Fever in the tropics: aetiology and case-fatality - a prospective observational study in a tertiary care hospital in South India.
The objective of this study was to describe aetiology and case fatality of fever among inpatients in a tertiary care hospital in South India. This was an observational, prospective study conducted in a tertiary care hospital in Vellore, Tamil Nadu, India. Between July 2nd 2007 and August 2nd in 2007, adult patients admitted to the hospital with temperature ≥ 38.0°C were included consecutively and followed during the hospitalisation period. Demographic and clinical data were collected and analysed for each patient. Associations were sought between death and various clinical and demographic variables. One hundred patients were included, 61 male and 39 female. Mean age was 37.5 (range: 16 to 84) years. Mean fever duration was 5.4 (range: 0.1 to 42.9) weeks.The following infectious aetiologies were recorded: tuberculosis (19%), lower respiratory infection (11%) including three with sepsis, urinary tract infection (10%) including three with E. coli sepsis, Plasmodium falciparum malaria (5%) including three patients with mixed P. vivax infection, scrub typhus (5%), typhoid fever (4%), cryptococcal meningitis (4%) including three HIV positive patients, endocarditis (3%) including two patients with Staphylococcus aureus sepsis, spleen abscess (2%), amoebic liver abscess (2%), sepsis undefined focus (1%), HIV infection (1%), hepatitis B (1%), rubella (1%), peritonitis (1%) and cholecystitis (1%).Non-infectious causes of fever were diagnosed in 15%, including systemic lupus erythematosus in four and malignancy in six patients. Cause of fever remained unknown in 13%.Case fatality during hospitalisation was 7% (7/100). Six of those who died were male. Five fatalities had bacterial sepsis, one spleen abscess and malignancy, and one had lymphomalignant disorder.Diabetes and increasing age were significant risk factors for fatal outcome in unadjusted analyses, but only increasing age was a risk factor for death in adjusted analysis. A high number of tuberculosis and bacterial infections and a high case fatality rate from sepsis were found in this cohort, underlining the importance of microbiological diagnostics and targeted antimicrobial treatment in the management of fever. P. falciparum was identified in all malaria cases, and this rapidly fatal infection should be considered in patients with acute undifferentiated fever in India.


Effects of underfeeding and of starvation on thermoregulatory responses to cooling in women.
1. The effects of acute alterations in energy intake on the thermoregulatory responses to a cooling stimulus were studied in healthy, normal weight, young female subjects. On separate occasions, seven subjects were underfed for 7 days at 60 kJ day-1 kg-1 ideal body weight and six subjects were starved for 48 h. The cooling stimulus was provided by a coverall perfused with water at 16 degrees C. 2. After the application of the cooling stimulus, central body (auditory canal) temperature rose initially in both studies. After underfeeding, the magnitude of this rise in temperature was not significantly different from that seen in the normally fed state. After 48 h starvation, the rise in temperature on cooling was reduced from 0.30 (SEM 0.03) to 0.10 (0.04) degrees C (P less than 0.01). In two subjects in whom central body temperature had been maintained in the normally fed state, a fall occurred after starvation. 3. Underfeeding for 7 days did not affect thermogenesis or the degree of vasoconstriction in the forearm or hand in response to cooling. 4. After 48 h starvation, the thermogenic response to cooling was abolished and blood flow in the forearm remained higher than in the normally fed state. 5. In normal weight young females, thermoregulatory responses to a cooling stimulus were therefore substantially affected by 48 h starvation but not by 7 days underfeeding.


Patient-derived xenografts undergo mouse-specific tumor evolution.
Patient-derived xenografts (PDXs) have become a prominent cancer model system, as they are presumed to faithfully represent the genomic features of primary tumors. Here we monitored the dynamics of copy number alterations (CNAs) in 1,110 PDX samples across 24 cancer types. We observed rapid accumulation of CNAs during PDX passaging, often due to selection of preexisting minor clones. CNA acquisition in PDXs was correlated with the tissue-specific levels of aneuploidy and genetic heterogeneity observed in primary tumors. However, the particular CNAs acquired during PDX passaging differed from those acquired during tumor evolution in patients. Several CNAs recurrently observed in primary tumors gradually disappeared in PDXs, indicating that events undergoing positive selection in humans can become dispensable during propagation in mice. Notably, the genomic stability of PDXs was associated with their response to chemotherapy and targeted drugs. These findings have major implications for PDX-based modeling of human cancer.


Obesity alters the collagen organization and mechanical properties of murine cartilage.
Osteoarthritis is a debilitating disease characterized by cartilage degradation and altered cartilage mechanical properties. Furthermore, it is well established that obesity is a primary risk factor for osteoarthritis. The purpose of this study was to investigate the influence of obesity on the mechanical properties of murine knee cartilage. Two-month old wild type mice were fed either a normal diet or a high fat diet for 16 weeks. Atomic force microscopy-based nanoindentation was used to quantify the effective indentation modulus of medial femoral condyle cartilage. Osteoarthritis progression was graded using the OARSI system. Additionally, collagen organization was evaluated with picrosirius red staining imaged using polarized light microscopy. Significant differences between diet groups were assessed using t tests with p < 0.05. Following 16 weeks of a high fat diet, no significant differences in OARSI scoring were detected. However, we detected a significant difference in the effective indentation modulus between diet groups. The reduction in cartilage stiffness is likely the result of disrupted collagen organization in the superficial zone, as indicated by altered birefringence on polarized light microscopy. Collectively, these results suggest obesity is associated with changes in knee cartilage mechanical properties, which may be an early indicator of disease progression.


Patterns of Infection and Patterns of Evolution: How a Malaria Parasite Brought "Monkeys and Man" Closer Together in the 1960s.
In 1960, American parasitologist Don Eyles was unexpectedly infected with a malariaparasite isolated from a macaque. He and his supervisor, G. Robert Coatney of the National Institutes of Health, had started this series of experiments with the assumption that humans were not susceptible to "monkey malaria." The revelation that a mosquito carrying a macaque parasite could infect a human raised a whole range of public health and biological questions. This paper follows Coatney's team of parasitologists and their subjects: from the human to the nonhuman; from the American laboratory to the forests of Malaysia; and between the domains of medical research and natural history. In the course of this research, Coatney and his colleagues inverted Koch's postulate, by which animal subjects are used to identify and understand human parasites. In contrast, Coatney's experimental protocol used human subjects to identify and understand monkey parasites. In so doing, the team repeatedly followed malaria parasites across the purported boundary separating monkeys and humans, a practical experience that created a sense of biological symmetry between these separate species. Ultimately, this led Coatney and his colleagues make evolutionary inferences, concluding "that monkeys and man are more closely related than some of us wish to admit." In following monkeys, men, and malaria across biological, geographical, and disciplinary boundaries, this paper offers a new historical narrative, demonstrating that the pursuit of public health agendas can fuel the expansion of evolutionary knowledge.


Screening of budesonide nanoformulations for treatment of inflammatory bowel disease in an inflamed 3D cell-culture model.
Drug formulation screenings for treatment of inflammatory bowel disease (IBD) are mostly conducted in chemically induced rodent models that represent acute injury-caused inflammation instead of a chronic condition. To accurately screen drug formulations for chronic IBD, a relevant model that mimics the chronic condition in vitro is urgently needed. In an effort to reduce and potentially replace this scientifically and ethically questionable animal testing for IBD drugs, our laboratory has developed an in vitro model for the inflamed intestinal mucosa observed in chronic IBD, which allows high-throughput screening of anti-inflammatory drugs and their formulations. The in vitro model consists of intestinal epithelial cells, human blood-derived macrophages, and dendritic cells that are stimulated by the inflammatory cytokine interleukin-1β. In this study, the model was utilized for evaluation of the efficacy and deposition of budesonide, an anti-inflammatory drug, in three different pharmaceutical formulations: (1) a free drug solution, (2) encapsulated into PLGA nanoparticles, and (3) encapsulated into liposomes. The in vitro model of the inflamed intestinal mucosa demonstrated its ability to differentiate therapeutic efficacy among the formulations while maintaining the convenience of conventional in vitro studies and adequately representing the complex pathophysiological changes observed in vivo.


Mortality Patterns in Patients with Multiple Trauma: A Systematic Review of Autopsy Studies.
A high percentage (50%-60%) of trauma patients die due to their injuries prior to arrival at the hospital. Studies on preclinical mortality including post-mortem examinations are rare. In this review, we summarized the literature focusing on clinical and preclinical mortality and studies included post-mortem examinations. A literature search was conducted using PubMed/Medline database for relevant medical literature in English or German language published within the last four decades (1980-2015). The following MeSH search terms were used in different combinations: "multiple trauma", "epidemiology", "mortality ", "cause of death", and "autopsy". References from available studies were searched as well. Marked differences in demographic parameters and injury severity between studies were identified. Moreover, the incidence of penetrating injuries has shown a wide range (between 4% and 38%). Both unimodal and bimodal concepts of trauma mortality have been favored. Studies have shown a wide variation in time intervals used to analyze the distribution of death. Thus, it is difficult to say which distribution is correct. We have identified variable results indicating bimodal or unimodal death distribution. Further more stundardized studies in this field are needed. We would like to encourage investigators to choose the inclusion criteria more critically and to consider factors affecting the pattern of mortality.


Glucocorticoid receptor signaling in leukocytes after early life adversity.
Early life adversity (ELA) has been associated with inflammation and immunosenescence, as well as hyporeactivity of the HPA axis. Because the immune system and the HPA axis are tightly intertwined around the glucocorticoid receptor (GR), we examined peripheral GR functionality in the EpiPath cohort among participants who either had been exposed to ELA (separation from parents and/or institutionalization followed by adoption; n = 40) or had been reared by their biological parents (n = 72).Expression of the strict GR target genes FKBP5 and GILZ as well as total and 1F and 1H GR transcripts were similar between groups. Furthermore, there were no differences in GR sensitivity, examined by the effects of dexamethasone on IL6 production in LPS-stimulated whole blood. Although we did not find differences in methylation at the GR 1F exon or promoter region, we identified a region of the GR 1H promoter (CpG 1-9) that showed lower methylation levels in ELA.Our results suggest that peripheral GR signaling was unperturbed in our cohort and the observed immune phenotype does not appear to be secondary to an altered GR response to the perturbed HPA axis and glucocorticoid (GC) profile, although we are limited in our measures of GR activity and time points.


The Newell Test for a theory of cognition.
Newell (1980; 1990) proposed that cognitive theories be developed in an effort to satisfy multiple criteria and to avoid theoretical myopia. He provided two overlapping lists of 13 criteria that the human cognitive architecture would have to satisfy in order to be functional. We have distilled these into 12 criteria: flexible behavior, real-time performance, adaptive behavior, vast knowledge base, dynamic behavior, knowledge integration, natural language, learning, development, evolution, and brain realization. There would be greater theoretical progress if we evaluated theories by a broad set of criteria such as these and attended to the weaknesses such evaluations revealed. To illustrate how theories can be evaluated we apply these criteria to both classical connectionism (McClelland & Rumelhart 1986; Rumelhart & McClelland 1986b) and the ACT-R theory (Anderson & Lebiere 1998). The strengths of classical connectionism on this test derive from its intense effort in addressing empirical phenomena in such domains as language and cognitive development. Its weaknesses derive from its failure to acknowledge a symbolic level to thought. In contrast, ACT-R includes both symbolic and sub-symbolic components. The strengths of the ACT-R theory derive from its tight integration of the symbolic component with the sub-symbolic component. Its weaknesses largely derive from its failure, as yet, to adequately engage in intensive analyses of issues related to certain criteria on Newell's list.


Binocular coordination of saccades in Duane Retraction Syndrome.
Disconjugate oculomotor adaptation is driven by the need to maintain binocular vision. Since binocular vision in Duane Retraction Syndrome (DRS) patients is normal in half of their horizontal field of gaze (i.e., sound-side of gaze), we wondered whether oculomotor adaptive capabilities are efficient despite such a severe impairment of eye motility towards the other half of the horizontal field of gaze (i.e., affected-side gaze). We compared properties of horizontal saccades of patients with congenital unilateral Duane Retraction Syndrome type I in binocular viewing and monocular viewing conditions by simultaneously recording both eyes with the search coil technique. Our results show a mismatch between the pulse and the step signal of the innervation for saccades. When tested in the affected eye viewing condition (sound eye covered), the eyes showed not only similarly-directed increases of the saccadic gain (pulse signal) in the two eyes but also disjunctive post-saccadic drifts (step signal). This behavior suggests that visuomotor errors presented only to the affected eye were transferred to the sound eye, producing conjugate changes of the saccadic command. The post-saccadic command remained unchanged, however, and controlled the final position of each eye separately. This suggests that monocular adaptation is possible only for the step of innervation (i.e., controlling the final eye position) but not for the pulse of innervation (i.e., controlling the saccadic gain), even though the peculiarity of unilateral DRS type I offers a clear advantage for separate pathways of control for the two eyes.


Association between influenza vaccination and the reduced risk of acute kidney injury among older people: A nested case-control study.
The objective of this study is to determine whether vaccination against influenza is associated with a reduced risk of acute kidney injury (AKI) in a nationwide cohort of adults aged ≥65 years. We investigated a total of 13,270 patients aged ≥65 years who were hospitalized for AKI between 2000 and 2013 from Taiwan's National Health Insurance Research Database. Each AKI case was matched with one control subject according to duration of follow-up, age, sex, monthly income, urbanization level, and baseline comorbidities. Odds ratios (ORs) for AKI associated with exposure to the influenza vaccine in the previous year were calculated in a nested case-control analysis. Influenza vaccination in the previous year was associated with a lower risk of AKI (adjusted OR 0.67, 95% confidence interval [CI] 0.63-0.72). Compared with a reference group of unvaccinated individuals with no influenza infection, vaccination with no influenza infection was associated with a lower risk of AKI (adjusted OR 0.68, 95% CI 0.64-0.73). Lack of vaccination and presence of influenza infection was associated with a higher risk of AKI (adjusted OR 1.78, 95% CI 1.57-2.01), whereas the risk of AKI was insignificant in vaccinated patients who developed influenza (adjusted OR 1.01, 95% CI 0.69-1.18). The risk of AKI was 37% lower among older people who received vaccination against influenza in a real-world setting. Further work is required to clarify causality.


Evaluation of oxidation and fatigue damage of retrieved crossfire polyethylene acetabular cups.
Crossfire cross-linked polyethylene is produced differently from other cross-linked polyethylene materials; a below-melt-temperature annealing process is used with the goal of avoiding compromised mechanical properties. The present study was performed to evaluate retrieved Crossfire acetabular cups to determine whether they had oxidized and to what extent oxidation might have influenced their clinical performance. Eleven acetabular cups were received at retrieval and a twelfth acetabular cup was received two years post-retrieval over a period of four years. None were retrieved because of polyethylene wear or fatigue. The cups had been in vivo from 0.1 to 5.3 years. Each was examined visually, clinical fatigue damage was rated, and oxidation was measured with use of Fourier transform infrared spectroscopy. The cups exhibited oxidation that varied with its location on the cup: the oxidation value was generally low on the articular surface but more than an order of magnitude higher value on the rim. Maximum rim oxidation correlated significantly with the time in vivo (Spearman rho = 0.734, p = 0.010). Oxidation was identified visually by a white band in thin sections on the rim of seven of the cups and on the articular surface of one of these seven cups. Six of the seven cups also exhibited clinical fatigue damage. Eight of the twelve cups exhibited evidence of impingement or dislocation. Acetabular cups made of Crossfire polyethylene oxidized to a measurable degree. The oxidation-related reduction of polyethylene mechanical properties was sufficient to allow the fatigue damage seen in these retrieved cups.


Design of a tobacco exon array with application to investigate the differential cadmium accumulation property in two tobacco varieties.
For decades the tobacco plant has served as a model organism in plant biology to answer fundamental biological questions in the areas of plant development, physiology, and genetics. Due to the lack of sufficient coverage of genomic sequences, however, none of the expressed sequence tag (EST)-based chips developed to date cover gene expression from the whole genome. The availability of Tobacco Genome Initiative (TGI) sequences provides a useful resource to build a whole genome exon array, even if the assembled sequences are highly fragmented. Here, the design of a Tobacco Exon Array is reported and an application to improve the understanding of genes regulated by cadmium (Cd) in tobacco is described. From the analysis and annotation of the 1,271,256 Nicotiana tabacum fasta and quality files from methyl filtered genomic survey sequences (GSS) obtained from the TGI and ~56,000 ESTs available in public databases, an exon array with 272,342 probesets was designed (four probes per exon) and tested on two selected tobacco varieties.Two tobacco varieties out of 45 accumulating low and high cadmium in leaf were identified based on the GGE biplot analysis, which is analysis of the genotype main effect (G) plus analysis of the genotype by environment interaction (GE) of eight field trials (four fields over two years) showing reproducibility across the trials. The selected varieties were grown under greenhouse conditions in two different soils and subjected to exon array analyses using root and leaf tissues to understand the genetic make-up of the Cd accumulation. An Affymetrix Exon Array was developed to cover a large (~90%) proportion of the tobacco gene space. The Tobacco Exon Array will be available for research use through Affymetrix array catalogue. As a proof of the exon array usability, we have demonstrated that the Tobacco Exon Array is a valuable tool for studying Cd accumulation in tobacco leaves. Data from field and greenhouse experiments supported by gene expression studies strongly suggested that the difference in leaf Cd accumulation between the two specific tobacco cultivars is dependent solely on genetic factors and genetic variability rather than on the environment.


Fronto-Parietal gray matter and white matter efficiency differentially predict intelligence in males and females.
While there are minimal sex differences in overall intelligence, males, on average, have larger total brain volume and corresponding regional brain volumes compared to females, measures that are consistently related to intelligence. Limited research has examined which other brain characteristics may differentially contribute to intelligence in females to facilitate equal performance on intelligence measures. Recent reports of sex differences in the neural characteristics of the brain further highlight the need to differentiate how the structural neural characteristics relate to intellectual ability in males and females. The current study utilized a graph network approach in conjunction with structural equation modeling to examine potential sex differences in the relationship between white matter efficiency, fronto-parietal gray matter volume, and general cognitive ability (GCA). Participants were healthy adults (n = 244) who completed a battery of cognitive testing and underwent structural neuroimaging. Results indicated that in males, a latent factor of fronto-parietal gray matter was significantly related to GCA when controlling for total gray matter volume. In females, white matter efficiency and total gray matter volume were significantly related to GCA, with no specificity of the fronto-parietal gray matter factor over and above total gray matter volume. This work highlights that different neural characteristics across males and females may contribute to performance on intelligence measures. Hum Brain Mapp 37:4006-4016, 2016. © 2016 Wiley Periodicals, Inc.


Human Milk Oligosaccharides Influence Neonatal Mucosal and Systemic Immunity.
The immune system of the infant is functionally immature and naïve. Human milk contains bioactive proteins, lipids, and carbohydrates that protect the newborn and stimulate innate and adaptive immune development. This review will focus on the role human milk oligosaccharides (HMO) play in neonatal gastrointestinal and systemic immune development and function. For the past decade, intense research has been directed at defining the complexity of oligosaccharides in the milk of many species and is beginning to delineate their diverse functions. These studies have shown that human milk contains a higher concentration as well as a greater structural diversity and degree of fucosylation than the milk oligosaccharides in other species, particularly bovine milk from which many infant formulae are produced. The commercial availability of large quantities of certain HMO has furthered our understanding of the functions of specific HMO, which include protecting the infant from pathogenic infections, facilitating the establishment of the gut microbiota, promoting intestinal development, and stimulating immune maturation. Many of these actions are exerted through carbohydrate-carbohydrate interactions with pathogens or host cells. Two HMOs, 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT), have recently been added to infant formula. Although this is a first step in narrowing the compositional gap between human milk and infant formula, it is unclear whether 1 or 2 HMO will recapitulate the complexity of actions exerted by the complex mixture of HMO ingested by breastfed infants. Thus, as more HMO become commercially available, either isolated from bovine milk or chemically or microbially synthesized, it is anticipated that more oligosaccharides will be added to infant formula either alone or in combination with other prebiotics.


Discrepancy in Frailty Identification: Move Beyond Predictive Validity.
To evaluate the discordance in frailty classification between the frailty index (FI) and the physical frailty phenotype (PFP) and identify factors discriminating those with discordant frailty classification from each other and from those for whom the assessments agree. A prospective observational study of older adults aged 65 and older selected from Medicare eligibility lists in four U.S. communities (n = 5,362). The PFP was measured by the Cardiovascular Health Study PFP. Participants meeting three or more of the five criteria were deemed frail. The FI was calculated as the proportion of deficits in an a priori selected set of 48 measures, and participants were classified as frail if FI is greater than 0.35. The prevalence of frailty was 7.0% by the PFP and 8.3% by the FI. Of the 730 deemed frail by either instrument, only 12% were in agreement, whereas 39% were classified as frail by the PFP, but not the FI, and 48% were classified as frail by the FI, but not the PFP. Participants aged 65-72 years or with greater disease burden were most likely to be characterized as being FI-frail, but not PFP-frail. The associations of frailty with age and mortality were stronger when frailty was measured by the PFP rather than the FI. Despite comparable frailty prevalence between the PFP and the FI, there was substantial discordance in individual-level classification, with highest agreement existing only in the most vulnerable subset. These findings suggest that there are clinically important contexts in which the PFP and the FI cannot be used interchangeably.


[Transcutaneous electrical nerve stimulation in the relief of pain related to physical therapy after abdominal surgery.].
There are few studies on transcutaneous electrical stimulation (TENS) as an adjunct to postoperative physical therapy. The objective of this study was to determine the efficacy of TENS on relieving pain related to physical therapy after abdominal surgery. A clinical, randomized, prospective study was undertaken with 48 patients on the first postoperative day, who presented a pain score > 3 on the visual analogic scale (VAS). Patients were divided in three groups: treated with the usual analgesic routine, without TENS, and with physical therapy; treated with the usual analgesic routine associated with TENS and physical therapy; Contrast-Placebo Group: treated with the usual analgesic routine associated with physical therapy but TENS was off. A visual analogic scale of pain was presented to the patients before (M1), after TENS (M2), and after physical therapy (M3) - cough, incentive spirometry, changing lateral decubitus and sitting - to quantify the efficacy of the analgesia. Electrical stimulation was done during 30 minutes. The STUDY GROUP presented a significant relief of the pain when compared to the other two groups only for coughing at M3 (p= 0.015). In this group, there was a significant reduction in pain associated with coughing (p= 0.003) [M1 versus M3]; with lateral decubitus (p= 0.025), sitting (p= 0.001), and with incentive spirometry (p= 0.017) [M1 versus M2]; and when changing to the lateral decubitus (p= 0.03) and sitting (p= 0.001) [M1xM3]. There were no significant differences in the Contrast-Placebo Group. The STUDY GROUP presented a reduction in pain in a few moments and parameters. Further studies are needed since TENS is indicated only as an adjuvant in controlling postoperative pain.


A framework for analysis of linear ultrasound videos to detect fetal presentation and heartbeat.
Confirmation of pregnancy viability (presence of fetal cardiac activity) and diagnosis of fetal presentation (head or buttock in the maternal pelvis) are the first essential components of ultrasound assessment in obstetrics. The former is useful in assessing the presence of an on-going pregnancy and the latter is essential for labour management. We propose an automated framework for detection of fetal presentation and heartbeat from a predefined free-hand ultrasound sweep of the maternal abdomen. Our method exploits the presence of key anatomical sonographic image patterns in carefully designed scanning protocols to develop, for the first time, an automated framework allowing novice sonographers to detect fetal breech presentation and heartbeat from an ultrasound sweep. The framework consists of a classification regime for a frame by frame categorization of each 2D slice of the video. The classification scores are then regularized through a conditional random field model, taking into account the temporal relationship between the video frames. Subsequently, if consecutive frames of the fetal heart are detected, a kernelized linear dynamical model is used to identify whether a heartbeat can be detected in the sequence. In a dataset of 323 predefined free-hand videos, covering the mother's abdomen in a straight sweep, the fetal skull, abdomen, and heart were detected with a mean classification accuracy of 83.4%. Furthermore, for the detection of the heartbeat an overall classification accuracy of 93.1% was achieved.


Validation and clinical application of an LC-MS/MS method for the quantification of everolimus using volumetric absorptive microsampling.
Everolimus is a mammalian target of rapamycin inhibitor approved for the treatment of various tumor types. Less invasive measurement of everolimus concentrations could facilitate pharmacokinetic studies and personalized dosing based on whole blood concentrations, known as therapeutic drug monitoring. Volumetric Absorptive Microsampling (VAMS) has been introduced as a patient friendly, less invasive sampling technique to obtain an accurate volume of whole blood regardless of hematocrit value. We describe the bioanalytical validation and clinical application of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to quantify everolimus using VAMS. For the quantification, 13C2D4-Everolimus was used as internal standard (IS). Everolimus and the IS were extracted with methanol from the VAMS device, which was evaporated after ultrasonification and shaking. The residue was reconstituted in 20 mM ammonium formate buffer and methanol (50%, v/v) of which 5 μL was injected into the LC-MS/MS system. Quantification was performed for the ammonium adduct of everolimus in positive electrospray ion mode. The VAMS method met all pre-defined validation criteria. Accuracy and precision were within 11.1% and ≤14.6%, respectively. Samples were shown to be stable on the VAMS device for at least 362 days at ambient temperatures. Considerable biases from -20 to 31% were observed over a 30-50% hematocrit range. Although the method fulfilled all validation criteria, the perceived advantage of VAMS over dried blood spot sampling could not be demonstrated. Despite the effect of hematocrit, using an empirically derived formula the whole blood everolimus concentration could be back calculated with reasonable accuracy in the clinical application study.


Return to swimming protocol for competitive swimmers: a post-operative case study and fundamentals.
A large percentage of swimmers report shoulder pain during their swimming career. Shoulder pain in swimmers has been attributed to duration of swim practice, total yardage, and break down in stroke technique. Rehabilitation programs are generally land-based and cannot adequately address the intricacies of the swimming strokes. Return to swimming protocols (RTSP) that address progression of yardage are scarce, yet needed. The purpose of this clinical commentary is to familiarize the clinician with the culture and vernacular of swimming, and to provide a suggested yardage based RTSP for high school and collegiate level swimmers. 5.


Stimulation of monokine production by lipoteichoic acids.
Lipoteichoic acids (LTAs) isolated from bacterial species, including Staphylococcus aureus, Streptococcus pyogenes A, Enterococcus faecalis, Streptococcus pneumoniae, and Listeria monocytogenes, were tested for their ability to stimulate the production of interleukin-1 beta (IL-1 beta), IL-6, and tumor necrosis factor alpha in cultured human monocytes. LTAs from S. aureus and S. pneumoniae failed to induce monokine production when applied in the concentration range of 0.05 to 5.0 micrograms/ml. However, LTAs from several enterococcal species (0.5 to 5 micrograms/ml) induced the release of all three monokines at levels similar to those observed after lipopolysaccharide stimulation. The kinetics of IL-1 beta and tumor necrosis factor alpha release elicited by LTAs closely resembled those observed following lipopolysaccharide application. Cytokine production occurred in the presence of both fetal calf serum and autologous human serum. Hence, it was not dependent on complement activation and could not be suppressed by naturally occurring human antibodies. Deacylation caused the total loss of monocyte stimulatory capacity. Deacylated LTAs were unable to prevent monocyte activation by intact LTAs, so primary binding of these molecules probably does not involve a simple interaction of a membrane receptor with the hydrophilic portion of the molecule. The results identify some species of LTAs as inducers of monokine production in human monocytes.


Structure and function of histone methyltransferases.
Histones are the major protein constituent of chromatin in the eukaryotic nucleus. These proteins undergo a host of different post-translational modifications, including phosphorylation, acetylation, and methylation, which have profound effects on the remodeling of chromatin. Histone modifications can function either individually or combinatorially to govern such processes as transcription, replication, DNA repair, and apoptosis. Recent studies have focused on histone arginine and lysine methylation and the roles of these modifications in transcriptional regulation and the establishment of heterochromatin. Concomitantly, several families of histone methyltransferases (HMTs) have been identified that catalyze the methylation of specific arginines or lysines in histones H3 and H4. Not surprisingly, many of these methyltransferase genes had been previously identified as important genetic regulators in organisms such as yeast and Drosophila, which underscores the importance of histone methylation in transcriptional control and chromatin remodeling. Structures of several representatives of these HMT families have recently been determined, yielding insight into their catalytic mechanism and histone substrate specificity. The focus of this review is to briefly summarize the roles of histone methylation in chromatin remodeling and to discuss the structures, substrate specificities, and mechanisms of the different classes of HMTs.


Targeting PPARalpha in Alzheimer's Disease.
The molecular mechanisms underlying Alzheimer's disease (AD) are yet to be fully elucidated. The so-called "amyloid cascade hypothesis" has long been the prevailing paradigm for causation of disease, and is today being revisited in relation to other pathogenic pathways, such as oxidative stress, neuroinflammation and energy dysmetabolism. The peroxisome proliferator-activated receptors (PPARs) are expressed in the central nervous system (CNS) and regulate many physiological processes, such as energy metabolism, neurotransmission, redox homeostasis, autophagy and cell cycle. Among the three isotypes (α, β/δ, γ), PPARγ role is the most extensively studied, while information on α and β/δ are still scanty. However, recent in vitro and in vivo evidence point to PPARα as a promising therapeutic target in AD. This review provides an update on this topic, focussing on the effects of natural or synthetic agonists in modulating pathogenetic mechanisms at AD onset and during its progression. Ligandactivated PPARα inihibits amyloidogenic pathway, Tau hyperphosphorylation and neuroinflammation. Concomitantly, the receptor elicits an enzymatic antioxidant response to oxidative stress, ameliorates glucose and lipid dysmetabolism, and stimulates autophagy.


Amino Acid Changes at Arginine 204 of Troponin I Result in Increased Calcium Sensitivity of Force Development.
Mutations in human cardiac troponin I (cTnI) have been associated with restrictive, dilated, and hypertrophic cardiomyopathies. The most commonly occurring residue on cTnI associated with familial hypertrophic cardiomyopathy (FHC) is arginine (R), which is also the most common residue at which multiple mutations occur. Two FHC mutations are known to occur at cTnI arginine 204, R204C and R204H, and both are associated with poor clinical prognosis. The R204H mutation has also been associated with restrictive cardiomyopathy (RCM). To characterize the effects of different mutations at the same residue (R204) on the physiological function of cTnI, six mutations at R204 (C, G, H, P, Q, W) were investigated in skinned fiber studies. Skinned fiber studies showed that all tested mutations at R204 caused significant increases in Ca2+ sensitivity of force development (ΔpCa50 = 0.22-0.35) when compared to wild-type (WT) cTnI. Investigation of the interactions between the cTnI mutants and WT cardiac troponin C (cTnC) or WT cardiac troponin T (cTnT) showed that all the mutations investigated, except R204G, affected either or both cTnI:cTnT and cTnI:cTnC interactions. The R204H mutation affected both cTnI:cTnT and cTnI:cTnC interactions while the R204C mutation affected only the cTnI:cTnC interaction. These results suggest that different mutations at the same site on cTnI could have varying effects on thin filament interactions. A mutation in fast skeletal TnI (R174Q, homologous to cTnI R204Q) also significantly increased Ca2+ sensitivity of force development (ΔpCa50 = 0.16). Our studies indicate that known cTnI mutations associated with poor prognosis (R204C and R204H) exhibit large increases in Ca2+ sensitivity of force development. Therefore, other R204 mutations that cause similar increases in Ca2+ sensitivity are also likely to have poor prognoses.


The multidimensional schizotypy scale-brief: Scale development and psychometric properties.
This article reports on the development and psychometric properties of a brief version of the Multidimensional Schizotypy Scale (MSS-B). The MSS-B contains 38 items that assess positive, negative, and disorganized schizotypy. The scale was derived from the full-length Multidimensional Schizotypy Scale, and the positive, negative, and disorganized subscales were designed to provide the same content coverage as the original subscales. Scale development involved a derivation sample (n = 6265) and a separate cross-validation sample (n = 1000), both drawn from four universities and Amazon Mechanical Turk. The MSS-B was derived using classical test theory, item response theory, and differential item functioning. The three subscales exhibited high internal-consistency reliability, good item- and model-fit, good test information functions, and expected patterns of intercorrelations and associations with neuroticism, sex, and race/ethnicity. This pattern of findings was almost identical between the derivation and cross-validation samples. Furthermore, the pattern of findings was closely comparable for MSS-B subscales and the full-length MSS subscales. The MSS-B appears to offer a promising brief measure for assessing schizotypy.


Selection of quorum quenching (QQ) bacteria for membrane biofouling control: effect of different Gram-staining QQ bacteria, Bacillus sp. T5 and Delftia sp. T6, on microbial population in membrane bioreactors.
This study aimed to address the gap in understanding how the microbial community present within quorum quenching-membrane bioreactor (QQ-MBRs) changes during the operations by investigating the behavior of two different types of QQ bacteria, Bacillus sp. T5 and Delftia sp. T6. The anti-biofouling effects of T5 and T6 in the QQ-MBR were 85% and 76%, respectively. According to the Illumina HiSeq results, when the QQ-MBR was operated with Gram-positive bacteria, T5, in the mixed liquor a reduction was observed in Gram-positive bacteria and Gram-negative bacteria population increased. In contrast, when the QQ-MBR was operated with Gram-negative bacteria, T6, Gram-negative bacteria population reduced and an increase in Gram-positive bacteria observed. As such, the outputs of the Illumina analysis revealed that use of Gram-negative QQ bacteria in the reactor induced a Gram-positive microbial community and vice versa. This indicates that a close interaction occurs between indigenous Gram-negative and positive bacterial phyla, and Bacillus sp. T5/Delftia sp. T6 is fundamental to the performance of MBRs. This is the first study demonstrating such a relationship and assistance selecting QQ bacteria/strategy in an effective way.


Characterization of human FSH isoforms reveals a nonglycosylated beta-subunit in addition to the conventional glycosylated beta-subunit.
Human FSH consists of a mixture of isoforms that can be separated on the basis of differences in negative charge conferred by variations in the numbers of sialic acid residues that terminate oligosaccharide branches. Western analysis of human FSH isoforms separated by chromatofocusing revealed the presence of two human FSHbeta isoforms that differed in size. A low mol wt human FSHbeta isoform was associated with all FSH isoform fractions. A high mol wt human FSHbeta isoform was associated with the more acidic fractions and increased in relative abundance as the pI decreased. Characterization of representative human FSHbeta isoforms by mass spectrometry and automated Edman degradation revealed a low mol wt isoform that was not glycosylated. A high mol wt isoform was N-glycosylated at Asn residues 7 and 24. These results indicate that pituitary human FSH consists of two classes of molecules: those that possess a nonglycosylated beta-subunit and those that possess a glycosylated beta-subunit. Glycoprotein hormones are known to be elliptical molecules, and the beta-subunit oligosaccharides project outward from the short diameter, thereby increasing it. It is interesting to speculate that this change in shape might affect ultrafiltration rates, leading to differences in delivery rates to target tissues and elimination by filtration in the kidney.


Pharmacologic irreversible narrowing in chronic cerebrovasospasm in rabbits is associated with functional damage.
We studied isolated basilar artery segments from a rabbit model of chronic cerebrovasospasm. Autologous blood placed around the basilar artery of rabbits killed 1, 2, 3, 4, 5, 6, 7, or 9 days later caused narrowing of the segments with a biphasic time course. The first (immediate) phase was reversed by intra-arterial papaverine; the second phase exhibited an increasing component of narrowing that was papaverine-insensitive. Based on the passive force/length curves, basilar artery segments became increasingly stiff over 9 days. By contrast, the segments' contractility decreased. Responses of the basilar artery segments were greater over the first few days, but then became less than that of saline-injected controls. Contractions in response to norepinephrine and potassium were reduced. Endothelium-based acetylcholine-induced vasodilation progressively diminished, as did the response to sympathetic nerve stimulation. There was a negative correlation between artery wall stiffness and contractility. The papaverine-insensitive component of angiographic narrowing correlated directly with loss of contractility and with artery wall stiffness. These results are consistent with the conclusion that increased artery wall stiffness is a primary determining factor in the arterial narrowing of chronic cerebrovasospasm.


CIB1 is essential for mouse spermatogenesis.
CIB1 is a 22-kDa calcium binding, regulatory protein with approximately 50% homology to calmodulin and calcineurin B. CIB1 is widely expressed and binds to a number of effectors, such as integrin alphaIIb, PAK1, and polo-like kinases, in different tissues. However, the in vivo functions of CIB1 are not well understood. To elucidate the function of CIB1 in whole animals, we used homologous recombination in embryonic stem cells to generate Cib1(-/-) mice. Although Cib1(-/-) mice grow normally, the males are sterile due to disruption of the haploid phase of spermatogenesis. This is associated with reduced testis size and numbers of germ cells in seminiferous tubules, increased germ cell apoptosis, and the loss of elongated spermatids and sperm. Cib1(-/-) testes also show increased mRNA and protein expression of the cell cycle regulator Cdc2/Cdk1. In addition, mouse embryonic fibroblasts (MEFs) derived from Cib1(-/-) mice exhibit a much slower growth rate compared to Cib1(+/+) MEFs, suggesting that CIB1 regulates the cell cycle, differentiation of spermatogenic germ cells, and/or differentiation of supporting Sertoli cells.


Biological prognostic markers in chronic lymphocytic leukemia.
Chronic lymphocytic leukemia (CLL) is the most frequent leukemic disease of adults in the Western world. It is remarkable by an extraordinary heterogeneity of clinical course with overall survival ranging from several months to more than 15 years. Classical staging sytems by Rai and Binet, while readily available and useful for initial assessment of prognosis, are not able to determine individual patient's ongoing clinical course of CLL at the time of diagnosis, especially in early stages. Therefore, newer biological prognostic parameters are currently being clinically evaluated. Mutational status of variable region of immunoglobulin heavy chain genes (IgVH), cytogenetic aberrations, and both intracellular ZAP-70 and surface CD38 expression are recognized as parameters with established prognostic value. Molecules regulating the process of angiogenesis are also considered as promising markers. The purpose of this review is to summarize in detail the specific role of these prognostic factors in chronic lymphocytic leukemia.


Central nervous pathways of insulin action in the control of metabolism and food intake.
Insulin acts on the CNS to modulate behaviour and systemic metabolism. Disturbances in brain insulin action represent a possible link between metabolic and cognitive health. Current findings from human research suggest that boosting central insulin action in the brain modulates peripheral metabolism, enhancing whole-body insulin sensitivity and suppressing endogenous glucose production. Moreover, central insulin action curbs food intake by reducing the salience of highly palatable food cues and increasing cognitive control. Animal models show that the mesocorticolimbic circuitry is finely tuned in response to insulin, driven mainly by the dopamine system. These mechanisms are impaired in people with obesity, which might increase their risk of developing type 2 diabetes and associated diseases. Overall, current findings highlight the role of insulin action in the brain and its consequences on peripheral metabolism and cognition. Hence, improving central insulin action could represent a therapeutic option for people at an increased risk of developing metabolic and cognitive diseases.


Determinants of sexual dysfunction and interventions for patients with obstructive sleep apnoea: a systematic review.
Obstructive sleep apnoea (OSA) may negatively affect a couple's sexual relationship. This systematic review evaluated what characteristics are determinants of sexual function and dysfunction in women and men with OSA, and what interventions are shown to be effective. A systematic literature review was conducted using PubMed, CINAHL, Cochrane and TRIP, and articles published between January 2004 and December 2014 in English; original research; adults ≥ 18 years; and both experimental and non-experimental designs. The Effective Public Health Practice Project Quality Assessment Tool for Quantitative Studies was used to assess study quality. Of 21 studies, six studies (no randomised control trials, RCTs) included women and 15 (with six RCTs) studies included men. Extracted data were scrutinised and adjusted until consensus was reached; suitable quantitative data were pooled in statistical meta-analysis. Sexual function was affected similarly in both genders, but effective interventions were reported only for men. In some studies, OSA severity and medications contributed to greater sexual dysfunction. In women, menopausal status, hormone levels and SaO2 < 90% were determinants of sexual dysfunction, while for men factors included BMI, hormonal status and inflammatory markers. Continuous positive airway pressure (CPAP) not only improved clinical measures such as excessive daytime sleepiness but also the erectile and orgasmic function. Nevertheless, sildenafil was superior CPAP with regard to erectile dysfunction. The findings illustrate important contributors to sexual dysfunction; however, firm generalisations cannot be made. There were limited RCTs and none for women, indicating further RCTs are needed to determine how OSA affects sexual function.


Anatomic modeling from unstructured samples using variational implicit surfaces.
We describe the use of variational implicit surfaces (level sets of an embedded generating function modeled using radial basis interpolants) in anatomic modeling. This technique allows the practitioner to employ sparsely and unevenly sampled data to represent complex biological surfaces, including data acquired as a series of non-parallel image slices. The method inherently accommodates interpolation across irregular spans. In addition, shapes with arbitrary topology are easily represented without interpolation or aliasing errors arising from discrete sampling. To demonstrate the medical use of variational implicit surfaces, we present the reconstruction of the inner surfaces of blood vessels from a series of endovascular ultrasound images.


Progressive multifocal leucoencephalopathy and viral antibody titres.
Progressive multifocal leucoencephalopathy (PML) is caused by a papovavirus but serum antibody titres are generally considered unhelpful in clinical diagnosis because antibodies to the commonest causal agent (JC virus) are frequently found in normal adults. There is little published information about CSF titres but usually they have not been useful. Two cases of PML, confirmed by autopsy, are described where CSF antibody to JC virus was measured. In one case the JC antibody titre was significantly higher in the CSF than the serum and we suggest that this finding is diagnostically useful. In this case there was a transient stabilization of the disease following treatment with cytarabine with a change in antibody titres suggestive of reduced viral replication in the central nervous system and a host response to the infection. In the other case, which was untreated, rising serum antibody levels indicated active infection with a host response.


HeLa human cervical cancer cell migration is inhibited by treatment with dibutyryl-cAMP.
Cyclic AMP (cAMP) activates both protein kinase A (PKA) and guanine-nucleotide exchange factor exchange protein directly activated by CAMP (EPAC)-mediated Ras-related Protein1 (RAP1) GTPase that regulates various cellular functions including cell migration. Herein, we investigated whether cAMP-mediated PKA and EPAC1/RAP1 pathways differentially control HeLa cervical cancer cell migration. Although HeLa cell migration was reduced by dibutyryl-cAMP, we observed an increase in cAMP/PKA, cAMP/EPAC1/RAP1-GTPase, and RAC1-GTPase. HeLa cell migration and RAC1-GTPase were increased by treatment with 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cAMP analogue to activate EPAC-specific signaling pathways. When HeLa cells were treated with H-89, a PKA inhibitor, cell migration was enhanced but RAC1-GTPase was inhibited. In addition, cell migration induced by dibutyryl-cAMP was reversed but the activity of Rac1-GTPase was inhibited by H-89 treatment. Taken together, these data demonstrate that cAMP/PKA and cAMP/EPAC1/RAP1-GTPase might inversely control cervical cancer cell migration, although both signaling pathways may up-regulate RAC1-GTPase. It also suggests that cAMP-mediated cancer cell migration was independent of RAC1-GTPase activation.


Horseradish peroxidase interacts with the cell wall peptidoglycans on oral bacteria.
Salivary peroxidase and myeloperoxidase are known to display antibacterial activity against oral microbes, and previous indications have pointed to the possibility that horseradish peroxidase (HRP) adsorbs onto the membrane of the major oral streptococci, Streptococcus mutans and Streptococcus sanguinis (S. sanguinis). However, the mechanism of interaction between HRP and the bacterial cell wall component is unclear. Dental plaques containing salivary glycoproteins and extracellular microbial products are visualized with 'dental plaque disclosing agent', and are controlled within dental therapy. However, current 'dental plaque disclosing agents' are difficult to evaluate with just dental plaques, since they stain and disclose not only dental plaques but also pellicle formed with salivary glycoproteins on a tooth surface. In this present study, we have demonstrated that HRP interacted with the cell wall component of the major gram-positive bacterial peptidoglycan, but not the major cell wall component of gram-negative bacteria lipopolysaccharide. Furthermore, we observed that the adsorbed HRP labeled with fluorescence was detected on the major oral gram-positive strains S. sanguinis and Streptococcus salivarius (S. salivarius), but not on a gram-negative strain, Escherichia coli (E. coli). Furthermore, we have demonstrated that the combination of HRP and chromogenic substrate clearly disclosed the dental plaques and the biofilm developed by S. sanguinis, S. salivarius and the major gram-postive bacteria Lactobacillus casei on tooth surfaces, and slightly disclosed the biofilm by E. coli. The combination of HRP and chromogenic substrate did not stain either the dental pellicle with the salivary glycoprotein mucin, or naked tooth surfaces. These results have suggested the possibility that the adsorption activity of HRP not only contributes to the evaluation of dental plaque, but that enzymatic activity of HRP may also contribute to improve dental hygiene.


Polyamine analogue induction of the p53-p21WAF1/CIP1-Rb pathway and G1 arrest in human melanoma cells.
Although polyamines are well recognized for their critical involvement in cell growth, the cell cycle specificity of this requirement has not yet been characterized with respect to the newly delineated regulatory pathways. We recently reported that polyamine analogues having close structural and functional similarities to the natural polyamines produce a distinct G1 and G2-M cell cycle arrest in MALME-3M human melanoma cells. To determine a molecular basis for this observation, we examined the effects of N1,N11-diethylnorspermine on cell cycle regulatory proteins associated with G1 arrest. The analogue is known to deplete polyamine pools by suppressing biosynthetic enzymes and potently inducing the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase. Treatment of MALME-3M cells with 10 microM N1,N11-diethylnorspermine caused an increase in hypophosphorylated Rb, which correlated temporally with the onset of G1 arrest at 16-24 h. Rb hypophosphorylation was preceded by an increase in wild-type p53 (approximately 100-fold at maximum) and a concomitant increase in the cyclin-dependent kinase inhibitor, p21WAF1/CIP1 (p21; approximately 5-fold at maximum). Another cyclin-dependent kinase inhibitor, p27KIP1, and cyclin D increased slightly, whereas proliferating cell nuclear antigen and p130 remained unchanged. Induction of p21 protein was accompanied by an increase in p21 mRNA, whereas induction of p53 protein was not, suggesting transcriptional activation of the former and posttranscriptional regulation of the latter. SK-MEL-28 human melanoma cells, which contain a mutated p53, failed to induce p53 or p21 and did not arrest in G1. Rather, these cells rapidly underwent programmed cell death within 48 h. Overall, these findings provide the first indication of the cell cycle regulatory pathways by which polyamine antagonists such as analogues might inhibit growth in cells containing wild-type p53 and further suggest a mechanistic basis for differential cellular responses to these agents.


Exopolysaccharides from olive brines could reduce the adhesion of ETEC K88 to intestinal epithelial cells.
This study aims to explore the biological functions of the isolated exopolysaccharides (EPSs) produced during the industrial fermentation of olives against enterotoxigenic E. coli (ETEC) K88. Exopolysaccharides were isolated from five industrial fermenters. Analysis of their monosaccharide composition by GLC revealed that the main components were glucose (27%-50%) and galactose (23%-33%) followed by rhamnose (4-23%) and arabinose (6-17%). The 1H NMR spectrum showed a very similar profile between samples, and a more in-depth analysis revealed the presence of an α-pyranose in the form of α-d-Glcp-(1→) and two different α-furanoses, with chemicals shift values, suggesting the presence of α-d-Glcf and α-d-Galf. Miniaturized in vitro tests demonstrated the ability of EPS samples to attach specifically to ETEC K88 (P < 0.05) with variable intensities. The competition test did not show the ability to block the ETEC K88 adhesion to IPEC-J2 cells; however, in the displacement test, all EPS samples were shown to effectively remove the pathogens attached to the cells (P < 0.01). These results suggest that the EPSs produced during the fermentation of table green olives could interfere with the attachment of opportunistic pathogens onto the intestinal epithelial cells. This would open the possibility of novel functional properties for this traditional Mediterranean fermented food and for the isolated EPSs as candidates for nutraceutics to be used in human and/or animal diets in the prevention and treatment of ETEC diarrhoea.


Nonparametric estimation of Küllback-Leibler divergence.
In this letter, we introduce an estimator of Küllback-Leibler divergence based on two independent samples. We show that on any finite alphabet, this estimator has an exponentially decaying bias and that it is consistent and asymptotically normal. To explain the importance of this estimator, we provide a thorough analysis of the more standard plug-in estimator. We show that it is consistent and asymptotically normal, but with an infinite bias. Moreover, if we modify the plug-in estimator to remove the rare events that cause the bias to become infinite, the bias still decays at a rate no faster than O(1/n). Further, we extend our results to estimating the symmetrized Küllback-Leibler divergence. We conclude by providing simulation results, which show that the asymptotic properties of these estimators hold even for relatively small sample sizes.


Simultaneous analysis of nanoparticles and small molecules by high-performance liquid chromatography using a silica monolithic column.
A high-performance liquid chromatography method using a commercially available silica monolithic column for the simultaneous analysis of nanoparticles and small molecules was developed. The method uses the micrometer-sized flow-through pores and nanometer-sized mesopores of the monolithic column for separation: first, size separation of nanoparticles was performed by the micrometer-sized pores using the hydrodynamic mode, and then small molecules were separated by the nanometer-sized pores using the normal-phase mode. The method was used to evaluate and compare three existing methods for purifying nanoparticles and to analyse nanoparticle stability. The bimodal structure of the monolithic column is promising for the simultaneous separation of nanoparticles and small molecules.


Physical layer secret key generation for fiber-optical networks.
We propose and experimentally demonstrate a method for generating and sharing a secret key using phase fluctuations in fiber optical links. The obtained key can be readily used to support secure communication between the parties. The security of our approach is based on a fundamental asymmetry associated with the optical physical layer: the sophistication of tools needed by an eavesdropping adversary to subvert the key establishment is significantly greater and more costly than the complexity needed by the legitimate parties to implement the scheme. In this sense, the method is similar to the classical asymmetric algorithms (Diffie-Hellman, RSA, etc.).


A practical guide to methods controlling false discoveries in computational biology.
In high-throughput studies, hundreds to millions of hypotheses are typically tested. Statistical methods that control the false discovery rate (FDR) have emerged as popular and powerful tools for error rate control. While classic FDR methods use only p values as input, more modern FDR methods have been shown to increase power by incorporating complementary information as informative covariates to prioritize, weight, and group hypotheses. However, there is currently no consensus on how the modern methods compare to one another. We investigate the accuracy, applicability, and ease of use of two classic and six modern FDR-controlling methods by performing a systematic benchmark comparison using simulation studies as well as six case studies in computational biology. Methods that incorporate informative covariates are modestly more powerful than classic approaches, and do not underperform classic approaches, even when the covariate is completely uninformative. The majority of methods are successful at controlling the FDR, with the exception of two modern methods under certain settings. Furthermore, we find that the improvement of the modern FDR methods over the classic methods increases with the informativeness of the covariate, total number of hypothesis tests, and proportion of truly non-null hypotheses. Modern FDR methods that use an informative covariate provide advantages over classic FDR-controlling procedures, with the relative gain dependent on the application and informativeness of available covariates. We present our findings as a practical guide and provide recommendations to aid researchers in their choice of methods to correct for false discoveries.


A Phase I First-in-Human Study of Enoticumab (REGN421), a Fully Human Delta-like Ligand 4 (Dll4) Monoclonal Antibody in Patients with Advanced Solid Tumors.
Enoticumab (REGN421) is a fully human IgG1 monoclonal antibody that binds human Dll4 and disrupts Notch-mediated signaling. The main objectives of this trial were to determine the safety, dose-limiting toxicities (DLT), pharmacokinetics (PK), and recommended phase II dose (RP2D) of enoticumab. Enoticumab was administered intravenously, with dose escalations from 0.25 to 4 mg/kg every 3 weeks (Q3W) and 0.75 to 3 mg/kg every 2 weeks (Q2W). Of 53 enrolled patients, 31 patients were treated Q3W and 22 patients were treated Q2W. Two DLTs occurred: grade 3 nausea (0.5 mg/kg Q3W) and grade 3 abdominal pain (1 mg/kg Q2W). An MTD was not reached on either schedule. The most frequent adverse events (AE) were fatigue, nausea, vomiting, hypertension, headache, and anorexia. Six treatment-related serious AEs were reported in 4 patients: brain natriuretic peptide (BNP) increase (0.25 mg/kg Q3W, Gr1), troponin I increase (4 mg/kg Q3W, Gr3), right ventricular dysfunction and pulmonary hypertension (1.5 mg/kg Q2W, both Gr3), and left ventricular dysfunction and pulmonary hypertension (3 mg/kg Q2W, both Gr3). Enoticumab was characterized by nonlinear, target-mediated PK, and had a terminal half-life of 8 to 9 days. With multiple Q2W or Q3W dosing, accumulation was not observed. Antitumor activity included two partial responses (non-small cell lung cancer bronchoalveolar-type with a β-catenin mutation, and ovarian cancer) and 16 patients with stable disease (3> 6 months). Enoticumab was tolerated, with RP2D of 4 mg/kg Q3W and 3 mg/kg Q2W based on PK profile and clinical activity. Responses and SD were noted in ovarian cancer and other solid tumors. Clin Cancer Res; 21(12); 2695-703. ©2015 AACR.


The mutational spectrum of human malignant autosomal recessive osteopetrosis.
Human malignant infantile osteopetrosis (arOP; MIM 259700) is a genetically heterogeneous autosomal recessive disorder of bone metabolism, which, if untreated, has a fatal outcome. Our group, as well as others, have recently identified mutations in the ATP6i (TCIRG1) gene, encoding the a3 subunit of the vacuolar proton pump, which mediates the acidification of the bone/osteoclast interface, are responsible for a subset of this condition. By sequencing the ATP6i gene in arOP patients from 44 unrelated families with a worldwide distribution we have now established that ATP6i mutations are responsible for approximately 50% of patients affected by this disease. The vast majority of these mutations (40 out of 42 alleles, including seven deletions, two insertions, 10 nonsense substitutions and 21 mutations in splice sites) are predicted to cause severe abnormalities in the protein product and are likely to represent null alleles. In addition, we have also analysed nine unrelated arOP patients from Costa Rica, where this disease is apparently much more frequent than elsewhere. All nine Costa Rican patients bore either or both of two missense mutations (G405R and R444L) in amino acid residues which are evolutionarily conserved from yeast to humans. The identification of ATP6i gene mutations in two families allowed us for the first time to perform prenatal diagnosis: both fetuses were predicted not to be affected and two healthy babies were born. This study contributes to the determination of genetic heterogeneity of arOP and allows further delineation of the other genetic defects causing this severe condition.


Articular cartilage in the knee: current MR imaging techniques and applications in clinical practice and research.
Magnetic resonance (MR) imaging is the most important imaging modality for the evaluation of traumatic or degenerative cartilaginous lesions in the knee. It is a powerful noninvasive tool for detecting such lesions and monitoring the effects of pharmacologic and surgical therapy. The specific MR imaging techniques used for these purposes can be divided into two broad categories according to their usefulness for morphologic or compositional evaluation. To assess the structure of knee cartilage, standard spin-echo (SE) and gradient-recalled echo (GRE) sequences, fast SE sequences, and three-dimensional SE and GRE sequences are available. These techniques allow the detection of morphologic defects in the articular cartilage of the knee and are commonly used in research for semiquantitative and quantitative assessments of cartilage. To evaluate the collagen network and proteoglycan content in the knee cartilage matrix, compositional assessment techniques such as T2 mapping, delayed gadolinium-enhanced MR imaging of cartilage (or dGEMRIC), T1ρ imaging, sodium imaging, and diffusion-weighted imaging are available. These techniques may be used in various combinations and at various magnetic field strengths in clinical and research settings to improve the characterization of changes in cartilage.


Scandcleft randomised trials of primary surgery for unilateral cleft lip and palate: 2. Surgical results.
Longstanding uncertainty surrounds the selection of surgical protocols for unilateral cleft lip and palate, and randomised trials have only rarely been performed. The Scandcleft Project consists of three trials commenced in 1997 involving ten centres in Denmark, Finland, Norway, Sweden, and the UK. Three groups of centres tested a newly-defined common technique for palatal repair (Arm A) against their local protocols (Arms B, C, D). Arm A was familiar to most of the surgeons in Trial 1, but not to the surgeons in the other Trials. To evaluate surgical events and complications of the 448 (293 boys, 155 girls) patients with complete unilateral cleft lip and palate (UCLP) enrolled in the three trials. The three trials were carried out in parallel in adherence with a fully developed, ethically approved protocol. Operative time, bleeding, complications, and major dehiscence during and after both primary surgeries were recorded by the surgeon. Rates of fistula and surgery for velopharyngeal incompetence (VPI) were assessed until the youngest patient of the study had reached the age of 9 years. Pearson Chi-square statistical analysis was used to compare the outcomes. No significant differences in bleeding, infection, anaesthetic complications or length of hospital stay between the different arms were found for Trial 1. However, in Trials 2 and 3 there were more airway problems in Arm A than with the traditional local protocols (Arms C or D). In Trial 3 fistula and VPI surgery rates were also higher in Arm A. The results do not provide statistical evidence that any technique is better than others, but indicate that surgery was more problematic for surgeons who were still gaining experience with an unfamiliar surgical protocol. ISRCTN29932826.


Light intensity and FOV-controlled adaptive fluidic iris.
In this paper, we propose a light intensity and field of view (FOV) controlled adaptive fluidic iris. A 90° twisted-nematic liquid crystal (TNLC) cell and two orthogonal polarizers are attached on the substrate. When a voltage is applied to TNLC, it can change the phase of the incident light, leading to change in the light intensity. A black mask is then placed in the middle of the chamber; when changing the hydraulic pressure from the inlet and outlet, the black mask can move within the chamber. It is equivalent to changing the iris position along the optical axis. The iris, therefore, can control the FOV in an optical system. The experiments show that the device can control the light intensity from 100% to 0% by applying a voltage of 9 V on the TNLC cell. The proposed device can be applied in imaging systems and optical attenuators.


Computational analysis of flow structure and particle deposition in a single asthmatic human airway bifurcation.
This paper aims to improve current understanding of flow structure and particle deposition in asthmatic human airways. A single, symmetric airway bifurcation, corresponding to generations 10-11 of Weibel's model, is investigated through validated numerical simulations. The parent airway segment is modelled as a smooth circular tube. The child segments are considered asthmatic and their cross-section is modelled as a constricted tube with sinusoidal folds uniformly distributed along the circumference. The flow structure and particle deposition pattern for normal (i.e., healthy) and asthmatic airway bifurcations are compared and discussed. The numerical results reveal that the secondary flow in the asthmatic airway bifurcation is much stronger than in the healthy one, resulting in higher particle deposition. The effects of size of the lumen area and number of folds on particle deposition and pressure drop are also investigated. It is found that particle deposition efficiency is significantly affected by lumen area of the asthmatic segment (the smaller the lumen area, the higher the particle deposition efficiency). The effect of number of folds is small. Particle deposition efficiency also increases with Reynolds number. The pressure drop in the asthmatic airway bifurcation depends mainly on size of the lumen area. The effect of number of folds becomes important for strongly collapsed airways.


Common genetic contributions to alcohol and cannabis use and dependence symptomatology.
Despite mounting evidence that use of and dependence on alcohol and cannabis are influenced by heritable factors, the extent to which heritable influences on these phenotypes overlap across the 2 substances has only rarely been explored. In the current study, we quantified cross-substance overlap in sources of variance and estimated the degree to which within-substance associations between use and dependence measures are attributable to common genetic and environmental factors for alcohol and cannabis. The sample was comprised of 6,257 individuals (2,761 complete twin pairs and 735 singletons) from the Australian Twin Registry, aged 24 to 36 years. Alcohol and cannabis use histories were collected via telephone diagnostic interviews and used to derive an alcohol consumption factor, a frequency measure for cannabis use, and DSM-IV alcohol and cannabis dependence symptom counts. Standard genetic analyses were conducted to produce a quadrivariate model that provided estimates of overlap in genetic and environmental influences across the 4 phenotypes. Over 60% of variance in alcohol consumption, cannabis use, and cannabis dependence symptoms, and just under 50% of variance in alcohol dependence (AD) symptoms were attributable to genetic sources. Shared environmental factors did not contribute significantly to the 4 phenotypes. Nearly complete overlap in heritable influences was observed for within-substance measures of use and dependence symptoms. Genetic correlations across substances were 0.68 and 0.62 for use and dependence symptoms, respectively. Common heritable influences were evident for alcohol and cannabis use and for AD and cannabis dependence symptomatology, but findings indicate that substance-specific influences account for the majority of the genetic variance in the cannabis use and dependence phenotypes. By contrast, the substantial correlations between alcohol use and AD symptoms and between cannabis use and cannabis dependence symptoms suggest that measures of heaviness of use capture much of the same genetic liability to alcohol- and cannabis-related problems as dependence symptomatology.


Evaluation of estrogenic/antiestrogenic activity of ellagic acid via the estrogen receptor subtypes ERalpha and ERbeta.
Ellagic acid is a plant-derived polyphenol, possessing antioxidant, antiproliferative, and antiatherogenic properties. Whether this compound has estrogenic/antiestrogenic activity, however, remains largely unknown. To answer this question, we first investigated the ability of ellagic acid to influence the activity of the estrogen receptor subtypes ERalpha and ERbeta in HeLa cells. Cells co-transfected with an estrogen response element (ERE)-driven luciferase (Luc) reporter gene and an ERalpha- or ERbeta-expression vector were exposed to graded concentrations of ellagic acid. At low concentrations (10(-7) to 10(-9) M), this compound displayed a small but significant estrogenic activity via ERalpha, whereas it was a complete estrogen antagonist via ERbeta. Further evaluation revealed that ellagic acid was a potent antiestrogen in MCF-7 breast cancer-derived cells, increasing, like the pure estrogen antagonist ICI182780, IGFBP-3 levels. Moreover, ellagic acid induced nodule mineralization in an osteoblastic cell line (KS483), an effect that was abolished by the estrogen antagonist. Endometrium-derived epithelial cells (Ishikawa) showed no response to the natural compound by using a cell viability assay (MTT). These findings suggest that ellagic acid may be a natural selective estrogen receptor modulator (SERM).


Limitations to estimating bacterial cross-species transmission using genetic and genomic markers: inferences from simulation modeling.
Cross-species transmission (CST) of bacterial pathogens has major implications for human health, livestock, and wildlife management because it determines whether control actions in one species may have subsequent effects on other potential host species. The study of bacterial transmission has benefitted from methods measuring two types of genetic variation: variable number of tandem repeats (VNTRs) and single nucleotide polymorphisms (SNPs). However, it is unclear whether these data can distinguish between different epidemiological scenarios. We used a simulation model with two host species and known transmission rates (within and between species) to evaluate the utility of these markers for inferring CST. We found that CST estimates are biased for a wide range of parameters when based on VNTRs and a most parsimonious reconstructed phylogeny. However, estimations of CST rates lower than 5% can be achieved with relatively low bias using as low as 250 SNPs. CST estimates are sensitive to several parameters, including the number of mutations accumulated since introduction, stochasticity, the genetic difference of strains introduced, and the sampling effort. Our results suggest that, even with whole-genome sequences, unbiased estimates of CST will be difficult when sampling is limited, mutation rates are low, or for pathogens that were recently introduced.


Dense and overlapping innervation of pyramidal neurons by chandelier cells.
Chandelier (or axo-axonic) cells are a distinct group of GABAergic interneurons that innervate the axon initial segments of pyramidal cells and thus could have an important role controlling the activity of cortical circuits. To understand their connectivity, we labeled upper layers chandelier cells (ChCs) from mouse neocortex with a genetic strategy and studied how their axons contact local populations of pyramidal neurons, using immunohistochemical detection of axon initial segments. We studied ChCs located in the border of layers 1 and 2 from primary somatosensory cortex and found that practically all ChC axon terminals contact axon initial segments, with an average of three to five boutons per cartridge. By measuring the number of putative GABAergic synapses in initial segments, we estimate that each pyramidal neuron is innervated, on average, by four ChCs. Additionally, each individual ChC contacts 35-50% of pyramidal neurons within the areas traversed by its axonal arbor, with pockets of very high innervation density. Finally, ChCs have similar innervation patterns at different postnatal ages (P18-P90), with only relatively small lateral expansions of their arbor and increases in the total number of their cartridges during the developmental period analyzed. We conclude that ChCs innervate neighboring pyramidal neurons in a dense and overlapping manner, a connectivity pattern that could enable ChCs to exert a widespread influence on their local circuits.


The iatrogenic pathology of percutaneous interventions in coronary arteries.
Acute coronary syndromes (ACS) represent the clinical manifestations of sudden flow limiting coronary artery disease leading to acute myocardial ischemia or necrosis. Treatment of progressive coronary stenosis or acute thrombotic occlusion by means of percutaneous coronary intervention (PCI) with balloon dilatation and stent placement aims to reduce the risk of myocardial ischemia or necrosis by restoring coronary flow. But, being an invasive technique, it is associated with a periprocedural and also eventually long-term risk of complications. Pathological examination of atherosclerotic coronary arteries after PCI treatment has been shown to be very helpful in providing insights in this iatrogenic pathology. Importantly, the pathological substrate of the treated coronary artery segment in patients with ACS differs significantly from coronary artery segments in patients with stable coronary artery disease. Such studies have shown that besides the physical trauma induced by a balloon or a stent also the specific histomorphological and biological properties of the treated coronary plaques play an important role in the risk of PCI related vascular complications. Major complications, which are thrombosis and restenosis, have reduced significantly over the past years. Still, late stent thrombosis remains a small but clinically important problem after placement of drug eluting stents DES, mainly related to delayed in stent wound healing and early withdrawal of antiplatelet therapy. Moreover, restenosis remains a problem in the still large group of patients treated with bare metal stents (BMS) worldwide. Both in case of BMS and DES emerging evidence from recent histopathological studies on coronary resected stents shows that the outcome of PCI can be influenced by the occurrence of in stent neo- atherosclerosis, in DES more frequent than in BMS, which in turn may stimulate both thrombosis and restenosis on the very long term.


Heterozygous missense mutations in NFATC1 are associated with atrioventricular septal defect.
Atrioventricular septal defect (AVSD) may occur as part of a complex disorder (e.g., Down syndrome, heterotaxy), or as isolate cardiac defect. Multiple lines of evidence support a role of calcineurin/NFAT signaling in AVSD, and mutations in CRELD1, a protein functioning as a regulator of calcineurin/NFAT signaling have been reported in a small fraction of affected subjects. In this study, 22 patients with isolated AVSD and 38 with AVSD and heterotaxy were screened for NFATC1 gene mutations. Sequence analysis identified three missense variants in three individuals, including a subject with isolated AVSD [p.(Ala367Val)], an individual with AVSD and heterotaxy [p.(Val210Met)], and a subject with AVSD, heterotaxy, and oculo-auriculo-vertebral spectrum (OAVS) [p.(Ala696Thr)], respectively. The latter was also heterozygous for a missense change in TBX1 [p.(Pro86Leu)]. Targeted resequencing of genes associated with AVSD, heterotaxy, or OAVS excluded additional hits in the three mutation-positive subjects. Functional characterization of NFATC1 mutants documented defective nuclear translocation and decreased transcriptional transactivation activity. When expressed in zebrafish, the three NFATC1 mutants caused cardiac looping defects and altered atrioventricular canal patterning, providing evidence of their functional relevance in vivo. Our findings support a role of defective NFATC1 function in the etiology of isolated and heterotaxy-related AVSD.


Solvent comparison in the isolation, solubilization, and toxicity of Stachybotrys chartarum spore trichothecene mycotoxins in an established in vitro luminescence protein translation inhibition assay.
It is well known that non-viable mold contaminants such as macrocyclic trichothecene mycotoxins of Stachybotrys chartarum are highly toxinigenic to humans. However, the method of recovering native mycotoxin has been without consensus. Inconsistencies occur in the methods of isolation, suspension, preparation, and quantitation of the mycotoxin from the spores. The purpose of this study was to provide quantitatively comparative data on three concurrent preparations of 10(6)S. chartarum spores. The experiments were designed to specifically evaluate a novel method of mycotoxin extraction, solubilization, and the subsequent inhibitory effect in an established in vitro luminescence protein translation assay from 30 day-old spores. The mycotoxin-containing spores swabbed from wallboard cultures were milled with and without glass beads in 100% methanol, 95% ethanol, or water. Milled spore lysates were cleared of cell debris by filter centrifugation followed by a second centrifugation through a 5000 MWCO filter to remove interfering proteins and RNases. Cleared lysate was concentrated by centrivap and suspended in either alcohol or water as described. The suspensions were used immediately in the in vitro luminescence protein translation assay with the trichothecene, T-2 toxin, as a control. Although, mycotoxin is reported to be alcohol soluble, the level of translation inhibition was not reliably satisfactory for either the methanol or ethanol preparations. In fact, the methanol and ethanol control reactions were not significantly different than the alcohol prepared spore samples. In addition, we observed that increasing amounts of either alcohol inhibited the reaction in a dose dependent manner. This suggests that although alcohol isolation of mycotoxin is desirable in terms of time and labor, the presence of alcohol in the luminescence protein translation reaction was not acceptable. Conversely, water extraction of mycotoxin demonstrated a dose dependent response, and there was significant difference between the water controls and the water extracted mycotoxin reactions. In our hands, water was the best extraction agent for mycotoxin when using this specific luminescence protein translation assay kit.


Parthenogenesis in birds: a review.
Parthenogenesis or 'virgin birth' is embryonic development in unfertilized eggs. It is a routine means of reproduction in many invertebrates. However, even though parthenogenesis occurs naturally in even more advanced vertebrates, like birds, it is mostly abortive in nature. In fact, multiple limiting factors, such as delayed and unorganized development as well as unfavorable conditions developing within the unfertilized egg upon incubation, are associated with termination of progressive development of parthenogenetic embryos. In birds, diploid parthenogenesis is automictic and facultative producing only males. However, the mechanisms controlling parthenogenesis in birds are not clearly elucidated. Additionally, it appears from even very recent research that these mechanisms may hinder the normal fertilization process and subsequent embryonic development. For instance, virgin quail and turkey hens exhibiting parthenogenesis have reduced reproductive performance following mating. Also, genetic selection and environmental factors, such as live virus vaccinations, are known to trigger the process of parthenogenesis in birds. Therefore, parthenogenesis has a plausible negative impact on the poultry industry. Hence, a better understanding of parthenogenesis and the mechanisms that control it could benefit commercial poultry production. In this context, the aim of this review is to provide a complete overview of the process of parthenogenesis in birds.


Biofilm formation, epiphytic fitness, and canker development in Xanthomonas axonopodis pv. citri.
The phytopathogenic bacterium Xanthomonas axonopodis pv. citri is responsible for the canker disease affecting citrus plants throughout the world. Here, we have evaluated the role of bacterial attachment and biofilm formation in leaf colonization during canker development on lemon leaves. Crystal violet staining and confocal laser scanning microscopy analysis of X. axonopodis pv. citri strains expressing the green fluorescent protein were used to evaluate attachment and biofilm formation on abiotic and biotic (leaf) surfaces. Wild-type X. axonopodis pv. citri attached to and formed a complex, structured biofilm on glass in minimal medium containing glucose. Similar attachment and structured biofilm formation also were seen on lemon leaves. An X. axonopodis pv. citri gumB mutant strain, defective in production of the extracellular polysaccharide xanthan, did not form a structured biofilm on either abiotic or biotic surfaces. In addition, the X. axonopodis pv. citri gumB showed reduced growth and survival on leaf surfaces and reduced disease symptoms. These findings suggest an important role for formation of biofilms in the epiphytic survival of X. axonopodis pv. citri prior to development of canker disease.


Risk factors for COVID-19-related in-hospital mortality in a high HIV and tuberculosis prevalence setting in South Africa: a cohort study.
The interaction between COVID-19, non-communicable diseases, and chronic infectious diseases such as HIV and tuberculosis is unclear, particularly in low-income and middle-income countries in Africa. South Africa has a national HIV prevalence of 19% among people aged 15-49 years and a tuberculosis prevalence of 0·7% in people of all ages. Using a nationally representative hospital surveillance system in South Africa, we aimed to investigate the factors associated with in-hospital mortality among patients with COVID-19. In this cohort study, we used data submitted to DATCOV, a national active hospital surveillance system for COVID-19 hospital admissions, for patients admitted to hospital with laboratory-confirmed SARS-CoV-2 infection between March 5, 2020, and March 27, 2021. Age, sex, race or ethnicity, and comorbidities (hypertension, diabetes, chronic cardiac disease, chronic pulmonary disease and asthma, chronic renal disease, malignancy in the past 5 years, HIV, and past and current tuberculosis) were considered as risk factors for COVID-19-related in-hospital mortality. COVID-19 in-hospital mortality, the main outcome, was defined as a death related to COVID-19 that occurred during the hospital stay and excluded deaths that occurred because of other causes or after discharge from hospital; therefore, only patients with a known in-hospital outcome (died or discharged alive) were included. Chained equation multiple imputation was used to account for missing data and random-effects multivariable logistic regression models were used to assess the role of HIV status and underlying comorbidities on COVID-19 in-hospital mortality. Among the 219 265 individuals admitted to hospital with laboratory-confirmed SARS-CoV-2 infection and known in-hospital outcome data, 51 037 (23·3%) died. Most commonly observed comorbidities among individuals with available data were hypertension in 61 098 (37·4%) of 163 350, diabetes in 43 885 (27·4%) of 159 932, and HIV in 13 793 (9·1%) of 151 779. Tuberculosis was reported in 5282 (3·6%) of 146 381 individuals. Increasing age was the strongest predictor of COVID-19 in-hospital mortality. Other factors associated were HIV infection (adjusted odds ratio 1·34, 95% CI 1·27-1·43), past tuberculosis (1·26, 1·15-1·38), current tuberculosis (1·42, 1·22-1·64), and both past and current tuberculosis (1·48, 1·32-1·67) compared with never tuberculosis, as well as other described risk factors for COVID-19, such as male sex; non-White race; underlying hypertension, diabetes, chronic cardiac disease, chronic renal disease, and malignancy in the past 5 years; and treatment in the public health sector. After adjusting for other factors, people with HIV not on antiretroviral therapy (ART; adjusted odds ratio 1·45, 95% CI 1·22-1·72) were more likely to die in hospital than were people with HIV on ART. Among people with HIV, the prevalence of other comorbidities was 29·2% compared with 30·8% among HIV-uninfected individuals. Increasing number of comorbidities was associated with increased COVID-19 in-hospital mortality risk in both people with HIV and HIV-uninfected individuals. Individuals identified as being at high risk of COVID-19 in-hospital mortality (older individuals and those with chronic comorbidities and people with HIV, particularly those not on ART) would benefit from COVID-19 prevention programmes such as vaccine prioritisation as well as early referral and treatment. South African National Government.


Postnatal inflammation following intrauterine inflammation exacerbates the development of atherosclerosis in ApoE-/- mice.
Atherosclerosis is a chronic inflammatory disease that has its origins in early life. Postnatal inflammation exacerbates atherosclerosis, but the possible effect of intrauterine inflammation is largely unexplored. Exposure to inflammation in utero is common, especially in infants born preterm, who have increased cardiovascular risk in adulthood. We hypothesised that exposure to inflammation before birth would accelerate the development of atherosclerosis, with the most severe atherosclerosis following exposure to both pre- and postnatal inflammation. Here we studied the effect of prenatal and postnatal inflammation on the development of atherosclerosis by combining established techniques for modelling histological chorioamnionitis and atherosclerosis using apolipoprotein E (ApoE) knockout mice. A single intra-amniotic (IA) injection of lipopolysaccharide (LPS) caused intrauterine inflammation, and increased atherosclerosis at 13 weeks of postnatal age. In mice exposed to postnatal LPS, chorioamnionitis modulated subsequent responses; atherosclerotic lesion size, number and severity were greatest for mice exposed to both intrauterine and postnatal inflammation, with a concomitant decrease in collagen content and increased inflammation of the atherosclerotic plaque. In conclusion, pre- and postnatal inflammation have additive and deleterious effects on the development of atherosclerosis in ApoE knockout mice. The findings are particularly relevant to preterm human infants, whose gestations are frequently complicated by chorioamnionitis and who are particularly susceptible to repeated postnatal infections. Human and mechanistic studies are warranted to guide preventative strategies.


Pharmacologic inhibition of cyclin-dependent kinases 4 and 6 arrests the growth of glioblastoma multiforme intracranial xenografts.
Activation of cyclin-dependent kinases 4 and 6 (cdk4/6) occurs in the majority of glioblastoma multiforme (GBM) tumors, and represents a promising molecular target for the development of small molecule inhibitors. In the current study, we investigated the molecular determinants and in vivo response of diverse GBM cell lines and xenografts to PD-0332991, a cdk4/6-specific inhibitor. In vitro testing of PD-0332991 against a panel of GBM cell lines revealed a potent G(1) cell cycle arrest and induction of senescence in each of 16 retinoblastoma protein (Rb)-proficient cell lines regardless of other genetic lesions, whereas 5 cell lines with homozygous inactivation of Rb were completely resistant to treatment. Short hairpin RNA depletion of Rb expression conferred resistance of GBM cells to PD-0332991, further demonstrating a requirement of Rb for sensitivity to cdk4/6 inhibition. PD-0332991 was found to efficiently cross the blood-brain barrier and proved highly effective in suppressing the growth of intracranial GBM xenograft tumors, including those that had recurred after initial therapy with temozolomide. Remarkably, no mice receiving PD-0332991 died as a result of disease progression while on therapy. Additionally, the combination of PD-0332991 and radiation therapy resulted in significantly increased survival benefit compared with either therapy alone. In total, our results support clinical trial evaluation of PD-0332991 against newly diagnosed as well as recurrent GBM, and indicate that Rb status is the primary determinant of potential benefit from this therapy.


Epstein-Barr virus-specific methylation of human genes in gastric cancer cells.
Epstein-Barr Virus (EBV) is found in 10% of all gastric adenocarcinomas but its role in tumor development and maintenance remains unclear. The objective of this study was to examine EBV-mediated dysregulation of cellular factors implicated in gastric carcinogenesis. Gene expression patterns were examined in EBV-negative and EBV-positive AGS gastric epithelial cells using a low density microarray, reverse transcription PCR, histochemical stains, and methylation-specific DNA sequencing. Expression of PTGS2 (COX2) was measured in AGS cells and in primary gastric adenocarcinoma tissues. In array studies, nearly half of the 96 human genes tested, representing 15 different cancer-related signal transduction pathways, were dysregulated after EBV infection. Reverse transcription PCR confirmed significant impact on factors having diverse functions such as cell cycle regulation (IGFBP3, CDKN2A, CCND1, HSP70, ID2, ID4), DNA repair (BRCA1, TFF1), cell adhesion (ICAM1), inflammation (COX2), and angiogenesis (HIF1A). Demethylation using 5-aza-2'-deoxycytidine reversed the EBV-mediated dysregulation for all 11 genes listed here. For some promoter sequences, CpG island methylation and demethylation occurred in an EBV-specific pattern as shown by bisulfite DNA sequencing. Immunohistochemistry was less sensitive than was western blot for detecting downregulation of COX2 upon EBV infection. Virus-related dysregulation of COX2 levels in vitro was not recapitulated in vivo among naturally infected gastric cancer tissues. EBV alters human gene expression in ways that could contribute to the unique pathobiology of virus-associated cancer. Furthermore, the frequency and reversability of methylation-related transcriptional alterations suggest that demethylating agents have therapeutic potential for managing EBV-related carcinoma.


IL-23 produced by CNS-resident cells controls T cell encephalitogenicity during the effector phase of experimental autoimmune encephalomyelitis.
CNS-resident cells, in particular microglia and macrophages, are a source of inflammatory cytokines during inflammation within the CNS. Expression of IL-23, a recently discovered cytokine, has been shown to be critical for the development of experimental autoimmune encephalomyelitis (EAE) in mice. Expression of the p40 subunit of IL-12 and IL-23 by microglia has been shown in situ and in vitro, but direct evidence for a functional significance of p40 expression by CNS cells during an immune response in vivo is still lacking. Here we report that p40 plays a critical role in maintaining encephalitogenicity during the disease course. By using irradiation bone marrow chimeras, we have generated mice in which p40 is deleted from the CNS parenchyma but not the systemic immune compartment. Our studies show that p40 expressed by CNS-endogenous cells is critical for the development of myelin oligodendrocyte glycoprotein-induced EAE. In spite of the reduced clinical disease, the absence of p40 from the CNS has little impact on the degree of inflammation. Expression profiles of the CNS lesions show an increase in Th2 cytokines when compared with mice that develop EAE in the presence of CNS IL-12 and/or IL-23. Taken together, our data demonstrate that p40 expression by CNS-resident cells forms the basis for the Th1 bias of the CNS.