Oxygen diffusion in ellipsoidal tumour spheroids.
Oxygen plays a central role in cellular metabolism, in both healthy and tumour tissue. The presence and concentration of molecular oxygen in tumours has a substantial effect on both radiotherapy response and tumour evolution, and as a result the oxygen micro-environment is an area of intense research interest. Multi-cellular tumour spheroids closely mimic real avascular tumours, and in particular they exhibit physiologically relevant heterogeneous oxygen distribution. This property has made them a vital part of in vitro experimentation. For ideal spheroids, their heterogeneous oxygen distributions can be predicted from theory, allowing determination of cellular oxygen consumption rate (OCR) and anoxic extent. However, experimental tumour spheroids often depart markedly from perfect sphericity. There has been little consideration of this reality. To date, the question of how far an ellipsoid can diverge from perfect sphericity before spherical assumptions break down remains unanswered. In this work, we derive equations governing oxygen distribution (and, more generally, nutrient and drug distribution) in both prolate and oblate tumour ellipsoids, and quantify the theoretical limits of the assumption that the spheroid is a perfect sphere. Results of this analysis yield new methods for quantifying OCR in ellipsoidal spheroids, and how this can be applied to markedly increase experimental throughput and quality.


Impact of Community Health Workers on Elderly Patients' Advance Care Planning and Health Care Utilization: Moving the Dial.
Advance care planning (ACP) is recommended for all persons to ensure that the care they receive aligns with their values and preferences. To evaluate an ACP intervention developed to better meet the needs and priorities of persons with chronic diseases, including mild cognitive impairment. A year-long, pre-post intervention using lay community health workers [care coordinator assistants (CCAs)] trained to conduct and document ACP conversations with patients during home health visits with pre-post evaluation. The 818 patients were 74.2 years old (mean); 78% women; 51% African American; 43% white. Documentation of ACP conversation in electronic health record fields and health care utilization outcomes. In this target population ACP documentation rose from 3.4% (pre-CCA training) to 47.9% (post) of patients who had at least 1 discussion about ACP in the electronic health record. In the 1-year preintervention period, there were no differences in admissions, emergency department (ED) visits, and outpatient visits between patients who did and did not have ACP discussion. After adjusting for prior hospitalization and ED use histories, ACP discussions were associated with a 34% less probability of hospitalization (hazard ratios, 0.66; 95% confidence interval, 0.45-0.97), and similar effects are apparent on ED use independent of age and prior ED use effects. Patients with chronic diseases including mild cognitive impairment can engage in ACP conversations with trusted home health care providers. Having ACP conversation is associated with significant reduction in seeking urgent health care and in hospitalizations.


Recurrent thrombosis due to compound heterozygosity for factor V Leiden and factor V deficiency.
A point mutation in the factor V gene (factor V Leiden) is the most common cause of familial thrombophilia. Patients with factor V Leiden have an increased risk of thrombosis, particularly those homozygous for the mutation. However, the phenotype in individuals with the mutation is variable, suggesting that other factors influence thrombotic risk. We describe for the first time a family in which two independent defects in factor V co-exist: heterozygosity for factor V Leiden and factor V deficiency. Compound heterozygosity for these two defects results in a phenotype similar to a homozygous factor V Leiden state with profound resistance to APC and recurrent thrombosis.


Gene silencing via DNA methylation in naturally occurring Tragopogon miscellus (Asteraceae) allopolyploids.
Hybridization coupled with whole-genome duplication (allopolyploidy) leads to a variety of genetic and epigenetic modifications in the resultant merged genomes. In particular, gene loss and gene silencing are commonly observed post-polyploidization. Here, we investigated DNA methylation as a potential mechanism for gene silencing in Tragopogon miscellus (Asteraceae), a recent and recurrently formed allopolyploid. This species, which also exhibits extensive gene loss, was formed from the diploids T. dubius and T. pratensis. Comparative bisulfite sequencing revealed CG methylation of parental homeologs for three loci (S2, S18 and TDF-44) that were previously identified as silenced in T. miscellus individuals relative to the diploid progenitors. One other locus (S3) examined did not show methylation, indicating that other transcriptional and post-transcriptional mechanisms are likely responsible for silencing that homeologous locus. These results indicate that Tragopogon miscellus allopolyploids employ diverse mechanisms, including DNA methylation, to respond to the potential shock of genome merger and doubling.


Antisense oligonucleotides targeting the miR-29b binding site in the GRN mRNA increase progranulin translation.
Heterozygous GRN (progranulin) mutations cause frontotemporal dementia (FTD) due to haploinsufficiency, and increasing progranulin levels is a major therapeutic goal. Several microRNAs, including miR-29b, negatively regulate progranulin protein levels. Antisense oligonucleotides (ASOs) are emerging as a promising therapeutic modality for neurological diseases, but strategies for increasing target protein levels are limited. Here, we tested the efficacy of ASOs as enhancers of progranulin expression by sterically blocking the miR-29b binding site in the 3' UTR of the human GRN mRNA. We found 16 ASOs that increase progranulin protein in a dose-dependent manner in neuroglioma cells. A subset of these ASOs also increased progranulin protein in iPSC-derived neurons and in a humanized GRN mouse model. In FRET-based assays, the ASOs effectively competed for miR-29b from binding to the GRN 3' UTR RNA. The ASOs increased levels of newly synthesized progranulin protein by increasing its translation, as revealed by polysome profiling. Together, our results demonstrate that ASOs can be used to effectively increase target protein levels by partially blocking miR binding sites. This ASO strategy may be therapeutically feasible for progranulin-deficient FTD as well as other conditions of haploinsufficiency.


Does long-term oxygen therapy affect quality of life in patients with chronic obstructive pulmonary disease and severe hypoxaemia?
Long-term oxygen therapy (LTOT) improves survival in patients with hypoxaemic chronic obstructive pulmonary disease (COPD), but previous studies using general health measures have shown no effect on quality of life (QoL). In this study, the effect of LTOT on QoL was assessed using a disease-specific health measure, the St George's Respiratory Questionnaire (SGRQ). Twenty three hypoxaemic COPD patients (15 females and 8 males) were studied before and after starting LTOT: median age 71 (range 47-82) yrs, mean (SD) forced expiratory volume in one second (FEV1) 0.75 (0.22) L, arterial oxygen tension (Pa,O2) 6.95 (0.75) kPa, arterial carbon dioxide tension (Pa,CO2) 6.52 (1.21) kPa. A control group comprised 18 COPD patients (6 females and 12 males) with less severe hypoxaemia: median age 72 (range 58-85) yrs, FEV1 0.94 (0.33) L, Pa,O2 8.17 (0.94) kPa, Pa,CO2 6.02 (0.75) kPa. QoL was measured at baseline, 2 weeks, 3 and 6 months. The LTOT group had higher SGRQ total scores than controls (p<0.05) at all visits, implying lower QoL. Repeated measures analysis of variance showed no effect of LTOT on QoL over 6 months (F=0.43, p=0.79). In this study we detected no change in quality of life using a disease-specific health measure in patients with severe chronic obstructive pulmonary disease using an oxygen concentrator to provide long-term oxygen therapy.


Prediction of metabolic drug clearance in humans: in vitro-in vivo extrapolation vs allometric scaling.
Previously in vitro-in vivo extrapolation (IVIVE) with the Simcyp Clearance and Interaction Simulator has been used to predict the clearance of 15 clinically used drugs in humans. The criteria for the selection of the drugs were that they are used as probes for the activity of specific cytochromes P450 (CYPs) or have a single CYP isoform as the major or sole contributor to their metabolism and that they do not exhibit non-linear kinetics in vivo. Where data were available for the clearance of the drugs in at least three animal species, the predictions from IVIVE have now been compared with those based on allometric scaling (AS). Adequate data were available for estimating oral clearance (CLp.o.) in 9 cases (alprazolam, sildenafil, caffeine, clozapine, cyclosporine, dextromethorphan, midazolam, omeprazole and tolbutamide) and intravenous clearance in 6 cases (CLi.v.) (cyclosporine, diclofenac, midazolam, omeprazole, theophylline and tolterodine). AS predictions were based on five different methods: (1) simple allometry (clearance versus body weight); (2) correction for maximum life-span potential (CL x MLP); (3) correction for brain weight (CL x BrW); (4) the use of body surface area; and (5) the rule of exponents. A prediction accuracy was indicated by mean-fold error and the Pearson product moment correlation coefficient. Predictions were considered successful if the mean-fold error was <or=2. IVIVE predictions were accurate in 14 of 15 cases (mean-fold error range: 1.02-4.00). All five AS methods were accurate in 13, 11, 10, 10 and 14 cases, respectively. However, in some cases the error of AS exceeded fivefold. On the basis of the current results, IVIVE is more reliable than AS in predicting human clearance values for drugs mainly metabolized by CYP450 enzymes. This suggests that the place of AS methods in pre-clinical drug development warrants further scrutiny.


Mobilisation of stored calcium in the neck region of human sperm--a mechanism for regulation of flagellar activity.
Calcium signalling plays a pivotal role in sperm physiology, being intimately involved in the regulation of acrosome reaction, chemotaxis and hyperactivation. Here we describe briefly the mechanisms of calcium regulation in somatic cells and the ways in which these mechanisms have been adapted to function in mature spermatozoa. We then consider recent data from this and other laboratories on the responses of sperm to three compounds: progesterone and nitric oxide (both products of the cumulus oophorus) and 4-aminopyridine. All of these compounds induce calcium signals in the posterior sperm head and neck region and, when applied at appropriate concentrations, modify flagellar activity, causing asymmetric bending of the proximal flagellum. We argue that these effects reflect a common mode of action, mobilisation of calcium stored in the sperm neck region. Finally we consider the nature of calcium signalling pathways in sperm. We suggest that this highly specialised and extremely polarised cell, though working with the same calcium signalling 'tools' as those of somatic cells, employs them to generate unusually 'hard-wired' calcium signals that do not act to integrate stimuli. 'Leakage' between these calcium signalling pathways will generate inappropriate responses, compromising functioning of the cell.


Role of human natural killer cells during Epstein-Barr virus infection.
Human natural killer (NK) cells have been suggested to restrict viral infections. However, the evidence for this notion is mostly circumstantial. Recent studies in mice with reconstituted human immune system components, children with symptomatic primary Epstein-Barr virus (EBV) infection, and in secondary lymphoid tissues of healthy EBV carriers have, however, shown that early differentiated human NK cells limit lytic EBV replication and thereby prevent the immunopathological expansion of lytic EBV antigen specific CD8+ T cells that is known as infectious mononucleosis (IM). These findings, which will be discussed in this review, might offer the opportunity to identify EBV negative adolescents at risk to develop IM, and also more generally provide a good example to document restriction of a viral infection by human NK cells.


Accuracy of Measuring Bladder Volumes With Ultrasound and Bladder Scanning.
Removal of urinary catheters depends on accurate noninvasive measurements of bladder volume. Patients with acute kidney injury often have low bladder volumes/ascites, possibly causing measurement inaccuracy. To evaluate the accuracy of bladder volumes measured with bladder scanning and 2-dimensional ultrasound (US) compared with urinary catheterization among different types of clinicians. Prospective correlational descriptive study of 73 adult critical care patients with low urine output receiving hemodialysis or unable to void. Bladder volumes were independently measured by (1) a physician and an advanced practice registered nurse using US, (2) an advanced practice registered nurse and a bedside nurse using bladder scanning, and (3) urinary catheterization (cath). Bland-Altman and χ2 analyses were conducted. Mean (SD) cath volume was 171.7 (269.7) mL (range, 0-1100 mL). Abdominal fluid was observed in 28% of patients. Bias was -1.3 mL for US vs cath and 3.3 mL for bladder scanning vs cath. For patients with abdominal fluid and cath volume less than 150 mL, decisions to not catheterize patients were accurate more often when based on US measurements (97%-100%) than when based on bladder scanning measurements (86%-89%; P = .02). In patients with cath volume of 300 mL or more, decisions to catheterize patients were accurate more often when based on bladder scanning measurements (94%-100%) than when based on horizontal US measurements (50%-56%; P = .001). Bladder volume can be measured accurately with bladder scanning or US, but abdominal fluid remains a confounding factor limiting accuracy of bladder scanning.


Can time-based decay explain temporal distinctiveness effects in task switching?
In task switching, extending the response-cue interval (RCI) reduces the switch cost--the detriment to performance when switching compared to repeating tasks. This reduction has been used as evidence for the existence of task-set decay processes. Recently, this has been challenged by the observation of sequential dependencies on the RCI effect: switch cost is only reduced at longer RCIs when the previous trial had a short RCI. This trial-wise variation of RCI is thought to affect the temporal distinctiveness (TD) of a previous task's episodic trace, affecting the probability of its automatic retrieval on the current trial; importantly, TD is thought to be independent of the current trial's RCI. The present study highlights a dependency between the current RCI and TD, and demonstrates that a decay model can reproduce some patterns of data attributed to TD. Further, the decay account makes a strong prediction when TD is held constant: repetition response times should slow as the RCI increases, and switch response times should be facilitated. This prediction was tested via re-analysis of extant data and three experiments. The re-analysis provided some evidence for the decay account, but Experiments 1 and 2 report slowing for task repetition and switch trials, which cannot be explained by a task-set decay process. Experiment 3, which utilized tasks requiring perceptual judgements, showed small evidence for decay. We conclude that the data are largely consistent with the TD account and that the evidence for decay of higher-level task-sets is not convincing.


Korean red ginseng improves glucose control in subjects with impaired fasting glucose, impaired glucose tolerance, or newly diagnosed type 2 diabetes mellitus.
This study was designed to evaluate the effect of Korean red ginseng (KRG) supplementation on glucose control in subjects with impaired fasting glucose (IFG), impaired glucose tolerance (IGT), or newly diagnosed type 2 diabetes mellitus (T2DM). The study was a 12-week randomized, double-blinded, placebo-controlled (5 g of KRG [n=21] or placebo [n=20] in tablet form) trial. Glucose-related biomarkers, including serum and whole blood levels of glucose, insulin, and C-peptide, were measured by 2-h oral glucose tolerance tests (OGTTs) at baseline and after the 12-week intervention. After the intervention, the test group showed a significant decrease in serum levels of glucose at 30 min (-22.24±10.77 mg/dL) and whole blood levels of glucose at 30 min (-17.52±5.22 mg/dL). In addition, the test group tended to have lower whole blood levels of glucose at 0 min and glucose area under curve (AUC). However, the placebo group did not show any changes in blood glucose-related indices. The changes (difference from baseline) in serum glucose levels at 30 min, whole blood glucose levels at 60 min, and glucose AUC during OGTTs in the test group exhibited a tendency toward a decrease from those in the placebo group. There were significant decreases or trends toward a decrease in both serum insulin and C-peptide concentrations at most time intervals in the test group. In conclusion, KRG supplementation (5 g/day) may be beneficial for controlling serum and whole blood glucose levels compared with placebo among patients with IFG, IGT, or T2DM.


Efficacy of single dose of a bivalent vaccine containing inactivated Newcastle disease virus and reassortant highly pathogenic avian influenza H5N1 virus against lethal HPAI and NDV infection in chickens.
Highly pathogenic avian influenza (HPAI) and Newcastle disease (ND) are 2 devastating diseases of poultry, which cause great economic losses to the poultry industry. In the present study, we developed a bivalent vaccine containing antigens of inactivated ND and reassortant HPAI H5N1 viruses as a candidate poultry vaccine, and we evaluated its immunogenicity and protective efficacy in specific pathogen-free chickens. The 6:2 reassortant H5N1 vaccine strain containing the surface genes of the A/Chicken/Korea/ES/2003(H5N1) virus was successfully generated by reverse genetics. A polybasic cleavage site of the hemagglutinin segment was replaced by a monobasic cleavage site. We characterized the reverse genetics-derived reassortant HPAI H5N1 clade 2.5 vaccine strain by evaluating its growth kinetics in eggs, minimum effective dose in chickens, and cross-clade immunogenicity against HPAI clade 1 and 2. The bivalent vaccine was prepared by emulsifying inactivated ND (La Sota strain) and reassortant HPAI viruses with Montanide ISA 70 adjuvant. A single immunization with this vaccine induced high levels of hemagglutination-inhibiting antibody titers and protected chickens against a lethal challenge with the wild-type HPAI and ND viruses. Our results demonstrate that the bivalent, inactivated vaccine developed in this study is a promising approach for the control of both HPAI H5N1 and ND viral infections.


Nurses as patient advocates in oncology care: activities based on literature.
This article will explore oncology nurses' patient advocacy activities and compare those activities with patient advocacy activities defined in an earlier study by the authors. Data were collected from 42 English-language peer-reviewed articles published from 2000-2010. Search terms used included cancer care and advocacy and oncology nursing and advocacy. According to the findings of the reviewed articles, oncology nurses promote the interests of their patients by analyzing patients' psychosocial and physical distress and care plans, particularly at the beginning of the illness trajectory. Oncology nurses also are instructed in the literature to educate patients about cancer management prior to the first treatment and during cancer management to promote informed consent, but not to analyze patients' information or self-determination preferences. Oncology nurses do, however, advocate for their patients by presenting and raising awareness of patients' needs and preferences in regard to the healthcare system. To some degree, this advocacy can be seen as responding to patients' care and self-determination preferences. Oncology nurses' patient advocacy activities are similar to advocacy activities defined in the context of procedural pain care but are more focused on the beginning of the illness trajectory. However, care and self-determination needs, information needs, and advocacy needs of patients with cancer vary during the illness trajectory. Those needs should be analyzed and responded to systematically.


Cryo-EM structure of the SARS-CoV-2 3a ion channel in lipid nanodiscs.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus that causes the coronavirus disease 2019 (COVID-19). SARS-CoV-2 encodes three putative ion channels: E, 8a, and 3a1,2. 3a is expressed in SARS patient tissue and anti-3a antibodies are observed in patient plasma3-6. 3a has been implicated in viral release7, inhibition of autophagy8, inflammasome activation9, and cell death10,11 and its deletion reduces viral titer and morbidity in mice1, raising the possibility that 3a could be an effective vaccine or therapeutic target3,12. Here, we present the first cryo-EM structures of SARS-CoV-2 3a to 2.1 Å resolution and demonstrate 3a forms an ion channel in reconstituted liposomes. The structures in lipid nanodiscs reveal 3a dimers and tetramers adopt a novel fold with a large polar cavity that spans halfway across the membrane and is accessible to the cytosol and the surrounding bilayer through separate water- and lipid-filled openings. Electrophysiology and fluorescent ion imaging experiments show 3a forms Ca2+-permeable non-selective cation channels. We identify point mutations that alter ion permeability and discover polycationic inhibitors of 3a channel activity. We find 3a-like proteins in multiple Alphacoronavirus and Betacoronavirus lineages that infect bats and humans. These data show 3a forms a functional ion channel that may promote COVID-19 pathogenesis and suggest targeting 3a could broadly treat coronavirus diseases.


Improved in planta expression of the human islet autoantigen glutamic acid decarboxylase (GAD65).
The smaller isoform of the enzyme glutamic acid decarboxylase (GAD65) is a major islet autoantigen in autoimmune type 1 diabetes mellitus (T1DM). Transgenic plants expressing human GAD65 (hGAD65) are a potential means of direct oral administration of the islet autoantigen in order to induce tolerance and prevent clinical onset of disease. We have previously reported the successful generation of transgenic tobacco and carrot that express immunoreactive, full-length hGAD65. In the present study, we tested the hypothesis that the expression levels of recombinant hGAD65 in transgenic plants can be increased by targeting the enzyme to the plant cell cytosol and by mediating expression through the potato virus X (PVX) vector. By substituting the NH2-terminal region of hGAD65 with a homologous region of rat GAD67, a chimeric GAD67(1-87)/GAD65(88-585) molecule was expressed in transgenic tobacco plants. Immunolocalization analysis showed that immunoreactive GAD67/65 was found in the plant cell cytosol. By using a radio-immuno assay with human serum from a GAD65 autoantibody-positive T1DM patient, the highest expression level of the recombinant GAD67/65 protein was estimated to be 0.19% of the total soluble protein, compared to only 0.04% of wild-type hGAD65. Transient expression of wild-type, full-length hGAD65 in N. benthamiana mediated by PVX infection was associated with expression levels of immunoreactive protein as high as 2.2% of total soluble protein. This substantial improvement of the expression of hGAD65 in plants paves the way for immunoprevention studies of oral administration of GAD65-containing transgenic plant material in animal models of spontaneous autoimmune diabetes.


Potentially Prolonged Psychological Distress from Postponed Olympic and Paralympic Games during COVID-19-Career Uncertainty in Elite Athletes.
The coronavirus disease 2019 (COVID-19) pandemic has had a significant impact on the world of sports due to periods of home quarantine, bans against public gatherings, travel restrictions, and a large number of postponed or canceled major sporting events. The literature hitherto is sparse, but early indications display signs of psychological impact on elite athletes due to the pandemic. However, beyond acute effects from lockdown and short-term interrupted athletic seasons, the postponed and still uncertain Olympic and Paralympic Games may represent a major career insecurity to many athletes world-wide, and may lead to severe changes to everyday lives and potentially prolonged psychological distress. Given the long-term perspective of these changes, researchers and stakeholders should address mental health and long-term job insecurity in athletes, including a specific focus on those with small financial margins, such as many female athletes, parasports athletes, athletes in smaller sports, and athletes from developing countries. Implications and the need for research are discussed.


Metabolic constraints drive self-organization of specialized cell groups.
How phenotypically distinct states in isogenic cell populations appear and stably co-exist remains unresolved. We find that within a mature, clonal yeast colony developing in low glucose, cells arrange into metabolically disparate cell groups. Using this system, we model and experimentally identify metabolic constraints sufficient to drive such self-assembly. Beginning in a uniformly gluconeogenic state, cells exhibiting a contrary, high pentose phosphate pathway activity state, spontaneously appear and proliferate, in a spatially constrained manner. Gluconeogenic cells in the colony produce and provide a resource, which we identify as trehalose. Above threshold concentrations of external trehalose, cells switch to the new metabolic state and proliferate. A self-organized system establishes, where cells in this new state are sustained by trehalose consumption, which thereby restrains other cells in the trehalose producing, gluconeogenic state. Our work suggests simple physico-chemical principles that determine how isogenic cells spontaneously self-organize into structured assemblies in complimentary, specialized states.


Hyperglycemia and xerostomia are key determinants of tooth decay in type 1 diabetic mice.
Insulin-dependent type 1 diabetes mellitus (DM) and oral diseases are closely interrelated. Poor metabolic control in diabetics is associated with a high risk of gingivitis, periodontitis and tooth loss. Salivary flow declines in diabetics and patients suffer from xerostomia. Reduced saliva predisposes to enamel hypomineralization and caries formation; however, the mechanisms that initiate and lead to progression of tooth decay and periodontitis in type 1 DM have not been explored. To address this issue, we analyzed tooth morphology in Akita ⁻/⁻ mice that harbor a point mutation in the Ins2 insulin gene, which leads to progressive hyperglycemia. Mandibles from Akita ⁻/⁻ and wild-type littermates were analyzed by microCT, scanning EM and histology; teeth were examined for amelogenin (Amel) and ameloblastin (Ambn) expression. Mice were injected with pilocarpine to assess saliva production. As hyperglycemia may alter pulp repair, the effect of high glucose levels on the proliferation/differentiation of cultured MD10-F2 pulp cells was also analyzed. Results showed that Akita ⁻/⁻ mice at 6 weeks of age showed chalky white incisors that correlated with marked hyperglycemia and impaired saliva production. MicroCT of Akita ⁻/⁻ teeth revealed excessive enamel wearing and hypomineralization; immunostaining for Amel and Ambn was decreased. A striking feature was invasion of dentinal tubules with Streptococcus mitis and microabcesses that originated in the coronal pulp and progressed to pulp necrosis and periapical periodontitis. High levels of glucose also inhibited MD10-F2 cell proliferation and differentiation. Our findings provide the first evidence that hyperglycemia in combination with reduced saliva in a model of type1 DM leads to decreased enamel mineralization/matrix proteins and predisposes to excessive wearing and decay. Importantly, hyperglycemia adversely affects enamel matrix proteins and pulp repair. Early detection and treatment of hyperglycemia and hyposalivation may provide a useful strategy for preventing the dental complications of diabetes and promoting oral health in this population.


Autophagy Proteins Promote Repair of Endosomal Membranes Damaged by the Salmonella Type Three Secretion System 1.
Salmonella Typhimurium (S.Tm) is an enteropathogen requiring multiple virulence factors, including two type three secretion systems (T1 and T2). T1 triggers epithelium invasion in which the bacteria are taken up into endosomes that mature into Salmonella-containing vacuoles (SCV) and trigger T2 induction upon acidification. Mechanisms controlling endosome membrane integrity or pathogen egress into the cytosol are incompletely understood. We screened for host factors affecting invasion and SCV maturation and identified a role for autophagy in sealing endosomal membranes damaged by T1 during host cell invasion. S.Tm-infected autophagy-deficient (atg5(-/-)) cells exhibit reduced SCV dye retention and lower T2 expression but no effects on steps preceding SCV maturation. However, in the absence of T1, autophagy is dispensable for T2 induction. These findings establish a role of autophagy at early stages of S.Tm infection and suggest that autophagy-mediated membrane repair might be generally important for invasive pathogens and endosomal membrane function.


Alpha1-syntrophin mutations identified in sudden infant death syndrome cause an increase in late cardiac sodium current.
Sudden infant death syndrome (SIDS) is a leading cause of death during the first 6 months after birth. About 5% to 10% of SIDS may stem from cardiac channelopathies such as long-QT syndrome. We recently implicated mutations in alpha1-syntrophin (SNTA1) as a novel cause of long-QT syndrome, whereby mutant SNTA1 released inhibition of associated neuronal nitric oxide synthase by the plasma membrane Ca-ATPase PMCA4b, causing increased peak and late sodium current (I(Na)) via S-nitrosylation of the cardiac sodium channel. This study determined the prevalence and functional properties of SIDS-associated SNTA1 mutations. Using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing of SNTA1's open reading frame, 6 rare (absent in 800 reference alleles) missense mutations (G54R, P56S, T262P, S287R, T372M, and G460S) were identified in 8 (approximately 3%) of 292 SIDS cases. These mutations were engineered using polymerase chain reaction-based overlap extension and were coexpressed heterologously with SCN5A, neuronal nitric oxide synthase, and PMCA4b in HEK293 cells. I(Na) was recorded using the whole-cell method. A significant 1.4- to 1.5-fold increase in peak I(Na) and 2.3- to 2.7-fold increase in late I(Na) compared with controls was evident for S287R-, T372M-, and G460S-SNTA1 and was reversed by a neuronal nitric oxide synthase inhibitor. These 3 mutations also caused a significant depolarizing shift in channel inactivation, thereby increasing the overlap of the activation and inactivation curves to increase window current. Abnormal biophysical phenotypes implicate mutations in SNTA1 as a novel pathogenic mechanism for the subset of channelopathic SIDS. Functional studies are essential to distinguish pathogenic perturbations in channel interacting proteins such as alpha1-syntrophin from similarly rare but innocuous ones.


Butyrate-induced colonic hypersensitivity is mediated by mitogen-activated protein kinase activation in rat dorsal root ganglia.
Increased faecal butyrate levels have been reported in irritable bowel syndrome. Rectal instillation of sodium butyrate (NaB) increases visceral sensitivity in rats by an unknown mechanism. We seek to examine the signal transduction pathways responsible for the enhanced neuronal excitability in the dorsal root ganglion (DRG) following NaB enemas and demonstrate that this is responsible for the colonic hypersensitivity reported in this animal model. Colorectal distention (CRD) studies were performed in rats treated with NaB rectal instillation with/without intrathecal or intravenous administration of mitogen-activated protein (MAP) kinase kinase inhibitor U0126. Western blot analysis and immunocytochemistry studies elucidated intracellular signalling pathways that modulate IA. Patch-clamp recordings were performed on isolated DRG neurons treated with NaB, with/without U0126. Visceromotor responses (VMR) were markedly enhanced in NaB-treated rats. Western blot analysis of DRG neurons from NaB-treated rats showed a 2.2-fold increase in phosphorylated ERK1/2 (pEKR1/2) and 1.9-fold increase in phosphorylated voltage-gated potassium channel subunit 4.2 (pKv4.2). Intrathecal or intravenous administration of U0126 reduced VMR to CRD in NaB-treated rats and prevented increases in pERK1/2 and pKv4.2. Patch-clamp recordings of isolated DRG neurons showed that NaB caused a reduction in IA to 48.9%±1.4% of control and an increase in neuronal excitability, accompanied by a twofold increase in pERK1/2 and pKv4.2. Concurrent U0126 administration prevented these changes. Visceral hypersensitivity induced by colonic NaB treatment is mediated by activation of the MAP kinase-ERK1/2 pathway, which phosphorylates Kv4.2. This results in a reduction in IA and an enhancement of DRG neuronal excitability.


Novel ensemble of optimized CNN and dynamic selection techniques for accurate Covid-19 screening using chest CT images.
The world is significantly affected by infectious coronavirus disease (covid-19). Timely prognosis and treatment are important to control the spread of this infection. Unreliable screening systems and limited number of clinical facilities are the major hurdles in controlling the spread of covid-19. Nowadays, many automated detection systems based on deep learning techniques using computed tomography (CT) images have been proposed to detect covid-19. However, these systems have the following drawbacks: (i) limited data problem poses a major hindrance to train the deep neural network model to provide accurate diagnosis, (ii) random choice of hyperparameters of Convolutional Neural Network (CNN) significantly affects the classification performance, since the hyperparameters have to be application dependent and, (iii) the generalization ability using CNN classification is usually not validated. To address the aforementioned issues, we propose two models: (i) based on a transfer learning approach, and (ii) using novel strategy to optimize the CNN hyperparameters using Whale optimization-based BAT algorithm + AdaBoost classifier built using dynamic ensemble selection techniques. According to our second method depending on the characteristics of test sample, the classifier is chosen, thereby reducing the risk of overfitting and simultaneously produced promising results. Our proposed methodologies are developed using 746 CT images. Our method obtained a sensitivity, specificity, accuracy, F-1 score, and precision of 0.98, 0.97, 0.98, 0.98, and 0.98, respectively with five-fold cross-validation strategy. Our developed prototype is ready to be tested with huge chest CT images database before its real-world application.


SHROOM3 is a novel candidate for heterotaxy identified by whole exome sequencing.
Heterotaxy-spectrum cardiovascular disorders are challenging for traditional genetic analyses because of clinical and genetic heterogeneity, variable expressivity, and non-penetrance. In this study, high-resolution SNP genotyping and exon-targeted array comparative genomic hybridization platforms were coupled to whole-exome sequencing to identify a novel disease candidate gene. SNP genotyping identified absence-of-heterozygosity regions in the heterotaxy proband on chromosomes 1, 4, 7, 13, 15, 18, consistent with parental consanguinity. Subsequently, whole-exome sequencing of the proband identified 26,065 coding variants, including 18 non-synonymous homozygous changes not present in dbSNP132 or 1000 Genomes. Of these 18, only 4--one each in CXCL2, SHROOM3, CTSO, RXFP1--were mapped to the absence-of-heterozygosity regions, each of which was flanked by more than 50 homozygous SNPs, confirming recessive segregation of mutant alleles. Sanger sequencing confirmed the SHROOM3 homozygous missense mutation and it was predicted as pathogenic by four bioinformatic tools. SHROOM3 has been identified as a central regulator of morphogenetic cell shape changes necessary for organogenesis and can physically bind ROCK2, a rho kinase protein required for left-right patterning. Screening 96 sporadic heterotaxy patients identified four additional patients with rare variants in SHROOM3. Using whole exome sequencing, we identify a recessive missense mutation in SHROOM3 associated with heterotaxy syndrome and identify rare variants in subsequent screening of a heterotaxy cohort, suggesting SHROOM3 as a novel target for the control of left-right patterning. This study reveals the value of SNP genotyping coupled with high-throughput sequencing for identification of high yield candidates for rare disorders with genetic and phenotypic heterogeneity.


NAADP links histamine H1 receptors to secretion of von Willebrand factor in human endothelial cells.
A variety of endothelial agonist-induced responses are mediated by rises in intracellular Ca(2+), suggesting that different Ca(2+) signatures could fine-tune specific inflammatory and thrombotic activities. In search of new intracellular mechanisms modulating endothelial effector functions, we identified nicotinic acid adenine dinucleotide phosphate (NAADP) as a crucial second messenger in histamine-induced Ca(2+) release via H1 receptors (H1R). NAADP is a potent intracellular messenger mobilizing Ca(2+) from lysosome-like acidic compartments, functionally coupled to the endoplasmic reticulum. Using the human EA.hy926 endothelial cell line and primary human umbilical vein endothelial cells, we show that selective H1R activation increases intracellular NAADP levels and that H1R-induced calcium release involves both acidic organelles and the endoplasmic reticulum. To assess that NAADP links H1R to Ca(2+)-signaling we used both microinjection of self-inactivating concentrations of NAADP and the specific NAADP receptor antagonist, Ned-19, both of which completely abolished H1R-induced but not thrombin-induced Ca(2+) mobilization. Interestingly, H1R-mediated von Willebrand factor (VWF) secretion was completely inhibited by treatment with Ned-19 and by siRNA knockdown of 2-pore channel NAADP receptors, whereas thrombin-induced VWF secretion failed to be affected. These findings demonstrate a novel and specific Ca(2+)-signaling mechanism activated through H1R in human endothelial cells, which reveals an obligatory role of NAADP in the control of VWF secretion.


Mitochondrial dynamics in plant male gametophyte visualized by fluorescent live imaging.
Visualization of organelles in living cells is a powerful method for studying their dynamic behavior. Here we attempted to visualize mitochondria in angiosperm male gametophyte (pollen grain from Arabidopsis thaliana) that are composed of one vegetative cell (VC) and two sperm cells (SCs). Combination of mitochondria-targeted fluorescent proteins with VC- or SC-specific expression allowed us to observe the precise number and dynamic behavior of mitochondria in the respective cell types. Furthermore, live imaging of SC mitochondria during double fertilization confirmed previous observations, demonstrated by electron microscopy in other species, that sperm mitochondria enter into the egg and central cells. We also attempted to visualize mutant mitochondria that were elongated due to a defect in mitochondrial division. This mutant phenotype was indeed detectable in VC mitochondria of a heterozygous F(1) plant, suggesting active mitochondrial division in male gametophyte. Finally, we performed mutant screening and isolated a putative mitochondrial protein transport mutant whose phenotype was detectable only in haploid cells. The transgenic materials presented in this work are useful not only for live imaging but also for studying mitochondrial functions by mutant analysis.


Longitudinal evidence that fatherhood decreases testosterone in human males.
In species in which males care for young, testosterone (T) is often high during mating periods but then declines to allow for caregiving of resulting offspring. This model may apply to human males, but past human studies of T and fatherhood have been cross-sectional, making it unclear whether fatherhood suppresses T or if men with lower T are more likely to become fathers. Here, we use a large representative study in the Philippines (n = 624) to show that among single nonfathers at baseline (2005) (21.5 ± 0.3 y), men with high waking T were more likely to become partnered fathers by the time of follow-up 4.5 y later (P < 0.05). Men who became partnered fathers then experienced large declines in waking (median: -26%) and evening (median: -34%) T, which were significantly greater than declines in single nonfathers (P < 0.001). Consistent with the hypothesis that child interaction suppresses T, fathers reporting 3 h or more of daily childcare had lower T at follow-up compared with fathers not involved in care (P < 0.05). Using longitudinal data, these findings show that T and reproductive strategy have bidirectional relationships in human males, with high T predicting subsequent mating success but then declining rapidly after men become fathers. Our findings suggest that T mediates tradeoffs between mating and parenting in humans, as seen in other species in which fathers care for young. They also highlight one likely explanation for previously observed health disparities between partnered fathers and single men.


Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase.
The crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase complexed with a 20-amino acid substrate analog inhibitor has been solved and partially refined at 2.7 A resolution to an R factor of 0.212. The magnesium adenosine triphosphate (MgATP) binding site was located by difference Fourier synthesis. The enzyme structure is bilobal with a deep cleft between the lobes. The cleft is filled by MgATP and a portion of the inhibitor peptide. The smaller lobe, consisting mostly of amino-terminal sequence, is associated with nucleotide binding, and its largely antiparallel beta sheet architecture constitutes an unusual nucleotide binding motif. The larger lobe is dominated by helical structure with a single beta sheet at the domain interface. This lobe is primarily involved in peptide binding and catalysis. Residues 40 through 280 constitute a conserved catalytic core that is shared by more than 100 protein kinases. Most of the invariant amino acids in this conserved catalytic core are clustered at the sites of nucleotide binding and catalysis.


Transporter hypothesis of drug-resistant epilepsy: challenges for pharmacogenetic approaches.
Drug resistance in epilepsy is considered a complex and multifactorial problem. Overexpression of efflux transporters at the blood-brain barrier is discussed as one factor that might limit brain penetration and efficacy of antiepileptic drugs. Whereas experimental data render support for this hypothesis, there is still a lack of sufficient clinical evidence indicating a functional role of efflux transporters. Pharmacogenetic analysis has been considered as one approach in the evaluation of a putative link between transporters and drug-resistant epilepsy. However, the likelihood of a multifactorial nature of drug resistance and the complexity of the events regulating transporters pose a major challenge to any attempt at linking selected genetic polymorphisms to the outcome of drug therapy. In this article, the evidence for an impact of efflux transporters on the response to antiepileptic drugs is discussed, focusing in particular on the different issues presenting a challenge for pharmacogenetic approaches in this field.


Mobility and subsistence economy: a diachronic comparison between two groups settled in the same geographical area (Liguria, Italy).
The purpose of this article is to investigate temporal shifts in skeletal robusticity to infer behavioral changes in two populations (Neolithic, NEOL and Medieval, MED) settled in the same geographic area but involved in different subsistence economies (pastoralism and coastal resources exploitation). This comparison allows us to test the hypothesis that occupational stress and mobility in the same environment produce predictable changes in the robusticity of both upper and lower limbs. Results show a lower degree of humeral robusticity and a similar degree of humeral asymmetry in the two sexes in the MED population. These results are consistent with the relatively less stressful subsistence economy documented in the MED population relative to that of the NEOL. Lower limb results suggest that femoral robusticity does not correlate directly with the level of logistical mobility, but is instead due to the summation of several diverse factors that place biomechanical loads on the hindlimb, particularly unevenness of the terrain. However, female femoral gracility seems to indicate that below a certain "threshold" of mobility, i.e., movement over the natural terrain, terrain conformation is no longer the main contributing factor to femoral robusticity. The femoral shape index I(x)/I(y) declines through time, particularly in males. This agrees with the expected mobility of the samples based on archaeological and historical data, providing further evidence on the reliability of this index in inferring terrestrial mobility.


Progress in DNA-based heterologous prime-boost immunization strategies for malaria.
An effective vaccine against malaria is urgently required to relieve the immense human suffering and mortality caused by this parasite. A successful subunit vaccine against the liver stage of malaria will require the induction of high levels of protective T cells. Despite success in small animal models, DNA vaccines fail to induce strong cellular immune responses in humans. However, DNA vaccines can induce a T-cell response that can be strongly boosted by recombinant viral vectors. We have evaluated this heterologous prime-boost approach using the Plasmodium berghei mouse model for immunogenicity and protective efficacy against malaria challenge using combinations of plasmid DNA, recombinant modified vaccinia virus Ankara, fowlpox virus, and non-replicating adenovirus. We have proceeded to test immunogenicity and efficacy of successful heterologous prime-boost vaccines in phase I/IIa trials in malaria naïve subjects in the UK and in semi-immune individuals in The Gambia. In these clinical trials, remarkably high levels of effector T-cell responses have been induced and significant protection documented in a human sporozoite challenge model. We summarize the preclinical design and development of these heterologous prime-boost vaccines and discuss the encouraging results that have been observed in vaccinated humans.


Validation of a combined corneal topographer and aberrometer based on Shack-Hartmann wave-front sensing.
A corneal aberrometer based on Shack-Hartmann wave-front sensing was developed and validated by using calibrated aspheric surfaces. The aberrometer was found to accurately measure corneal reflective aberrations, from which corneal topography and corneal refractive aberrations were derived. Measurements of reflective aberrations correlated well with theory (R2 = 0.964 to 0.994). The sag error root mean square (RMS) was small, ranging from 0.1 to 0.17 microm for four of the five calibrated surfaces with the fifth at 0.36 microm as a result of residual defocus. Measured refractive aberrations matched with theory and whole-eye aberrometry to within a small fraction of a wavelength. Measurements on three human corneas revealed very large refractive astigmatism (0.65-1.2 microm) and appreciable levels of trefoil (0.08-0.47 microm), coma (0.14-0.19 microm), and spherical aberration (0.18-0.25 microm). The mean values of these aberrations were significantly larger than the RMS in repeated measurements.


Risk factors for lead exposure in adult population in southern Brazil.
In Brazil there is no systematic evaluation to access blood lead levels (BLL) in the general population and few studies with adults have been published. The aim of this study was to examine the socioeconomic, environmental, and lifestyle determinants of BLL in the adult Brazilian population. In total, 959 adults, aged 40 years or more, were randomly selected in a city in southern Brazil. Information on socioeconomic, dietary, lifestyle, and occupational background was obtained by interviews. A spatial analysis was conducted to discern whether there were any identifiable sources of exposure. BLL were measured by inductively coupled plasma-mass spectrometry. There was an adjustment for gender, age, race, education, income class, smoking status, alcohol consumption, occupation, and red meat or cow milk consumption (Model 1), and for occupation and gender (Model 2). The geometric mean of BLL was 1.97 μg/dl (95% CI: 1.9-2.04 μg/dl). In Model 1, BLL were positively associated with male gender, older age, and drinking and smoking habits, and less frequently with milk consumption. In Model 2, data showed higher BLL in non-white than white participants, in former smokers and individuals with current or former employment in lead (Pb) industries. The participants living in the area with more Pb industries had higher BLL (3.3 μg/dl) compared with those residing in other areas with no or fewer Pb industries (1.95 μg/dl). Despite the low BLL found in adults living in an urban area, Pb industries need to be monitored and regulatory laws implemented to prevent metal contamination in urban settings.


Adenovirus-mediated ING4/IL-24 double tumor suppressor gene co-transfer enhances antitumor activity in human breast cancer cells.
Cancer gene therapy represents a new and promising therapeutic modality for various types of cancer. Two or more anti-oncogenes carried by a single vector could theoretically improve treatment efficacy, reduce side-effects from vectors, and have a satisfactory clinical application prospect; however, this has seldom been studied in breast cancer. The inhibitor of growth 4 (ING4), as a member of the inhibitor of growth tumor suppressor family has potent inhibitory effects on a variety of tumors. Interleukin‑24 (IL-24) has also shown broad spectrum and tumor-specific antitumor activities. In this study, we aimed to prove the enhanced antitumor activity of adenovirus-mediated ING4/IL-24 double tumor suppressor gene co-transfer in human breast cancer cells. We assessed the combined effect of the ING4/IL-24 bicistronic adenovirus (Ad-ING4-IL-24) in vitro and in vivo on MDA-MB-231 human breast cancer cells by detecting and comparing the apoptotic status in the bicistronic anti-oncogene group (Ad-ING4-IL-24) and in the ING4 or IL-24 single anti-oncogene groups, and also investigated the possible underlying mechanism. Our results showed that the bicistronic adenovirus-mediated ING4 and IL-24 co-expression induced additive growth suppression and apoptosis as well as an overlapping effect on the upregulation of p21, p27 and Bax, and the downregulation of Bcl-2 and survivin in MDA-MB‑231 human breast cancer cells in vitro or in vivo. Moreover, Ad-ING4-IL-24 treatment additively reduced CD34 expression and the microvessel density in MDA-MB-231 xenografted tumors in athymic nude mice, which correlated with the decreased expression of the vascular endothelial growth factor. The enhanced antitumor activity on breast cancer elicited by Ad-ING4-IL-24 was closely associated with the activation of the apoptotic pathways and the additive inhibition of tumor angiogenesis.


Derangements in bone mineral parameters and bone mineral density in south Indian subjects on antiepileptic medications.
Although there are reports describing the association of alternations of bone and mineral metabolism in epileptic patients with long-term anticonvulsant therapy, there are only limited Indian studies which have looked at this aspect. This study was done to compare the prevalence of changes in bone mineral parameters and bone mineral density (BMD) in ambulant individuals on long-term anticonvulsant therapy with age- and body mass index (BMI)-matched healthy controls. There were 55 men (on medications for more than 6 months) and age- and BMI-matched 53 controls. Drug history, dietary calcium intake (DCI), and duration of sunlight exposure were recorded. Bone mineral parameters and BMD were measured. The control group had a significantly higher daily DCI with mean ± SD of 396 ± 91 mg versus 326 ± 101 mg (P = 0.007) and more sunlight exposure of 234 ± 81 vs 167 ± 69 min (P = 0.05). BMD at the femoral neck was significantly lower in cases (0.783 ± 0.105 g/cm(2)) when compared to controls (0.819 ± 0.114 g/cm(2)). Majority of the patients (61%) had low femoral neck BMD (P = 0.04). There was no significant difference in the proportion of subjects with vitamin D deficiency (<20 ng/mL) between cases (n = 32) and controls (n = 37) (P = 0.234). Vitamin D deficiency was seen in both the groups in equal proportions, highlighting the existence of a high prevalence of this problem in India. Low femoral neck BMD found in cases may stress the need for supplementing calcium and treating vitamin D deficiency in this specific group. However, the benefit of such intervention has to be studied in a larger proportion of epileptic patients.


SU-E-T-584: Dosimetric Comparison Between Static IMRT and VMAT for a Four- Lesion Brain Treatment.
Sparing brain volume is the goal when designing plans for multiple brain tumors. We compared dose distributions for tumor and normal tissues using VAMT and static IMRT. A patient presented with recurrent meningioma with 4 lesions identified. The greatest dimensions for the tumors were 0.4 to 2.0 cm. The tumor sizes and locations can be treated with a single plan with 1.8 Gy/fraction, 30 fractions. A 6-field non-coplanar IMRT with the gantry(G) and couch(C) in IEC scale were used: G0C0, G45C0, G330C0, G240C0, G50C90, and G120C90. IMRT was performed using iPlan sliding-window. For VMAT, four arcs were used; two using 350 degree from G175 to G185 and the other two using 175 degree from G0 to G175. Two arcs were designed with couch=0 and the rest two using couch=90 degree. VMAT was designed with Eclipse system. Tumors and normal brain were contoured in the iPlan and then exported to Eclipse to maintain identical volume. DVH for normal brain was compared for the same tumor coverage from the two plans. Either static IMRT or VMAT generated an acceptable coverage for these four tumors. The conformity of tumor coverage was better in VMAT than that using IMRT; the range of min.-max. doses were: 57.5-63.5 Gy from VMAT vs. 54.1-64.9 Gy from IMRT. For normal brain, DVH did not show a clear difference between the two plans. For doses 5-15 Gy, VMAT delivered 1- 10% more brain volume (1040 cc) but 1-2 % less volume in 30-40 Gy than that from static IMRT. Either static IMRT or VMAT can adequately be used to treat multiple lesions with a single isocenteric treatment. VMAT plan demonstrated improved tumor coverage, spared 1- 2% brain tissue at 30-40 Gy but irradiated up to 10% more brain in 2-7 Gy. The patient was treated with VMAT.


Atezolizumab plus nab-paclitaxel as first-line treatment for unresectable, locally advanced or metastatic triple-negative breast cancer (IMpassion130): updated efficacy results from a randomised, double-blind, placebo-controlled, phase 3 trial.
Immunotherapy in combination with chemotherapy has shown promising efficacy across many different tumour types. We report the prespecified second interim overall survival analysis of the phase 3 IMpassion130 study assessing the efficacy and safety of atezolizumab plus nab-paclitaxel in patients with unresectable, locally advanced or metastatic triple-negative breast cancer. In this randomised, placebo-controlled, double-blind, phase 3 trial, done in 246 academic centres and community oncology practices in 41 countries, patients aged 18 years or older, with previously untreated, histologically documented, locally advanced or metastatic triple-negative breast cancer, and Eastern Cooperative Oncology Group performance status of 0 or 1 were eligible. Patients were randomly assigned (1:1) using a permuted block method (block size of four) and an interactive voice-web response system. Randomisation was stratified by previous taxane use, liver metastases, and PD-L1 expression on tumour-infiltrating immune cells. Patients received atezolizumab 840 mg or matching placebo intravenously on day 1 and day 15 of every 28-day cycle and nab-paclitaxel 100 mg/m2 of body surface area intravenously on days 1, 8, and 15 until progression or unacceptable toxicity. Investigators, patients, and the funder were masked to treatment assignment. Coprimary endpoints were investigator-assessed progression-free survival per Response Evaluation Criteria in Solid Tumors version 1.1 and overall survival, assessed in the intention-to-treat population and in patients with PD-L1 immune cell-positive tumours (tumours with ≥1% PD-L1 expression). The final progression-free survival results were previously reported at the first interim overall survival analysis. The prespecified statistical testing hierarchy meant that overall survival in the subgroup of PD-L1 immune cell-positive patients could only be formally tested if overall survival was significantly different between the treatment groups in the intention-to-treat population. This study is registered with ClinicalTrials.gov, NCT02425891. Between June 23, 2015, and May 24, 2017, 902 patients were enrolled, of whom 451 were randomly assigned to receive atezolizumab plus nab-paclitaxel and 451 were assigned to receive placebo plus nab-paclitaxel (the intention-to-treat population). Six patients from each group did not receive treatment. At the second interim analysis (data cutoff Jan 2, 2019), median follow-up was 18·5 months (IQR 9·6-22·8) in the atezolizumab group and 17·5 months (8·4-22·4) in the placebo group. Median overall survival in the intention-to-treat patients was 21·0 months (95% CI 19·0-22·6) with atezolizumab and 18·7 months (16·9-20·3) with placebo (stratified hazard ratio [HR] 0·86, 95% CI 0·72-1·02, p=0·078). In the exploratory overall survival analysis in patients with PD-L1 immune cell-positive tumours, median overall survival was 25·0 months (95% CI 19·6-30·7) with atezolizumab versus 18·0 months (13·6-20·1) with placebo (stratified HR 0·71, 0·54-0·94]). As of Sept 3, 2018 (the date up to which updated safety data were available), the most common grade 3-4 adverse events were neutropenia (38 [8%] of 453 patients in the atezolizumab group vs 36 [8%] of 437 patients in the placebo group), peripheral neuropathy (25 [6%] vs 12 [3%]), decreased neutrophil count (22 [5%] vs 16 [4%]), and fatigue (17 [4%] vs 15 [3%]). Treatment-related deaths occurred in two (<1%) patients in the atezolizumab group (autoimmune hepatitis related to atezolizumab [n=1] and septic shock related to nab-paclitaxel [n=1]) and one (<1%) patient in the placebo group (hepatic failure). No new treatment-related deaths have been reported since the primary clinical data cutoff date (April 17, 2018). Consistent with the first interim analysis, this second interim overall survival analysis of IMpassion130 indicates no significant difference in overall survival between the treatment groups in the intention-to-treat population but suggests a clinically meaningful overall survival benefit with atezolizumab plus nab-paclitaxel in patients with PD-L1 immune cell-positive disease. However, this positive result could not be formally tested due to the prespecified statistical testing hierarchy. For patients with PD-L1 immune cell-positive metastatic triple-negative breast cancer, atezolizumab plus nab-paclitaxel is an important therapeutic option in a disease with high unmet need. F Hoffmann-La Roche and Genentech.


Mucin gene expression in immortalized human corneal-limbal and conjunctival epithelial cell lines.
The corneal and conjunctival epithelia, which cover the ocular surface, play an important role in preventing pathogen penetrance into the eye and maintaining a wet-surface phenotype by producing highly hydrophilic mucin molecules for their apical surfaces. Ocular surface infections, wounding, and pathologies resulting in dry eye threaten sight and can cause blindness. Understanding the ocular surface defense mechanisms that mucins provide has been hampered by the lack of immortalized human corneal and conjunctival epithelial cell lines that retain mucin gene expression patterns of the native tissue. The purpose of this work was to characterize newly developed immortalized corneal and conjunctival cell lines using mucin gene expression as markers of differentiation. The cell lines were derived as described by a previously published process. Primary cultures of corneal-limbal and conjunctival epithelia were sequentially transduced to express a dominant negative p53 protein and a p16(INK4A/Rb)-resistant, mutant cdk4 protein, which enabled the cells to bypass a senescence mechanism recently identified for primary cultures of keratinocytes. These cells were then transduced to express the catalytic subunit of telomerase to permit them to retain their telomeres and divide indefinitely. Cellular morphology and expression of mucin genes in the two cell lines, designated HCLE for the human corneal-limbal line and HCjE for the human conjunctival cell line, were determined after culture on plastic, type I collagen, or Matrigel, in coculture with fibroblasts, and in severe combined immunodeficient (SCID) mice. Expression of the epithelial cell mucins was assayed by reverse transcription, real-time polymerase chain reaction, immunoblot analysis, or immunohistochemistry and compared with expression in native cornea and conjunctiva. When grown in high-calcium medium on plastic and type I collagen, cells of both lines stratified, exhibiting multiple cell layers. In Matrigel, both cell lines formed cell aggregates that contained lumens. In the SCID mice, the conjunctival cell line formed stratified layers under the kidney capsule. The corneal cell line expressed keratins K3 and K12, the keratins that are corneal-epithelial-specific, and both cell lines expressed K19. As in native tissue, the HCLE and HCjE cell lines expressed the membrane-associated mucins, MUC1, -4, and -16, although their levels were generally lower. Levels of MUC4 and -16 mRNA were the most comparable to native tissue, particularly when cultured on plastic. Apical cells of the stratified cultures were the cells that expressed the membrane-associated mucins MUC1 and -16. Goblet-cell-specific MUC5AC mRNA and protein was detected in a small population of HCjE cells only when using type I collagen as a substrate or when cells were cocultured with fibroblasts. Both cell lines produced glycosylated mucins as indicated by binding of H185 antibody, an antibody that recognizes a carbohydrate epitope on mucins. The immortalized corneal (HCLE) and conjunctival (HCjE) cell lines exhibit the mucin gene expression repertoire of their native epithelia. These cell lines will be useful in determining regulation of ocular surface mucin gene expression and, potentially, goblet cell differentiation.


Sphingosine-1-Phosphate Mediates Fibrosis in Orbital Fibroblasts in Graves' Orbitopathy.
To investigate the effect of sphingosine-1-phosphate (S1P) on fibrosis in orbital fibroblasts in Graves' orbitopathy (GO). Orbital fibroblasts were cultured from orbital adipose/connective tissues of patients with GO and healthy control subjects. Effects of treatment with TGF-β and cigarette smoke extract (CSE) on S1P receptor (S1PR) messenger RNA (mRNA) and S1P expression were evaluated by real-time polymerase chain reaction and Western blotting. To evaluate the role of S1P in fibrosis, cells were pretreated with W146 (S1PR1 antagonist); JTE013 (S1PR2 antagonist); FTY720 (S1PR1 modulator); or 5C (sphingosine kinase-1 blocker) for 1 hour before stimulation with TGF-β, CSE, or IL-1β. Expression of fibrosis-related proteins (collagen Iα, fibronectin, and α-smooth muscle actin [SMA]) and tissue remodeling-related proteins (matrix metalloproteinases [MMPs] and tissue inhibitor of metalloproteinase [TIMP]-1) was then evaluated by Western blotting. Expression levels of S1PR mRNA and S1P in GO orbital fibroblasts increased upon TGF-β and CSE treatment. Treatment with S1PR blockers and 5C inhibited TGF-β and CSE-induced expression of collagen Iα, fibronectin, and α-SMA, as well as IL-1β-induced expression of MMP-1, MMP-2, MMP-9, and TIMP-1. Exogenous S1P treatment without profibrotic stimulants upregulated collagen Iα, fibronectin, α-SMA, MMP-1, MMP-2, MMP-9, and TIMP-1 expression in a dose-dependent manner. Blocking of S1PR activity and inhibition of S1P synthesis led to decreased expression of fibrosis and tissue remodeling-related proteins in primary cultures of orbital fibroblasts derived from patients with GO. Thus, modulation of S1P activity might have therapeutic potential in the suppression of fibrosis in GO.


Cholesterol potentiates the coronary artery response to norepinephrine in anesthetized and conscious dogs.
We investigated the effect of hypercholesterolemia on coronary and cardiac hemodynamic responses to intracoronary norepinephrine (NE) (0.01 to 10.0 micrograms/min as the bitartrate) in a Gregg cannula autoperfusion system. Coronary blood flow was measured by the radioactive microsphere technique in two groups of open-chest dogs anesthetized with pentobarbital: 10 controls and 8 that were fed a cholesterol-rich diet (CD) which doubled the serum cholesterol level. In the control dogs, NE in doses of 0.01 to 1.0 micrograms/min had no effect on coronary vascular resistance (CVR) but 10 micrograms/min caused a significant decrease to 0.58 +/- 0.12 of control. In the CD dogs, NE at doses of 1.0 and 10.0 micrograms/min significantly reduced CVR, to 0.72 +/- 0.06 and 0.52 +/- 0.11 of control, respectively. There was no consistent effect of NE, at these doses, on myocardial oxygen uptake, left ventricular stroke work index, or maximal positive dP/dt. In a second series of experiments we measured coronary flow with electromagnetic flowmeters in 11 chronically instrumented conscious dogs, 5 controls, and 6 CD. In the control dogs, intravenously administered NE hydrochloride, 0.01 microgram/min, reduced CVR to 0.74 +/- 0.07 of control, and 1.0 microgram/min increased CVR to 1.26 +/- 0.09 of control. In the CD animals, these effects were seen at a 10-fold lower NE dose, 0.001 microgram/min (0.83 +/- 0.11 of control) and 0.1 microgram/min (1.32 +/- 0.06 of control). The vasodilation was blocked by propranolol, and vasoconstriction by phentolamine. We conclude that NE at low doses activates beta-adrenoreceptors to reduce CVR and at higher doses activates alpha-adrenoreceptors to increase CVR; the vasoconstrictor response is inhibited in pentobarbital anesthetized dogs, and hypercholesterolemia sensitizes coronary vessels to both the dilator and constrictor effects of NE.


Errors in a busy emergency department.
We describe errors occurring in a busy ED. This is a prospective, observational study of reported errors at an academic emergency department (ED) with 100000 annual visits. Trained personnel interviewed all ED staff with direct patient contact, during and at the end of every shift, by using standardized data sheets. One thousand nine hundred thirty-five ED patients registered during the 7-day study period in the summer of 2001. Four hundred error reports were generated, identifying 346 nonduplicative errors (18 per 100 registered patients; 95% confidence interval [CI] 15.9 to 20.0). Forty percent of errors were reported by nurses, 25% by providers, 19% by clerical staff, 13% by technicians and orderlies, and 3% multiple reporters. Errors reported for every 100 hours worked were similar for all groups (5.5; 95% CI 5.2 to 5.9). Errors were categorized as 22% diagnostic studies, 16% administrative procedures, 16% pharmacotherapy, 13% documentation, 12% communication, 11% environmental, and 9% other. Patients involved in errors were more likely to be older (P <.0001) and more likely to have higher visit level intensity (P <.0001) than registered ED patients. Ninety-eight percent of errors did not have a significant adverse outcome. Seven errors (0.36 per 100 registered patients; 95% CI 0.14 to 0.72) were associated with an adverse outcome. Reported errors occurred in almost every aspect of emergency care. Ninety-eight percent of errors in the ED do not result in adverse outcomes. System changes need to be implemented to reduce ED errors.


Cardiac myoglobin participates in the metabolic pathway of selenium in rats.
As an essential micronutrient, selenium deficiency is a leading cause of cardiovascular diseases. The heart is continuously beating to deliver blood to the entire body, and this requires a high amount of energy. An adult heart normally obtains 50-70% of its adenosine 5'-triphosphate from fatty acid β-oxidation. An increase in fatty acid oxidation activity induces the generation of larger amounts of by-products (reactive oxygen species, ROS) from mitochondrial oxidative phosphorylation. Selenium-dependent glutathione peroxidases play a critical role in the removal of these ROS, especially organic hydroperoxides, from the heart. The definitive transport and/or detailed metabolic pathways from the selenium-source compounds to the selenoproteins in the heart still remain unclear. We explored the selenium-binding proteins in a rat cardiac cell lysate using its reactive metabolic intermediate, selenotrisulfide (STS), and MALDI TOF-mass spectrometry. Several proteins with a free cysteine (Cys) thiol were found to be reactive with STS through a thiol-exchange reaction. The most distinctive Cys-containing protein in the cardiac cell lysate was identified as myoglobin (Mb) from a rat protein database search and tryptic fragmentation experiments. When separately examined in selenium adequate rats, selenium-binding to the cardiac Mb was verified using selenium-specific fluorometry. Cardiac Mb is thought to participate in the selenium metabolic pathway in the heart.


Topical ciprofloxin versus topical framycetin-gramicidin-dexamethasone in Australian aboriginal children with recently treated chronic suppurative otitis media: a randomized controlled trial.
Chronic suppurative otitis media (CSOM) affects many children in disadvantaged populations. The most appropriate topical antibiotic treatment in children with persistent disease is unclear. Children with CSOM despite standard topical treatment were randomized to 6-8 weeks of topical ciprofloxacin (CIP) versus topical framycetin-gramicidin-dexamethasone (FGD). Otoscopic, audiologic, and microbiologic outcomes were measured using standardized assessments and blinding. Ninety-seven children were randomized. Ear discharge failed to resolve at the end of therapy in 70% children regardless of allocation [risk difference = -2%; (95% CI: -20 to 16)]. Healing of the tympanic membrane occurred in one of 50 children in the CIP group and none of 47 children in the FGD group. Severity of discharge failed to improve in more than 50% children in each group, and mean hearing threshold (38 dB and 35 dB) and proportion of children with greater than 25 dB hearing loss (98% and 88%) were not significantly different between the CIP and FGD groups. Side effects were rare. This study showed a similarly low rate of improvement or cure in children with persistent CSOM for both CIP and FGD topical therapies. Complications and side effects were insufficient to cease therapy or inform prescribing of either therapy.


[Use of drugs to treat respiratory tract infections in the community].
This study describes the utilization of drugs to treat respiratory tract infections in a community setting. The description of antimicrobial and non-antimicrobial drugs use is important to design interventions aimed at improving treatment strategies for these common illnesses. In a population-based cross-sectional study, 6145 individuals living in an urban area in southern Brazil were inquired about the use of drugs for the treatment of respiratory tract infections in the 30 days previous to the interview. The Pearson chi-square test was used for statistical analyses. The global prevalence of respiratory infections treated with drugs was 6.3%. The prevalence was higher for children under 4 years of age (18%) and lower for the elderly (3%; p<0.001). Fifty-nine percent of the individuals used antimicrobials to treat respiratory infections. "Sore throat" was the main clinical condition associated with drug use (41%). Amoxicilin was the antimicrobial drug most frequently used (38%), while non-steroidal anti-inflamatory drugs were the most frequent among the non-antimicrobial drugs (27%). Antimicrobials were used in nearly 60% of the respiratory tract infections treated with some drug, even though they are mostly viral in their origin. The indiscriminate use of these drugs not only raises the costs of treatments, but may also lead to the emergence of bacterial resistance against the antimicrobials.


Localization of glucagon-like peptide-2 receptor mRNA expression at different sites in the small intestine of rats.
Preproglucagon is known to be processed into glucagon-like peptide (GLP)-1, GLP-2, and glicentin in the L cells of the intestinal tract. GLP-2 has been shown to possess intestinotrophic activity in rodents. However, the ligand-binding mechanisms of GLP-2 receptor (GLP-2R) have not been extensively investigated. The present study sought to determine the localization of GLP-2R in the small intestine by analyzing GLP-2R mRNA expression using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. GLP-2R mRNA expression was detected at all sites in the small intestine; its expression levels were particularly high in the jejunum. Moreover, GLP-2R mRNA expression was detected in both non-mucosal intestinal tissues and intestinal mucosa. These findings suggest that in addition to the intestinal mucosa, the functional sites of GLP-2 may be present in the non-mucosal tissues. These results imply that GLP-2 peptide acts as an intestinotrophic factor that may affect the intestines from outside the mucosa. The intestinotrophic effect of GLP-2 from outside the mucosa may be a function of the enteric neurons transmitting several growth signals to mucosal cells.


In vivo activation of chicken macrophages by infectious bursal disease virus.
Infectious bursal disease virus (IBDV) infects and replicates in the dividing B lymphocytes of chickens. In the present study, the in vivo effect of IBDV infection on chicken macrophage populations and macrophage activation were examined. Specific-pathogen-free chickens were exposed to virulent IBDV and splenic macrophages were recovered during the acute phase of the disease. At 3 and 5 days post-infection (dpi), spleens of virus-exposed chickens had fewer macrophages than those of virus-free controls (p < 0.05). Confocal microscopic examination revealed cells that were positive for both KUL01 (macrophage surface marker) and R63 (IBDVVP2), indicating presence of the virus in macrophages. MQ-NCSU cells, an avian macrophage cell line, were susceptible to replication of IBDV. In addition, splenic macrophages were activated and had temporarily increased levels of mRNA transcripts of pro-inflammatory mediators, including IL-1beta, IL-6, IL-18, and iNOS. The robust expression of proinflammatory cytokine transcripts, along with a decrease in macrophage numbers, suggest that IBDV activates and may lead to a reduction of resident macrophages in vivo.


Health-related quality of life in children and adolescents with syndromic craniosynostosis.
Syndromic craniosynostosis is a congenital disorder characterised by premature fusion of calvarial sutures combined with other anomalies. The facial appearance is different and patients may show physical impairment, mental or developmental disabilities, elevated intracranial pressure and obstructive sleep apnoea. The impact of this condition on daily functioning has not been studied before. The aim of this study is to assess the health-related quality of life in children and adolescents with syndromic or complex craniosynostosis and to determine the impact of these syndromes on parents. A prospective study was performed in 111 children. Health-related quality of life was measured by international standardised quality-of-life questionnaires, the Infant Toddler Quality of Life Questionnaire (ITQoL), Child Health Questionnaire Parental Form 50 (CHQ-PF50), Child Health Questionnaire Child Form 87 (CHQ-CF87) and Short-Form Health Survey (SF-36). For comparison, we used Dutch population norms of health-related quality-of-life-scores. Parents' scores for patients with syndromic or complex craniosynostosis were significantly lower than those for the norm population. Apert syndrome had the largest impact on the different domains. Scores on the CHQ-PF50 scales for 'physical functioning', 'parental impact emotional' and 'family activities' for these patients were significantly lower than scores for patients with other syndromes, possibly due to the complexity of the syndrome, which includes complex syndactyly, cognitive impairment and behaviour problems. Parents reported a reduced health-related quality of life for themselves, mostly psychosocial with clearly significantly lower general health perceptions. In conclusion, syndromic craniosynostosis has a large impact on the health-related quality of life of these children and their parents, both physical and psychosocial.


Conscientious objection in medical students: a questionnaire survey.
To explore attitudes towards conscientious objections among medical students in the UK. Medical students at St George's University of London, Cardiff University, King's College London and Leeds University were emailed a link to an anonymous online questionnaire, hosted by an online survey company. The questionnaire contained nine questions. A total of 733 medical students responded. Nearly half of the students in this survey stated that they believed in the right of doctors to conscientiously object to any procedure. Demand for the right to conscientiously object is greater in Muslim medical students when compared with other groups of religious medical students. Abortion continues to be a contentious issue among medical students and this may contribute to the looming crisis in abortion services over the coming years. This project sheds some light on how future doctors view some of their ethical rights and obligations. Using empirical evidence, it reveals that conscientious objection is an issue in the UK medical student body today. These data could help anticipate problems that may arise when these medical students qualify and practise medicine in the community. Clearer guidance is needed for medical students about the issue of conscientious objection at medical school.


Functional brain networks develop from a "local to distributed" organization.
The mature human brain is organized into a collection of specialized functional networks that flexibly interact to support various cognitive functions. Studies of development often attempt to identify the organizing principles that guide the maturation of these functional networks. In this report, we combine resting state functional connectivity MRI (rs-fcMRI), graph analysis, community detection, and spring-embedding visualization techniques to analyze four separate networks defined in earlier studies. As we have previously reported, we find, across development, a trend toward 'segregation' (a general decrease in correlation strength) between regions close in anatomical space and 'integration' (an increased correlation strength) between selected regions distant in space. The generalization of these earlier trends across multiple networks suggests that this is a general developmental principle for changes in functional connectivity that would extend to large-scale graph theoretic analyses of large-scale brain networks. Communities in children are predominantly arranged by anatomical proximity, while communities in adults predominantly reflect functional relationships, as defined from adult fMRI studies. In sum, over development, the organization of multiple functional networks shifts from a local anatomical emphasis in children to a more "distributed" architecture in young adults. We argue that this "local to distributed" developmental characterization has important implications for understanding the development of neural systems underlying cognition. Further, graph metrics (e.g., clustering coefficients and average path lengths) are similar in child and adult graphs, with both showing "small-world"-like properties, while community detection by modularity optimization reveals stable communities within the graphs that are clearly different between young children and young adults. These observations suggest that early school age children and adults both have relatively efficient systems that may solve similar information processing problems in divergent ways.


Relationship between musculoskeletal disorders, job demands, and burnout among emergency nurses.
Musculoskeletal disorders (MSDs) represent one of the most common occupational problems in nursing. MSDs can negatively impact one's quality of life. The aim of the study was to investigate the relationship between MSDs, job demands, and burnout among emergency nurses. The researchers hypothesized that increased job demands were associated with more MSDs and consequently higher levels of burnout. The study was conducted on a convenience sample of 58 nurses working in the emergency departments of Zagazig University Hospital and Al-Ahrar, Hospital Egypt from October to December 2010, using a cross-sectional analytic design. Data were collected using a self-administered questionnaire that included the Standardized Nordic Questionnaire, the Job Content Questionnaire, and the Maslach Burnout Inventory. The results revealed that 32.8% of the nurses were overweight and 17.2% were obese. The most common sites of pain were the neck (67.2%), shoulder (65.5%), and lower back (63.8%). Lower back pain was the most common site affected (72.4%) with a mean 5.1 on a scale ranging from 0 to 13. A positive correlation existed between the scores of job demand and burnout (r = 0.340, p < 0.01), and the number of reported MSDs with the score of job demand (r = 0.33, p < 0.05). Multiple linear stepwise regression analysis identified the score of job demand and the severity of lower back pain as positive independent predictors of the burnout whereas the job demand score was the independent predictor of the number of MSDs. This study documents an increased prevalence of MSDs among emergency nurses, as predicted by increased job demand and associated with a higher level of burnout. Hence, it is important for hospital and nursing administrators to address the factors contributing to job stress and burnout, with emphasis on job satisfaction and work organization to alleviate the burden of psychosocial factors in this setting.


WRN is required for ATM activation and the S-phase checkpoint in response to interstrand cross-link-induced DNA double-strand breaks.
Werner syndrome (WS) is a human genetic disorder characterized by extensive clinical features of premature aging. Ataxia-telengiectasia (A-T) is a multisystem human genomic instability syndrome that includes premature aging in some of the patients. WRN and ATM, the proteins defective in WS and A-T, respectively, play significant roles in the maintenance of genomic stability and are involved in several DNA metabolic pathways. A role for WRN in DNA repair has been proposed; however, this study provides evidence that WRN is also involved in ATM pathway activation and in a S-phase checkpoint in cells exposed to DNA interstrand cross-link-induced double-strand breaks. Depletion of WRN in such cells by RNA interference results in an intra-S checkpoint defect, and interferes with activation of ATM as well as downstream phosphorylation of ATM target proteins. Treatment of cells under replication stress with the ATM kinase inhibitor KU 55933 results in a S-phase checkpoint defect similar to that observed in WRN shRNA cells. Moreover, gamma H2AX levels are higher in WRN shRNA cells than in control cells 6 and 16 h after exposure to psoralen DNA cross-links. These results suggest that WRN and ATM participate in a replication checkpoint response, in which WRN facilitates ATM activation in cells with psoralen DNA cross-link-induced collapsed replication forks.


Aerobactin genes in Shigella spp.
Aerobactin, a hydroxamate iron transport compound, is synthesized by some, but not all, Shigella species. Conjugation and hybridization studies indicated that the genes for the synthesis and transport of aerobactin are linked and are found on the chromosome of Shigella flexneri, S. boydii, and S. sonnei. The genes were not found in S. dysenteriae. A number of aerobactin synthesis mutants and transport mutants have been isolated. The most common mutations are deletions of the biosynthesis or biosynthesis and transport genes. The Shigella aerobactin genes share considerable homology with the E. coli ColV aerobactin genes. On the ColV plasmid and in the Shigella chromosome, the aerobactin genes are associated with a repetitive sequence which has been identified as IS1.


Reliability of maternal-reports regarding the use of household pesticides: experience from a case-control study of childhood leukemia.
Self-reported household pesticide use has been associated with higher risk of childhood leukemia in a number of case-control studies. The aim of this study is to assess the reliability of self-reported household use of pesticides and potential differences in reliability by case-control status, and by socio-demographic characteristics. Analyses are based on a subset of the Northern California Childhood Leukemia Study population. Eligible households included those with children less than 8 years old who lived in the same residence since diagnosis (reference date for controls). The reliability was based on two repeated in-person interviews. Kappa, percent positive and negative agreements were used to assess reliability of responses to ever/never use of six pesticides categories. Kappa statistics ranged from 0.31 to 0.61 (fair to substantial agreement), with 9 out of the 12 tests indicating moderate agreement. The percent positive agreement ranged from 46 to 80% and the percent negative agreement from 54 to 95%. Reliability for all pesticide types as assessed by the three reliability measures did not differ significantly for cases and controls as confirmed by bootstrap analysis. For most pesticide types, Kappa and percent positive agreement were higher for non-Hispanics than Hispanics and for households with higher income vs. lower income. Reproducibility of maternal-reported pesticide use was moderate to high and was similar among cases and controls suggesting that differential recall is not likely to be a major source of bias.


Splice-correction strategies for treatment of X-linked agammaglobulinemia.
X-linked agammaglobulinemia (XLA) is a primary immunodeficiency disease caused by mutations in the gene coding for Bruton's tyrosine kinase (BTK). Deficiency of BTK leads to a developmental block in B cell differentiation; hence, the patients essentially lack antibody-producing plasma cells and are susceptible to various infections. A substantial portion of the mutations in BTK results in splicing defects, consequently preventing the formation of protein-coding mRNA. Antisense oligonucleotides (ASOs) are therapeutic compounds that have the ability to modulate pre-mRNA splicing and alter gene expression. The potential of ASOs has been exploited for a few severe diseases, both in pre-clinical and clinical studies. Recently, advances have also been made in using ASOs as a personalized therapy for XLA. Splice-correction of BTK has been shown to be feasible for different mutations in vitro, and a recent proof-of-concept study demonstrated the feasibility of correcting splicing and restoring BTK both ex vivo and in vivo in a humanized bacterial artificial chromosome (BAC)-transgenic mouse model. This review summarizes the advances in splice correction, as a personalized medicine for XLA, and outlines the promises and challenges of using this technology as a curative long-term treatment option.


MECT1-MAML2 fusion transcript defines a favorable subset of mucoepidermoid carcinoma.
Mucoepidermoid carcinoma is the most common primary malignancy of the salivary gland. Mucoepidermoid carcinoma translocated gene 1-mastermind-like gene family (MECT1-MAML2) gene fusion was identified from a recurring t(11;19)(q21;p13) translocation, which is often the sole cytogenetic alteration in this disease. This fusion transcript has been frequently detected in mucoepidermoid carcinoma and shown to be involved in the transformation of epithelial cells. However, its clinicopathologic significance remains unclear. Seventy-one cases of mucoepidermoid carcinoma and 51 cases of nonmucoepidermoid carcinoma salivary gland tumors (including 26 Warthin tumor cases) were retrospectively analyzed. RNA was extracted from archival materials: histologic paraffin specimens in all cases and cytologic specimens in 10 mucoepidermoid carcinoma cases. The MECT1-MAML2 fusion transcript was detected by a reverse transcription-PCR assay, which can be applied to both histologic and cytologic specimens. The presence of the fusion transcript was correlated with relevant clinicopathologic and survival data of the mucoepidermoid carcinoma patients. The MECT1-MAML2 fusion transcript was detected in 27 of the 71 (38%) mucoepidermoid carcinoma cases but not in any case of nonmucoepidermoid carcinoma tumors. The reverse transcription-PCR results showed no difference between histologic and cytologic specimens. Detection of the MECT1-MAML2 fusion transcript was associated with a less advanced clinical stage and a low-grade tumor histology. The presence of the transcript was associated with longer disease-free and overall survivals on univariate analysis and emerged as an independent prognostic factor for longer overall survival on multivariate analysis. The MECT1-MAML2 fusion transcript may be specific to mucoepidermoid carcinoma and associated with a distinct mucoepidermoid carcinoma subset that exhibits favorable clinicopathologic features and an indolent clinical course.


High-resolution MR imaging of sequestered lumbar intervertebral disks.
We retrospectively reviewed the MR examinations of 20 patients with surgically documented sequestered lumbar disks (free fragments). Sixteen of 20 cases demonstrated extradural masses that were distinct from the interspace of origin and had intermediate signal on T1-weighted images but increased signal on T2-weighted images. In 12 cases there was migration of the sequestered fragment. Sequestered disks that migrated away from the interspace had an irregular, oblong appearance, while those near the interspace were round in configuration. Additionally, the interspace of origin consistently demonstrated loss of signal on T2-weighted images when compared with intact lumbar disks. Sagittal 12-weighted images best depicted the absence of a high-signal pedicle contiguous with the interspace of origin in sequestered disks. These findings were applied to a prospective group of 20 patients undergoing lumbar diskectomy. There was an 89% sensitivity, 82% specificity, and 85% accuracy for MR in distinguishing sequestered disks from other forms of lumbar disk herniation. We conclude that high-resolution MR imaging is sensitive in detecting disk disease and specific in characterizing various subtypes of extradural defects. MR uses morphology as well as changes in signal intensity to make these distinctions.


Hereditary aspects of occipital bone hypoplasia and syringomyelia (Chiari type I malformation) in cavalier King Charles spaniels.
A database of over 1300 cavalier King Charles spaniels spanning 20 generations was established by obtaining pedigree information from 45 dogs with syringomyelia secondary to occipital bone hypoplasia. These data were supplemented with published information from the breed club. The incidence of syringomyelia was very high in certain families and lines which had been extensively inbred. The affected dogs could be traced back to one bitch born in 1956 and the two offspring from her single litter. Four key dogs representing four major breeding lines consistently occurred within the individual pedigrees. If a dog had more than five of its eight great-grandparents descended from these four lines there was a greater chance of it having syringomyelia. The data from this preliminary study suggest that occipital bone hypoplasia is hereditary in the cavalier King Charles spaniel and that its inheritance is more likely to be autosomal recessive because both dam and sire must be inbred descendants from certain lines. However, the inheritance is more likely to be of variable penetrance or oligogenic than simple.


Rapid determination of biosynthetic pathways using fractional isotope enrichment and high-resolution dynamic nuclear polarization enhanced NMR.
Carbon-13 NMR has traditionally been a method of choice for the determination of metabolic pathways. Through fractional labeling, (13)C spectra allow the identification of fragments incorporated as a unit into a biosynthesized molecule. The low sensitivity of (13)C spectroscopy is an impediment to such studies, especially if compounded with an often limited availability of biosynthesized molecules. Dynamic nuclear polarization (DNP) can increase the signal-to-noise ratio in magnetic resonance by several orders of magnitude, and in combination with high-resolution spectroscopy has the potential to increase the reach of this technique for metabolic profiling. Here, we present an application of high-resolution DNP enhanced NMR to the study of the biosynthetic pathways for membrane lipids. We show that fatty acid methyl esters are readily hyperpolarized in organic solvent. The resulting spectra resolve the various structural features of the chains, including atoms near the termini, as well as unsaturated and cyclopropyl groups. Peak patterns observed in fractionally labeled samples are explained by the way feed molecules are incorporated into fatty acid chains during synthesis. Differences in multiplet intensity between samples made from glucose and acetate feedstock mixtures further reveal metabolic preferences for these precursors in the biosynthesis of the product. In addition to the present study of lipid biosynthesis, high-resolution DNP-NMR of fractionally (13)C-labeled metabolites may present itself for the rapid determination of biosynthetic pathways in various biomedical applications, especially in cases of limited availability of the products of interest.


Atializumab, a humanized anti-aminoacyl-tRNA synthetase-interacting multifunctional protein-1 (AIMP1) antibody significantly improves nephritis in (NZB/NZW) F1 mice.
Aminoacyl-tRNA synthetase (ARS)-interacting multifunctional protein 1 (AIMP1) enhances the expression of proinflammatory cytokines. In our previous study, we have shown that serum AIMP1 in patients with SLE was significantly higher than that of healthy controls. To address whether neutralization of AIMP1 could ameliorate nephritis in lupus-prone mice, we generated atializumab, a humanized antibody against AIMP1 and investigated its therapeutic efficacy. ELISA showed that serum AIMP1 at 23 weeks old was significantly higher than that at 13 weeks old in lupus-prone mice. Therefore, lupus-prone mice were randomly assigned to 5 groups (vehicle, methylprednisolone and 0.5, 2, and 5 mg/kg atializumab). After treatment, disease severity was assessed using a variety of phenotypes, including proteinuria, histological damages, renal deposition of immune-complex. In addition, serum cytokines, anti-dsDNA and IgG subclasses were determined. T cell subsets were analyzed using a fluorescence-activated cell sorter. Atializumab significantly diminished proteinuria, improved glomerular and tubular damages and reduced the renal deposition of immune-complexes. Moreover, atializumab significantly decreased serum interferon (IFN)-γ, interleukin (IL)-17A, and IL-6, whereas it increased serum IL-10. Similarly, atializumab reduced the numbers of TH1, TH2 and TH17 cells in a dose-dependent manner, while atializumab enhanced the number of regulatory T (Treg) cells. Furthermore, atializumab decreased not only splenic plasma cells and serum anti-dsDNA but also pathogenic IgG subclasses for nephritis. It suppressed NF-κB activation by inhibiting IκBα degradation in a dose-dependent manner in vitro. Atializumab alleviated nephritis by inhibiting autoreactive T, B, and plasma cells and decreasing NF-κB-related proinflammatory cytokines in lupus-prone mice. These results suggest that treatment targeting AIMP1 could be a novel and highly immune-modulating therapeutic strategy in lupus nephritis.