlantic herring (Clupea harengus), one of the most abundant marine fishes in the world, has historically been a critical food source in Northern Europe. It is one of the few marine species that can reproduce throughout the brackish salinity gradient of the Baltic Sea. Previous studies based on few genetic markers have revealed a conspicuous lack of genetic differentiation between geographic regions, consistent with huge population sizes and minute genetic drift. Here, we present a cost-effective genome-wide study in a species that lacks a genome sequence. We first assembled a muscle transcriptome and then aligned genomic reads to the transcripts, creating an "exome assembly," capturing both exons and flanking sequences. We then resequenced pools of fish from a wide geographic range, including the Northeast Atlantic, as well as different regions in the Baltic Sea, aligned the reads to the exome assembly, and identified 440,817 SNPs. The great majority of SNPs showed no appreciable differences in allele frequency among populations; however, several thousand SNPs showed striking differences, some approaching fixation for different alleles. The contrast between low genetic differentiation at most loci and striking differences at others implies that the latter category primarily reflects natural selection. A simulation study confirmed that the distribution of the fixation index F(ST) deviated significantly from expectation for selectively neutral loci. This study provides insights concerning the population structure of an important marine fish and establishes the Atlantic herring as a model for population genetic studies of adaptation and natural selection.


Transmission processes in random patterns of subwavelength holes.
The optical transmission of random patterns of holes is believed to depend on the transmission of the independent holes only. By comparing the transmission spectra of random patterns with different densities, we show that the quasi-cylindrical wave plays an important role in the transmission of samples with large hole densities. Furthermore, we report on a speckle pattern seen in the transmission of these arrays. By studying the degree of depolarization in this speckle pattern, as a function of hole density, we are able to quantify the role of surface plasmons to the transmission.


Toward metabolic phenomics: analysis of genomic data using flux balances.
Small genome sequencing and annotations are leading to the definition of metabolic genotypes in an increasing number of organisms. Proteomics is beginning to give insights into the use of the metabolic genotype under given growth conditions. These data sets give the basis for systemically studying the genotype-phenotype relationship. Methods of systems science need to be employed to analyze, interpret, and predict this complex relationship. These endeavors will lead to the development of a new field, tentatively named phenomics. This article illustrates how the metabolic characteristics of annotated small genomes can be analyzed using flux balance analysis (FBA). A general algorithm for the formulation of in silico metabolic genotypes is described. Illustrative analyses of the in silico Escherichia coli K-12 metabolic genotypes are used to show how FBA can be used to study the capabilities of this strain.


Plant-derived compounds in treatment of leishmaniasis.
Leishmaniasis is a neglected public health problem caused by the protozoan species belonging to the genus Leishmania affecting mostly the poor populations of developing countries. The causative organism is transmitted by female sandflies. Cutaneous, mucocutaneous, and visceral clinical manifestations are the most frequent forms of leishmaniasis. Chemotherapy still relies on the use of pentavalent antimonials, amphotericin B, paromomycin, miltefosin and liposomal amphotericin B. However, the application of these drugs is limited due to low efficacy, life-threatening side effects, high toxicity, induction of parasite resistance, length of treatment and high cost. Given the fact that antileishmanial vaccines may not become available in the near future, the search for better drugs should be continued. Natural products may offer an unlimited source of chemical diversity to identify new drug modules. New medicines should be less toxic or non-toxic, safe, more efficient, less expensive and readily available antileishmanial agents, especially for low-income populations. In the present review, special focus is on medicinal plants used against leishmanaiasis. The bioactive phytocompounds present in the plant derivatives including the crude extracts, essential oils, and other useful compounds can be a good source for discovering and producing new antileishmanial medicines.


Temporal trends and long term follow-up of implantable cardioverter defibrillator therapy for secondary prevention: A 15-year single-centre experience.
The aim of this study was to determine overall and aetiology-related incidence of secondary prevention ICD implantation over the last 15years in Canton Ticino and to assess clinical outcome according to time period of implantation. Consecutive patients treated by implantation of an ICD for secondary prevention from 2000 to 2015 were included in the current study and compared between 5-year cohorts (2000/2004; 2005/2009; 2010/2015). Yearly implantation rate, changing in clinical presentation over years and events during follow-up were evaluated. One-hundred fifty six patients were included. ICD implantation rate increased from 2.1 in 2000-2005 to 5.1 in 2010-2015, respectively (p 0.001). There was an increase in the proportion of non-ischaemic patients and of ventricular tachycardia (VT) as presenting rhythm. No differences in appropriate ICD interventions were observed according to aetiology, presenting arrhythmia or type of device. Reverse remodelling was observed more often in non-ischaemic patients, without any influence on the occurrence of appropriate interventions. Previous myocardial infarction (MI), atrial fibrillation (AF), NYHA class 2-3 and left ventricular ejection fraction (LVEF)<35% were predictors of appropriate therapies during follow-up. Rate of implants for secondary prevention indication has almost doubled during the last 15years. Importantly, there has been a progressive increase of non-ischaemic patients receiving an ICD, and of VT as presenting rhythm. Patients had an overall good survival and a relatively low incidence of appropriate therapies. Improvement of ejection fraction did not correlate with risk reduction of ventricular arrhythmias.


Orientation Tuning Depends on Spatial Frequency in Mouse Visual Cortex.
The response properties of neurons to sensory stimuli have been used to identify their receptive fields and to functionally map sensory systems. In primary visual cortex, most neurons are selective to a particular orientation and spatial frequency of the visual stimulus. Using two-photon calcium imaging of neuronal populations from the primary visual cortex of mice, we have characterized the response properties of neurons to various orientations and spatial frequencies. Surprisingly, we found that the orientation selectivity of neurons actually depends on the spatial frequency of the stimulus. This dependence can be easily explained if one assumed spatially asymmetric Gabor-type receptive fields. We propose that receptive fields of neurons in layer 2/3 of visual cortex are indeed spatially asymmetric, and that this asymmetry could be used effectively by the visual system to encode natural scenes.


Who Chooses STEM Careers? Using A Relative Cognitive Strength and Interest Model to Predict Careers in Science, Technology, Engineering, and Mathematics.
Career aspirations in science, technology, engineering, and mathematics (STEM) are formulated in adolescence, making the high school years a critical time period for identifying the cognitive and motivational factors that increase the likelihood of future STEM employment. While past research has mainly focused on absolute cognitive ability levels in math and verbal domains, the current study tested whether relative cognitive strengths and interests in math, science, and verbal domains in high school were more accurate predictors of STEM career decisions. Data were drawn from a national longitudinal study in the United States (N = 1762; 48 % female; the first wave during ninth grade and the last wave at age 33). Results revealed that in the high-verbal/high-math/high-science ability group, individuals with higher science task values and lower orientation toward altruism were more likely to select STEM occupations. In the low-verbal/moderate-math/moderate-science ability group, individuals with higher math ability and higher math task values were more likely to select STEM occupations. The findings suggest that youth with asymmetrical cognitive ability profiles are more likely to select careers that utilize their cognitive strengths rather than their weaknesses, while symmetrical cognitive ability profiles may grant youth more flexibility in their options, allowing their interests and values to guide their career decisions.


Frustration and fidelity in influenza genome assembly.
The genome of the influenza virus consists of eight distinct single-stranded RNA segments, each encoding proteins essential for the viral life cycle. When the virus infects a host cell, these segments must be replicated and packaged into new budding virions. The viral genome is assembled with remarkably high fidelity: experiments reveal that most virions contain precisely one copy of each of the eight RNA segments. Cell-biological studies suggest that genome assembly is mediated by specific reversible and irreversible interactions between the RNA segments and their associated proteins. However, the precise inter-segment interaction network remains unresolved. Here, we computationally predict that tree-like irreversible interaction networks guarantee high-fidelity genome assembly, while cyclic interaction networks lead to futile or frustrated off-pathway products. We test our prediction against multiple experimental datasets. We find that tree-like networks capture the nearest-neighbour statistics of RNA segments in packaged virions, as observed by electron tomography. Just eight tree-like networks (of a possible 262 144) optimally capture both the nearest-neighbour data and independently measured RNA-RNA binding and co-localization propensities. These eight do not include the previously proposed hub-and-spoke and linear networks. Rather, each predicted network combines hub-like and linear features, consistent with evolutionary models of interaction gain and loss.


Study of the impact of long-duration space missions at the International Space Station on the astronaut microbiome.
Over the course of a mission to the International Space Station (ISS) crew members are exposed to a number of stressors that can potentially alter the composition of their microbiomes and may have a negative impact on astronauts' health. Here we investigated the impact of long-term space exploration on the microbiome of nine astronauts that spent six to twelve months in the ISS. We present evidence showing that the microbial communities of the gastrointestinal tract, skin, nose and tongue change during the space mission. The composition of the intestinal microbiota became more similar across astronauts in space, mostly due to a drop in the abundance of a few bacterial taxa, some of which were also correlated with changes in the cytokine profile of crewmembers. Alterations in the skin microbiome that might contribute to the high frequency of skin rashes/hypersensitivity episodes experienced by astronauts in space were also observed. The results from this study demonstrate that the composition of the astronauts' microbiome is altered during space travel. The impact of those changes on crew health warrants further investigation before humans embark on long-duration voyages into outer space.


Decreased serotonin function in bulimia nervosa.
Evidence that serotonin-active antidepressant medications decrease binge eating in patients with bulimia nervosa has fueled interest in the hypothesis that abnormal serotonergic neurotransmitter function contributes to symptoms of the disorder. To evaluate this hypothesis, we employed pharmacological challenge testing to compare serotonin function in patients with bulimia nervosa and healthy controls. Neuroendocrine response patterns were compared for 15 nonhospitalized, medication-free, normal-weight women with bulimia nervosa and 14 age-matched healthy female controls. Behavioral assessment included ratings of eating disorder symptoms, depression, and anxiety. Serotonergic response patterns were assessed by measuring the increase in serum prolactin concentration during 5 hours following single-dose, oral administration of 60 mg of d,l-fenfluramine hydrochloride (Pondimin). For patients with bulimia nervosa, the fenfluramine-stimulated increase in serum prolactin concentration was significantly less than for controls. Within the patient group, the frequency of binge eating episodes during the 4 weeks prior to the study exhibited a significant inverse correlation with serotonin-stimulated prolactin secretion. Our study provides new evidence that impaired central nervous system serotonergic responsiveness may contribute to the onset or maintenance of abnormal eating patterns in patients with bulimia nervosa.


Large-scale neurochemical metabolomics analysis identifies multiple compounds associated with methamphetamine exposure.
Methamphetamine (MA) is an illegal stimulant drug of abuse with serious negative health consequences. The neurochemical effects of MA have been partially characterized, with a traditional focus on classical neurotransmitter systems. However, these directions have not yet led to novel drug treatments for MA abuse or toxicity. As an alternative approach, we describe here the first application of metabolomics to investigate the neurochemical consequences of MA exposure in the rodent brain. We examined single exposures at 3 mg/kg and repeated exposures at 3 mg/kg over 5 days in eight common inbred mouse strains. Brain tissue samples were assayed using high-throughput gas and liquid chromatography mass spectrometry, yielding quantitative data on >300 unique metabolites. Association testing and false discovery rate control yielded several metabolome-wide significant associations with acute MA exposure, including compounds such as lactate (p = 4.4 × 10-5, q = 0.013), tryptophan (p = 7.0 × 10-4, q = 0.035) and 2-hydroxyglutarate (p = 1.1 × 10-4, q = 0.022). Secondary analyses of MA-induced increase in locomotor activity showed associations with energy metabolites such as succinate (p = 3.8 × 10-7). Associations specific to repeated (5 day) MA exposure included phosphocholine (p = 4.0 × 10-4, q = 0.087) and ergothioneine (p = 3.0 × 10-4, q = 0.087). Our data appear to confirm and extend existing models of MA action in the brain, whereby an initial increase in energy metabolism, coupled with an increase in behavioral locomotion, gives way to disruption of mitochondria and phospholipid pathways and increased endogenous antioxidant response. Our study demonstrates the power of comprehensive MS-based metabolomics to identify drug-induced changes to brain metabolism and to develop neurochemical models of drug effects.


Cluster headache and the hypocretin receptor 2 reconsidered: a genetic association study and meta-analysis.
Cluster headache is a severe neurological disorder with a complex genetic background. A missense single nucleotide polymorphism (rs2653349; p.Ile308Val) in the HCRTR2 gene that encodes the hypocretin receptor 2 is the only genetic factor that is reported to be associated with cluster headache in different studies. However, as there are conflicting results between studies, we re-evaluated its role in cluster headache. We performed a genetic association analysis for rs2653349 in our large Leiden University Cluster headache Analysis (LUCA) program study population. Systematic selection of the literature yielded three additional studies comprising five study populations, which were included in our meta-analysis. Data were extracted according to predefined criteria. A total of 575 cluster headache patients from our LUCA study and 874 controls were genotyped for HCRTR2 SNP rs2653349 but no significant association with cluster headache was found (odds ratio 0.91 (95% confidence intervals 0.75-1.10), p = 0.319). In contrast, the meta-analysis that included in total 1167 cluster headache cases and 1618 controls from the six study populations, which were part of four different studies, showed association of the single nucleotide polymorphism with cluster headache (random effect odds ratio 0.69 (95% confidence intervals 0.53-0.90), p = 0.006). The association became weaker, as the odds ratio increased to 0.80, when the meta-analysis was repeated without the initial single South European study with the largest effect size. Although we did not find evidence for association of rs2653349 in our LUCA study, which is the largest investigated study population thus far, our meta-analysis provides genetic evidence for a role of HCRTR2 in cluster headache. Regardless, we feel that the association should be interpreted with caution as meta-analyses with individual populations that have limited power have diminished validity.


Gender-based impact of epidermal growth factor receptor mutation in patients with nonsmall cell lung cancer and previous tuberculosis.
The association between tuberculosis (TB) and lung cancer is well known. However, carcinogenesis of TB and its connection with epidermal growth factor receptor (EGFR) mutation remains unclear. This study aimed to determine this connection to see if TB can affect the outcome of patients with lung cancer. This is a retrospective cohort study of patients with lung cancer receiving EGFR-tyrosine kinase inhibitors (TKIs) between 1996 and 2010 using the National Health Insurance Research Database of Taiwan. Because therapeutic response was required to apply EGFR-TKIs for >90 days, only patients with a follow-up of >120 days were studied and a responder was defined as intake of EGFR-TKIs >90 days. Predictors of EGFR-TKI response and survival were identified using logistic and Cox regression analyses, respectively. There were 8265 patients analyzed, including 6073 (73.5%) EGFR-TKI responder and 2192 (26.5%) nonresponder. A history of TB was found in 1.2% and 1.8% of the 2 groups, respectively. Comparing to male with pulmonary TB history, female with or without pulmonary TB history and male without pulmonary TB history all had a better EGFR-TKI response and 1-year progression-free survival (PFS). Gender and TB history were not independent prognostic factors of 2-year overall survival. The findings were similar in the subpopulation without chronic obstructive pulmonary disease, malignancies other than lung cancer, and low-income status. TB has a gender-dependent impact, with better EGFR-TKI response and 1-year PFS in female patients with lung cancer. The carcinogenesis and inflammation of TB may be different between genders.


Production and Concentration of Lentivirus for Transduction of Primary Human T Cells.
Lentiviral vectors have emerged as efficient tools for investigating T cell biology through their ability to efficiently deliver transgene expression into both dividing and nondividing cells. Such lentiviral vectors have the potential to infect a wide variety of cell types. However, despite this advantage, the ability to transduce primary human T cells remains challenging and methods to achieve efficient gene transfer are often time consuming and expensive. We describe a method for generating lentivirus that is simple to perform and does not require the purchase of non-standard equipment to transduce primary human T cells. Therefore, we provide an optimized protocol that is easy to implement and allow transduction with high efficiency and reproducibility.


Enhanced inhibition of murine tumor and human breast tumor xenografts using targeted delivery of an antibody-endostatin fusion protein.
Endostatin can inhibit angiogenesis and tumor growth in mice. A potential limitation of endostatin as an antitumor agent in humans is the short serum half-life of the protein that may decrease effective concentration at the site of tumor and necessitate frequent dosing. In an effort to improve antitumor activity, endostatin was fused to an antibody specific for the tumor-selective HER2 antigen to create an antibody-endostatin fusion protein (anti-HER2 IgG3-endostatin). Normal endostatin rapidly cleared from serum in mice (T(1/2)(2), = 0.6-3.8 hours), whereas anti-HER2 IgG3-endostatin had a prolonged half-life (90% intact; T(1/2)(2), 40.2-44.0 hours). Antigen-specific targeting of anti-HER2 IgG3-endostatin was evaluated in BALB/c mice implanted with CT26 tumors or CT26 tumors engineered to express the HER2 antigen (CT26-HER2). Radio-iodinated anti-HER2 IgG3-endostatin preferentially localized to CT26-HER2 tumors relative to CT26 tumors. Administration of anti-HER2 IgG3-endostatin to mice showed preferential inhibition of CT26-HER2 tumor growth compared with CT26. Anti-HER2 IgG3-endostatin also markedly inhibited the growth of human breast cancer SK-BR-3 xenografts in severe combined immunodeficient mice. Anti-HER2 IgG3-endostatin inhibited tumor growth significantly more effectively than endostatin, anti-HER2 IgG3 antibody, or the combination of antibody and endostatin. CT26-HER2 tumors treated with the endostatin fusion protein had decreased blood vessel density and branching compared with untreated CT26-HER2 or CT26 treated with the fusion protein. The enhanced effectiveness of anti-HER2 IgG3-endostatin may be due to a longer half-life, improved serum stability, and selective targeting of endostatin to tumors, resulting in decreased angiogenesis. Linking of an antiangiogenic protein, such as endostatin, to a targeting antibody represents a promising and versatile approach to antitumor therapy.


2020 French recommendations on the management of septic arthritis in an adult native joint.
Septic arthritis (SA) in an adult native joint is a rare condition but a diagnostic emergency due to the morbidity and mortality and the functional risk related to structural damage. Current management varies and the recommendations available are dated. The French Rheumatology Society (SFR) Bone and Joint Infection Working Group, together with the French Language Infectious Diseases Society (SPILF) and the French Orthopaedic and Trauma Surgery Society (SOFCOT) have worked according to the HAS methodology to devise clinical practice recommendations to diagnose and treat SA in an adult native joint. One new focus is on the importance of microbiological documentation (blood cultures and joint aspiration) before starting antibiotic treatment, looking for differential diagnoses (microcrystal detection), the relevance of a joint ultrasound to guide aspiration, and the indication to perform a reference X-ray. A cardiac ultrasound is indicated only in cases of SA involving Staphylococcus aureus, oral streptococci, Streptococcus gallolyticus or Enterococcus faecalis, or when infective endocarditis is clinically suspected. Regarding treatment, we stress the importance of medical and surgical collaboration. Antibiotic therapies (drugs and durations) are presented in the form of didactic tables according to the main bacteria in question (staphylococci, streptococci and gram-negative rods). Probabilistic antibiotic therapy should only be used for patients with serious symptoms. Lastly, non-drug treatments such as joint drainage and early physical therapy are the subject of specific recommendations.


Long-term experimental evolution in Escherichia coli. XII. DNA topology as a key target of selection.
The genetic bases of adaptation are being investigated in 12 populations of Escherichia coli, founded from a common ancestor and serially propagated for 20,000 generations, during which time they achieved substantial fitness gains. Each day, populations alternated between active growth and nutrient exhaustion. DNA supercoiling in bacteria is influenced by nutritional state, and DNA topology helps coordinate the overall pattern of gene expression in response to environmental changes. We therefore examined whether the genetic controls over supercoiling might have changed during the evolution experiment. Parallel changes in topology occurred in most populations, with the level of DNA supercoiling increasing, usually in the first 2000 generations. Two mutations in the topA and fis genes that control supercoiling were discovered in a population that served as the focus for further investigation. Moving the mutations, alone and in combination, into the ancestral background had an additive effect on supercoiling, and together they reproduced the net change in DNA topology observed in this population. Moreover, both mutations were beneficial in competition experiments. Clonal interference involving other beneficial DNA topology mutations was also detected. These findings define a new class of fitness-enhancing mutations and indicate that the control of DNA supercoiling can be a key target of selection in evolving bacterial populations.


Commentary. A picture of Mitf in melanoma immortality.
The Mitf gene has a key role in melanocytes and melanoma by regulating cell cycle progression, survival and differentiation. Two papers in this issue of Oncogene (Cheli et al., 2011; Strub et al., 2011) reveal that low-Mitf cells can initiate tumors with high efficiency, and that Mitf blocks senescence by regulating genes implicated in S-phase progression and mitosis.


Prolonged standing at work and hospitalisation due to varicose veins: a 12 year prospective study of the Danish population.
Recent studies suggest that prolonged standing at work is associated with the development of diseases of varicose veins (VV). To assess the risk of hospitalisation due to VV in the lower extremities prospectively in workers standing or walking at least 75% of their time at work. A representative random sample of 9653 working age adults was drawn from the Central Population Register of Denmark in 1991. Of these, 8664 accepted to be interviewed by telephone (response rate 90%). Respondents (2939 men and 2708 women) were 20-59 years old and employed in 1990. Risk ratios for VV were estimated by log-linear Poisson regression models separately for men and women with adjustment for smoking status, body mass index (BMI), heavy lifting, and, for females only, number of children at baseline. During 12 years of follow up, 40 hospitalisations due to VV were observed among the men and 71 among the women. For employees with jobs that require prolonged standing or walking compared to all other employees, the relative risk was 1.75 (95% CI 0.92 to 3.34) for men and 1.82 (95% CI 1.12 to 2.95) for women. The pooled estimate of the relative risk was 1.78 (95% CI 1.19 to 2.68). The aetiological fraction of prolonged standing or walking at work was estimated as 22.5% for men and 22.6% for women. This prospective study confirms that prolonged standing at work constitutes an excess risk of hospital treatment due to varicose veins and accounts for more than one fifth of all cases of working age.


Disrupting evolutionary processes: the effect of habitat fragmentation on collared lizards in the Missouri Ozarks.
Humans affect biodiversity at the genetic, species, community, and ecosystem levels. This impact on genetic diversity is critical, because genetic diversity is the raw material of evolutionary change, including adaptation and speciation. Two forces affecting genetic variation are genetic drift (which decreases genetic variation within but increases genetic differentiation among local populations) and gene flow (which increases variation within but decreases differentiation among local populations). Humans activities often augment drift and diminish gene flow for many species, which reduces genetic variation in local populations and prevents the spread of adaptive complexes outside their population of origin, thereby disrupting adaptive processes both locally and globally within a species. These impacts are illustrated with collared lizards (Crotaphytus collaris) in the Missouri Ozarks. Forest fire suppression has reduced habitat and disrupted gene flow in this lizard, thereby altering the balance toward drift and away from gene flow. This balance can be restored by managed landscape burns. Some have argued that, although human-induced fragmentation disrupts adaptation, it will also ultimately produce new species through founder effects. However, population genetic theory and experiments predict that most fragmentation events caused by human activities will facilitate not speciation, but local extinction. Founder events have played an important role in the macroevolution of certain groups, but only when ecological opportunities are expanding rather than contracting. The general impact of human activities on genetic diversity disrupts or diminishes the capacity for adaptation, speciation, and macroevolutionary change. This impact will ultimately diminish biodiversity at all levels.


Suppression of inflammatory mediators by cruciferous vegetable-derived indole-3-carbinol and phenylethyl isothiocyanate in lipopolysaccharide-activated macrophages.
This study was aimed to examine the effects of indole-3-carbinol (I3C) and beta-phenylethyl isothiocyanate (PEITC), bioactive components present in cruciferous vegetable, on the production of inflammatory mediators, including nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10), in lipopolysaccharide- (LPS-) stimulated RAW 264.7 macrophages. Possible mechanisms of the NO-inhibitory effects were also explored. The results indicated that I3C and PEITC inhibited NO production, and this suppression was associated with decreased production of TNF-alpha and IL-10 by activated macrophages. In addition, I3C suppressed NO production even after the inducible nitric oxide synthase (iNOS) protein had been produced, but such an inhibitory effect was not observed in cells treated with PEITC. Furthermore, both compounds reduced the NO contents generated from an NO donor in a cell-free condition, suggesting that the increased NO clearance may have contributed to the NO-inhibitory effects. In summary, both I3C and PEITC possessed antiinflammatory effects by inhibiting the productions of NO, TNF-alpha, and IL-10, although the NO-inhibitory effects may have involved in different mechanisms.


Evaluation of the effects of silver nanoparticles on Danio rerio cornea: Morphological and ultrastructural analysis.
Although in recent years there has been intensification in the use of nanoparticles (NPs) for the production of many commercial products, few studies have been carried out to assess the risks associated with its use. Among the most used NPs, silver nanoparticles (AgNPs) have a preponderant position as they have various applications. In this study, 40 adult zebrafish were exposed to increasing concentrations of AgNPs (8, 45, and 70 μg/L) for 30 days to evaluate the effects on eyes after chronic exposure to AgNPs with an average diameter of 50 nm. From the morphological and ultrastructural analysis performed, no alteration or lesions of the corneal epithelium were detected.


Whither the acute medical patient?
The continual change in NHS structure demands reappraisal of both the services provided and the allocation of resources to ensure appropriate standards of care. Unfortunately this equation never seems to balance. Although the overall goal is to have an excellent standard of care, the necessary resources nearly always are lacking. As a consequence "we" often have to critically analyse and change clinical practice to achieve this goal. Nowhere in medicine is this point more pertinent than in the management of acute medical emergencies. This article examines not only how the changing face of medicine influences this situation but also possible solutions to the question who will manage these patients in the future.