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The green peach aphid [Myzus persicae (Sulzer)] is an important pest of amaranth grown for leaf consumption (i.e., leafy amaranth) in the tropics. Aphids reduce the amount of fresh leaf yield of amaranth and the value of leafy amaranth as aphid-infested leaves are not marketable. Our objective was to evaluate Amaranthus species selected by a breeding program in East Africa to develop cultivars for leaf consumption with resistance to M. persicae. We focused on antibiosis to determine whether varieties of Amaranthus spp. could be grown without producing an aphid population. Artificial infestations of aphids were placed on multiple selections of three species of Amaranthus: two selections of A. blitum, four selections of A. hybridus and one selection of A. hypochondriacus. Aphid populations were assessed over a 5-wk period. Evaluations of vegetative yield, leaf damage symptoms, and specific leaf area (SLA) were made of the seven selections at the end of this experiment. Aphid populations assessed 49 d after planting differed significantly (P ≤ 0.001) among the amaranth species and within selections of the same species. The selections of A. blitum had the lowest aphid populations, and A. hybridus had the highest populations. Selections of A. hybridus produced the most marketable leaves (i.e., aphid free). The fresh weight of A. blitum were the lowest of the seven selections, whereas A. hybridus had the greatest fresh leaf weight. Implications of these finding for further promotion of amaranth breeding are discussed related to pest management for leaf production.

The Carboniferous lungfish genusSagenodusis reviewed from all available British specimens and described in detail for the first time. We identify two species exclusive to the UK:Sagenodusinaequalis, the type species, deriving from the late Carboniferous (=Pennsylvanian); andSagenodus quinquecostatusderived from the early Carboniferous (=Mississippian). The genus is probably the most widespread of the known Carboniferous lungfish genera, but the British species have not been formally described since their discovery in the mid–late 19th Century. This work will provide data to help resolve existing questions about the position ofSagenodusin the phylogeny of Palaeozoic lungfishes, and provide a template for the recognition of isolated elements in museum collections and the finds from recent and future field work. The early Carboniferous species,S. quinquecostatus, shows a so far unique functional mechanism in which the lower tooth plates appear to rotate relative to the upper plates during jaw closure, implying a kinetic function at the symphysis or jaw joint.

TRIM21 (Ro52/SSA1) is an E3 ubiquitin ligase with key roles in immune host defence, signal transduction, and possibly cell cycle regulation. It is also an autoantibody target in Sjögren’s syndrome, systemic lupus erythematosus, and other rheumatic autoimmune diseases. Here, we summarise the structure and function of this enzyme, its roles in innate immunity, adaptive immunity and cellular homeostasis, the pathogenesis of autoimmunity against TRIM21, and the potential impacts of autoantibodies to this intracellular protein.

The noradrenergic systems play a key role in stress triggered disorders such as post-traumatic stress disorder (PTSD). We hypothesized that traumatic stress will alter expression of norepinephrine transporter (NET) in locus coeruleus (LC) and its target brain regions which could be related to hyperarousal. Male Sprague-Dawley rats were subjected to single prolonged stress (SPS) and several weeks later the LC was isolated. NET mRNA levels in LC, determined by RT-PCR, displayed variable response with high and low responsive subgroups. In different cohort, acoustic startle response (ASR) was measured 2 weeks after SPS and levels of NET mRNA and protein in LC determined. The high NET responsive subgroup had greater hyperarousal. Nevertheless, NET protein levels, as determined by western blots, were lower than unstressed controls in LC, ventral hippocampus and medial prefrontal cortex and displayed considerable variability. Hypermethylation of specific CpG region in promoter of SLC6A2 gene, encoding NET, was present in the low, but not high, NET mRNA responsive subgroup. Taken together, the results demonstrate variability in stress elicited changes in NET gene expression and involvement of epigenetic changes. This may underlie mechanisms of susceptibility and resilience to traumatic stress triggered neuropsychiatric symptoms, especially hyperarousal.

Although recent epidemiological data suggest that pneumococci may contribute to the risk of SARS-CoV-2 disease, secondary pneumococcal pneumonia has been reported as infrequent. This apparent contradiction may be explained by interactions of SARS-CoV-2 and pneumococcus in the upper airway, resulting in the escape of SARS-CoV-2 from protective host immune responses. Here, we investigated the relationship of these two respiratory pathogens in two distinct cohorts of a) healthcare workers with asymptomatic or mildly symptomatic SARS-CoV-2 infection identified by systematic screening and b) patients with moderate to severe disease who presented to hospital. We assessed the effect of co-infection on host antibody, cellular and inflammatory responses to the virus. In both cohorts, pneumococcal colonisation was associated with diminished anti-viral immune responses, which affected primarily mucosal IgA levels among individuals with mild or asymptomatic infection and cellular memory responses in infected patients. Our findings suggest that S. pneumoniae modulates host immunity to SARS-CoV-2 and raises the question if pneumococcal carriage also enables immune escape of other respiratory viruses through a similar mechanism and facilitates reinfection occurrence.

Streptococcus pneumoniae (Spn) is a common cause of respiratory infection, but also frequently colonizes the nasopharynx in the absence of disease. We used mass cytometry to study immune cells from nasal biopsy samples collected following experimental human pneumococcal challenge in order to identify immunological mechanisms of control of Spn colonization. Using 37 markers, we characterized 293 nasal immune cell clusters, of which 7 were associated with Spn colonization. B cell and CD8+CD161+ T cell clusters were significantly lower in colonized than in non-colonized subjects. By following a second cohort before and after pneumococcal challenge we observed that B cells were depleted from the nasal mucosa upon Spn colonization. This associated with an expansion of Spn polysaccharide-specific and total plasmablasts in blood. Moreover, increased responses of blood mucosal associated invariant T (MAIT) cells against in vitro stimulation with pneumococcus prior to challenge associated with protection against establishment of Spn colonization and with increased mucosal MAIT cell populations. These results implicate MAIT cells in the protection against pneumococcal colonization and demonstrate that colonization affects mucosal and circulating B cell populations.

Traumatic brain injury (TBI) results in heterogeneous pathology affecting multiple cells and tissue types in the brain. It is likely that assessment of such complexity will require simultaneous measurement of multiple molecular biomarkers in a single sample of biological fluid. We measured glial fibrillary acidic protein (GFAP), ubiquitin c-terminal hydrolase L1 (UCH-L1), neurofilament light chain (NF-L) and total tau in plasma samples obtained from 107 subjects enrolled in the Transforming Research and Clinical Knowledge in Traumatic Brain Injury Pilot (TRACK-TBI Pilot) Study using the Quanterix Simoa 4-Plex assay. We also measured NF-L using the Simoa singleplex assay. We computed the correlation between the different biomarkers and calculated the discriminative value of each biomarker for distinguishing between subjects with abnormal versus normal head computed tomography (CT). We found a strong correlation between NF-L values derived from the multiplex and singleplex assays (correlation coefficient = 0.997). Among biomarker values derived from the multiplex assay, the strongest correlation was between the axonal and neuronal markers, NF-L and UCH-L1 (coefficient = 0.71). The weakest correlation was between the glial marker GFAP and the axonal marker tau (coefficient = 0.06). The areas under the curves for distinguishing between subjects with/without abnormal head CT for multiplex GFAP, UCH-L1, NF-L, and total tau were: 0.88 (95% confidence interval 0.81-0.95), 0.86 (0.79-0.93), 0.84 (0.77-0.92), and 0.77 0.67-0.86), respectively. We conclude that the multiplex assay provides simultaneous quantification of GFAP, UCH-L1, NF-L, and tau, and may be clinically useful in the diagnosis of TBI as well as identifying different types of cellular injury.

Background Previous studies have suggested that the pneumococcal niche changes from the nose to the oropharynx with age. We use an Experimental Human Pneumococcal Challenge model to investigate pneumococcal colonisation in each anatomical niche with age. Methods Healthy adults (n = 112) were intranasally inoculated with Streptococcus pneumoniae serotype 6B (Spn6B) and were categorised as young 18-55yrs (n = 57) or older > 55yrs (n = 55). Colonisation status (frequency and density) was determined by multiplex qPCR targeting the lytA and cpsA-6A/B genes in both raw and culture-enriched nasal wash and oropharyngeal swab samples collected at 2-, 7- and 14-days post-exposure. For older adults, raw and culture-enriched saliva samples were also assessed. Results 64% of NW samples and 54% of OPS samples were positive for Spn6B in young adults, compared to 35% of NW samples, 24% of OPS samples and 6% of saliva samples in older adults. Many colonisation events were only detected in culture-enriched samples. Experimental colonisation was detected in 72% of young adults by NW and 63% by OPS. In older adults, this was 51% by NW, 36% by OPS and 9% by saliva. Conclusions The nose is the best niche for detection of experimental pneumococcal colonisation in both young and older adults.

Systemic inflammation impacts outcome after traumatic brain injury (TBI), but most TBI biomarker studies have focused on brain-specific proteins. C-reactive protein (CRP) is a widely used biomarker of inflammation with potential as a prognostic biomarker after TBI. The Transforming Research and Clinical Knowledge in Traumatic Brain Injury (TRACK-TBI) study prospectively enrolled TBI patients within 24 h of injury, as well as orthopedic injury and uninjured controls; biospecimens were collected at enrollment. A subset of hospitalized participants had blood collected on day 3, day 5, and 2 weeks. High-sensitivity CRP (hsCRP) and glial fibrillary acidic protein (GFAP) were measured. Receiver operating characteristic analysis was used to evaluate the prognostic ability of hsCRP for 6-month outcome, using the Glasgow Outcome Scale-Extended (GOSE). We included 1206 TBI subjects, 122 orthopedic trauma controls (OTCs), and 209 healthy controls (HCs). Longitudinal biomarker sampling was performed in 254 hospitalized TBI subjects and 19 OTCs. hsCRP rose between days 1 and 5 for TBI and OTC subjects, and fell by 2 weeks, but remained elevated compared with HCs (p

RationalePneumococcal colonisation is key to the pathogenesis of invasive disease, but is also immunogenic in young adults, protecting against re-colonisation. Colonisation is rarely detected in older adults, despite high rates of pneumococcal disease.ObjectivesTo establish experimental human pneumococcal colonisation in healthy adults aged 50—84 years, to measure the immune response to pneumococcal challenge, and to assess the protective effect of prior colonisation against autologous strain rechallenge.MethodsSixty-four participants were inoculated with Streptococcus pneumoniae (serotype 6B, 80,000CFU in each nostril). Colonisation was determined by bacterial culture of nasal wash, serum anti-6B capsular IgG responses by ELISA, and anti-protein immune responses by multiplex electrochemiluminescence.Measurements and Main ResultsExperimental colonisation was established in 39% of participants (25/64) with no adverse events. Colonisation occurred in 47% (9/19) of participants aged 50—59 compared with 21% (3/14) in those aged ≥70 years. Previous pneumococcal polysaccharide vaccination did not protect against colonisation. Colonisation did not confer serotype-specific immune boosting: GMT (95% CI) 2.7μg/mL (1.9—3.8) pre-challenge versus 3.0 (1.9—4.7) four weeks post-colonisation (p = 0.53). Furthermore, pneumococcal challenge without colonisation led to a drop in specific antibody levels from 2.8μg/mL (2.0—3.9) to 2.2μg/mL (1.6—3.0) post-challenge (p = 0.006). Anti-protein antibody levels increased following successful colonisation. Rechallenge with the same strain after a median of 8.5 months (IQR 6.7—10.1) led to recolonisation in 5/16 (31%).ConclusionsIn older adults, experimental pneumococcal colonisation is feasible and safe, but demonstrates different immunological outcomes compared with younger adults in previous studies.

Older adults are at increased risk of pneumococcal disease. This work aims to evaluate whether there is any decrease in serum IgG against variants of the antigens Pneumococcal surface protein A (PspA) and Pneumococcal surface protein C (PspC) in healthy adults with increasing age. Levels of IgG against PspA and PspC variants were determined by ELISA in serum samples comparing volunteers 18–30 years of age with volunteers who were 50–70+ before and after an experimental pneumococcal colonization challenge. The serotype 6B strain used in the challenge belongs to a minor group of pneumococcal isolates expressing two PspC variants. There was a decrease in levels of IgG with increasing age for the most common PspA variants and for all PspC variants analyzed. No correlation was found between basal levels of IgG against these antigens and protection against colonization. There was an increase in levels of IgG against PspA variants that are more cross-reactive with the variant expressed by the challenge strain post challenge in younger individuals who became colonized. Since the challenge strain used in our study expresses two different PspC variants, an increase in serum IgG against all PspC variants tested was observed in younger individuals who became colonized. For some of the antigen variants tested, a decrease in serum IgG was observed in young volunteers who were challenged but did not become colonized. Serum IgG antibodies against PspA and PspC variants thus decrease with age in healthy adults, but there is no correlation between levels of IgG against these antigens and protection against human experimental colonization. Though no correlation between naturally induced serum IgG antibodies against PspA and PspC and protection against colonization was observed, these results do not rule out the protective potential of these antigens as vaccines against pneumococcal infections.

Objectives: The management of women with cervical squamous intraepithelial lesions (SIL) is fundamental to prevention of cervical cancer in an organized cervical screening programme. Clinical guidance should improve quality of care and clinical effectiveness if developed and implemented appropriately. This survey provides an update on the current situation of national guidelines for management of cervical SIL amongst member countries of European Federation for Colposcopy (EFC). Study design: A questionnaire was sent to representatives of each member country of EFC. The questionnaire contained questions on: guidelines for management of cervical SIL of the National Societies/Associations of Colposcopy or others national societies/associations including the development and the consultation processes, guidelines for management of lower genital tract diseases and the regulations in each country for colposcopy practice. Results: We received responses from all 34 member countries. Thirty countries reported a national guideline for management of cervical SIL that were developed by, or in conjunction with, their national societies or associations of colposcopy. In most cases there was adherence to the recommended steps for guideline development: they were developed by a multi-disciplinary group of specialists (29 countries) and society members were consulted before publication (21 countries). A small number of countries (8) reported to have guidelines for the management of lower genital tract dysplasia (e.g. vulval disease) developed by other national societies. In most countries (26) the colposcopists are obliged to follow the guidelines but this is regulated in only 6 and in 12 countries the colposcopists need to be certified by the national society of colposcopy in order to practice. Conclusion: There are advances in the development and provision of country specific guidance on the management of cervical SIL. Most EFC member countries have appropriate national guidelines that were developed using a clear methodology, are updated according to progresses in the field and are accessible online to current practitioners. These guidelines support colposcopists to follow evidence-based practice and provides understanding of best practice in guideline development and access. (C) 2020 Elsevier B.V. All rights reserved.

Glial fibrillary acidic protein (GFAP) is cleared by the Food and Drug Administration (FDA) to determine need for head computed tomography (CT) within 12 h after mild traumatic brain injury (TBI) (Glasgow Coma Score [GCS] 13-15); S100 calcium-binding protein B (S100B) serves this function in Europe. This phase 1 biomarker cohort analysis of the multi-center, observational Transforming Research and Clinical Knowledge in Traumatic Brain Injury (TRACK-TBI) study compares GFAP's diagnostic performance, measured on a rapid point-of-care platform, against protein S100B to predict intracranial abnormalities on CT within 24 h post-injury across the spectrum of TBI (GCS 3-15). Head CT scan performed in TBI subjects and blood was collected for all consenting subjects presenting to 18 United States level 1 trauma centers. Plasma was analyzed on a point-of-care device prototype assay for GFAP and serum was analyzed for S100B. In 1359 patients with TBI (GCS 3-15), mean (standard deviation [SD]) age = 40.1 (17.0) years; 68% were male. Plasma GFAP levels were significantly higher in CT+ TBI subjects (median = 1358 pg/mL, interquartile range [IQR]: 472-3803) than in CT- TBI subjects (median = 116 pg/mL, IQR: 26-397) or orthopedic trauma controls (n = 122; median = 13 pg/mL, IQR: 7-20), p

BackgroundPneumococcal pneumonia is a leading cause of death, particularly affecting those with chronic respiratory disease. Observational studies suggest increased nasopharyngeal colonisation rates withS.pneumoniaein asthma, and lower specific antibody levels.ObjectivesUsing experimental human pneumococcal challenge, we examined the acquisition and kinetics of nasopharyngeal colonisation ofStreptococcus pneumoniaeserotype 6B. We also aimed to dissect associated mucosal and systemic immune responses and immunizing effect of carriage.MethodsFifty participants with asthma well-controlled on moderate inhaled corticosteroid doses were challenged with pneumococcus, and a subset of colonized individuals were re-challenged 6–11 months later with the same pneumococcal isolate. Colonisation rates (from nasal wash), systemic antibody levels and mucosal cellular and cytokine responses were compared to 151 healthy controls.Measurements and Main ResultsColonisation rates were 28/50 (56%) and 68/151 (45%) in those with asthma and controls respectively, p=0.17. Duration of colonisation was shorter in people with asthma (median 14 days vs 29 days, p=0.03) but of similar density. Body mass index was higher in colonised compared with non-colonised asthma individuals (median 24.7 [IQR 24.1–29.0] and 23.5 [20.1–26.4] respectively, p=0.019). Despite an increase in pneumococcal capsular and protein antibodies after colonisation, 4/12 asthmatic individuals became colonised again upon re-challenge. Nasal neutrophil and T cell levels, in particular mucosa associated invariant T (MAIT) cells were decreased in people with asthma compared to healthy controls (median 9.4, [IQR 5.0–13.3 %] of CD8+T cells) vs median 15.8, [IQR 9.9–25.9 %] of CD8+T cells respectively (p=0.0047). Most nasal cytokines were also reduced in asthmatics. In both groups, colonisation led to recruitment of monocytes and granulocytes to the nasal mucosa.ConclusionsNasopharyngeal colonisation was of shorter duration in those with asthma compared to controls, although acquisition rates were not different. Rates of colonisation were higher with increasing BMI in individuals with asthma. Despite a baseline reduction in mucosal immune cells and cytokines in asthmatics with corticosteroids, colonisation led to cellular recruitment in both groups. Colonisation was not associated with protection from homologous re-challenge in individuals with asthma, in contrast to healthy volunteers.Clinical Implication: (single sentence)People with asthma on inhaled corticosteroids have an increased likelihood of pneumococcal infection secondary to reduced mucosal immune responses from nasopharyngeal colonisation and a lack of protection from re-exposure.Capsule SummaryEpidemiological studies show that people with asthma are more likely to have nasal colonisation withS. pneumoniae, which may proceed to infection such as pneumonia and invasive pneumococcal disease. This study investigates the mechanisms underlying pneumococcal colonisaion and its effect on subsequent pneumococcal encounters.

Background Typhoid fever, caused by S. enterica ser. Typhi, continues to be a substantial health burden in developing countries. Little is known of the genotypic diversity of S. enterica ser. Typhi in Zimbabwe, but this is key for understanding the emergence and spread of this pathogen and devising interventions for its control. Objectives To report the molecular epidemiology of S. enterica ser. Typhi outbreak strains circulating from 2012 to 2019 in Zimbabwe, using comparative genomics. Methods A review of typhoid cases records from 2012 to 2019 in Zimbabwe was performed. The phylogenetic relationship of outbreak isolates from 2012 to 2019 and emergence of antibiotic resistance was investigated by whole-genome sequence analysis. Results A total 22 479 suspected typhoid cases, 760 confirmed cases were reported from 2012 to 2019 and 29 isolates were sequenced. The majority of the sequenced isolates were predicted to confer resistance to aminoglycosides, β-lactams, phenicols, sulphonamides, tetracycline and fluoroquinolones (including qnrS detection). The qnrS1 gene was associated with an IncN (subtype PST3) plasmid in 79% of the isolates. Whole-genome SNP analysis, SNP-based haplotyping and resistance determinant analysis showed that 93% of the isolates belonged to a single clade represented by multidrug-resistant H58 lineage I (4.3.1.1), with a maximum pair-wise distance of 22 SNPs. Conclusions This study has provided detailed genotypic characterization of the outbreak strain, identified as S. Typhi 4.3.1.1 (H58). The strain has reduced susceptibility to ciprofloxacin due to qnrS carried by an IncN (subtype PST3) plasmid resulting from ongoing evolution to full resistance.

Lesch-Nyhan disease (LND), caused by a deficient salvage purine pathway, is characterized by severe neurological manifestations and uric acid overproduction. However, uric acid is not responsible for brain dysfunction, and it has been suggested that purine nucleotide depletion, or accumulation of other toxic purine intermediates, could be more relevant. Here we show that purine alterations in LND fibroblasts depend on the level of folic acid in the culture media. Thus, physiological levels of folic acid induce accumulation of 5-aminoimidazole-4-carboxamide riboside 5'-monophosphate (ZMP), an intermediary of de novo purine biosynthetic pathway, and depletion of ATP. Additionally, Z-nucleotide derivatives (AICAr, AICA) are detected at high levels in the urine of patients with LND and its variants (hypoxanthine-guanine phosphoribosyltransferase [HGprt]-related neurological dysfunction and HGprt-related hyperuricemia), and the ratio of AICAr/AICA is significantly increased in patients with neurological problems (LND and HGprt-related neurological dysfunction). Moreover, AICAr is present in the cerebrospinal fluid of patients with LND, but not in control individuals. We hypothesize that purine alterations detected in LND fibroblasts may also occur in the brain of patients with LND.

SummaryColonization efficiency varies considerably between Streptococcus pneumoniae (pneumococcus) strains. The microbial characteristics that influence those differences are still largely unknown. Here, we report rates and kinetics of colonization of four pneumococcal strains upon experimental human pneumococcal challenge. Healthy adults were intranasally challenged with one of four pneumococcal strains (serotype/clonal name: 6B/BHN418, 15B/SH8286, 23F/P1121 and 23F/P833) over a range of doses. Maximum colonization achieved was 60%, 31%, 16% and 10%, respectively. Density and duration of colonization did not differ significantly between the tested strains. We further evaluated murine colonization, non-opsonic neutrophil mediated killing, epithelial cell adherence and average chain length of these four pneumococcal strains. Of these, only chain length was found to be associated with colonization efficiency in the human challenge model. Our data demonstrate that colonization rates following experimental challenge vary with the strain used and suggest that efficiency in colonization is related to pneumococcal chain length.

Plasma tau and glial fibrillary acidic protein (GFAP) are promising biomarkers for identifying traumatic brain injury (TBI) patients with intracranial trauma on computed tomography (CT). Accuracy in older adults with mild TBI (mTBI), the fastest growing TBI population, is unknown. Our aim was to assess for age-related differences in diagnostic accuracy of plasma tau and GFAP for identifying intracranial trauma on CT. Samples from 169 patients (age

The locus coeruleus (LC)-amygdala circuit is implicated in playing a key role in responses to emotionally arousing stimuli and in the manifestation of post-traumatic stress disorder (PTSD). Here, we examined changes in gene expression of a number of important mediators of the LC-amygdala circuitry in the inhibition avoidance model of PTSD. After testing for basal acoustic startle response (ASR), rats were exposed to a severe footshock (1.5 mA for 10 s) in the inhibitory avoidance apparatus. They were given contextual situational reminders every 5 day for 25 days. Controls were treated identically but with the footshock inactivated. Animals were re-tested on second ASR and decapitated 1 h later. The shock group had enhanced hyperarousal and several changes in gene expression compared to controls. In the LC, mRNA levels of norepinephrine (NE) biosynthetic enzymes (TH, DBH), NE transporter (NET), NPY receptors (Y1R, Y2R), and CB1 receptor of endocannabinoid system were elevated. In the basolateral amygdala (BLA), there were marked reductions in gene expression for CB1, and especially Y1R, with rise for corticotropin-releasing hormone (CRH) system (CRH, CRH receptor 1), and no significant changes in the central amygdala. Our results suggest a fast forward mechanism in the LC-amygdala circuitry in the shock group.

Amaranth (Amaranthus L.) species are grown for their grain or leaves and contribute to farmers’ livelihoods and nutritional food security. Leafy amaranth (LA) is consumed widely as a vegetable in Kenya. An assessment of current farmers’ knowledge of pest management practices provides information about future educational needs. Six-hundred LA farmers were interviewed, focus group discussions with farmers, and interviews with key informants were completed in four Kenyan counties. The majority (71%) of survey respondents grew LA on less than 0.25 acre (

ObjectivesTo identify differences in the mutational profile of endometrial tumours between British White (BW) and South Asian (BSA) women.MethodsWe analysed primary tumours from matched cohorts of British White (BW) and British South Asian (BSA) women resident in Leicestershire diagnosed with EC. Next Generation Sequencing was performed to investigate mutational differences in a panel of 10 genes previously identified as being commonly mutated in EC. The presence of somatic Mismatch Repair (MMR) gene deficiencies was determined by immunohistochemistry.ResultsIn total, 57 tumours (27 BSA and 30 BW) were sequenced. There was no significant difference in the overall mutation frequency of the 10 genes analysed; however, numerous differences were observed between the groups. There was a positive association between PIK3CA and PTEN mutations in the BSA group, with 78% of PIK3CA-mutant tumours harbouring a PTEN mutation, whereas only 11% of PIK3CA wild-type (wt) tumours were PTEN mutant positive (p = 0.0012). In BW women, 90% of ARID1A mutant tumours had co-existent PI3K pathway mutations versus 50% of wild-type (wt) ARID1A patients (p = 0.0485). This trend was not significant in the BSA group (p = 0.66). The age at diagnosis was significantly higher in the BW group with a somatic MMR gene deficiency compared to those with no deficiency (72.8 years versus 59.6 years, p = 0.007), whereas this difference was not seen in the BSA group (64 years versus 60 years, p = 0.37).ConclusionWe have identified differences in the mutational profile of primary EC tumours from BW and BSA women. Further research is needed to confirm these findings and to explore their potential implications for early detection, treatment response and prognosis.

The circadian clock relies on posttranslational modifications to set the timing for degradation of core regulatory components, which drives clock progression. Ubiquitin-modifying enzymes that target clock components for degradation mainly recognize phosphorylated substrates. Degradation of the circadian clock component PERIOD 2 (PER2) is mediated by its phospho-specific recognition by β-transducin repeat-containing proteins (β-TrCPs), which are F-box-containing proteins that function as substrate recognition subunits of the SCFβ-TRCP ubiquitin ligase complex. However, this mode of regulating PER2 stability falls short of explaining the persistent oscillatory phenotypes reported in biological systems lacking functional elements of the phospho-dependent PER2 degradation machinery. We identified PER2 as a previously uncharacterized substrate for the ubiquitin ligase mouse double minute 2 homolog (MDM2) and found that MDM2 targeted PER2 for degradation in a manner independent of PER2 phosphorylation. Deregulation of MDM2 plays a major role in oncogenesis by contributing to the accumulation of genomic and epigenomic alterations that favor tumor development. MDM2-mediated PER2 turnover was important for defining the circadian period length in mammalian cells, a finding that emphasizes the connection between the circadian clock and cancer. Our results not only broaden the range of specific substrates of MDM2 beyond the cell cycle to include circadian components but also identify a previously unknown regulator of the clock as a druggable node that is often found to be deregulated during tumorigenesis.

Summary Background Outbreak response efforts for the 2014–15 Ebola virus disease epidemic in west Africa brought widespread transmission to an end. However, subsequent clusters of infection have occurred in the region. An Ebola virus disease cluster in Liberia in November, 2015, that was identified after a 15-year-old boy tested positive for Ebola virus infection in Monrovia, raised the possibility of transmission from a persistently infected individual. Methods Case investigations were done to ascertain previous contact with cases of Ebola virus disease or infection with Ebola virus. Molecular investigations on blood samples explored a potential linkage between Ebola virus isolated from cases in this November, 2015, cluster and epidemiologically linked cases from the 2014–15 west African outbreak, according to the national case database. Findings The cluster investigated was the family of the index case (mother, father, three siblings). Ebola virus genomes assembled from two cases in the November, 2015, cluster, and an epidemiologically linked Ebola virus disease case in July, 2014, were phylogenetically related within the LB5 sublineage that circulated in Liberia starting around August, 2014. Partial genomes from two additional individuals, one from each cluster, were also consistent with placement in the LB5 sublineage. Sequencing data indicate infection with a lineage of the virus from a former transmission chain in the country. Based on serology and epidemiological and genomic data, the most plausible scenario is that a female case in the November, 2015, cluster survived Ebola virus disease in 2014, had viral persistence or recurrent disease, and transmitted the virus to three family members a year later. Interpretation Investigation of the source of infection for the November, 2015, cluster provides evidence of Ebola virus persistence and highlights the risk for outbreaks after interruption of active transmission. These findings underscore the need for focused prevention efforts among survivors and sustained capacity to rapidly detect and respond to new Ebola virus disease cases to prevent recurrence of a widespread outbreak. Funding US Centers for Disease Control and Prevention, Defense Threat Reduction Agency, and WHO.

BackgroundHuman papillomavirus (HPV) is a common sexually transmitted infection that is implicated in 99.7% of cervical cancers and several other cancers that affect both men and women. Despite the role that HPV plays in an estimated 5% of all cancers and the evolving role of HPV vaccination and testing in protecting the public against these cancers, preliminary research in New Zealand health care professionals suggest knowledge about HPV may not be sufficient.MethodsA total of 230 practice nurses, smear takers and other clinical and laboratory staff who attended a range of training events completed a cross-sectional survey between April 2016 and July 2017. The survey explored four broad areas: demographics and level of experience, HPV knowledge (general HPV knowledge, HPV triage and test of cure (TOC) knowledge and HPV vaccine knowledge), attitudes towards the HPV vaccine and self-perceived adequacy of HPV knowledge.ResultsThe mean score on the general HPV knowledge questions was 13.2 out of 15, with only 25.2% of respondents scoring 100%. In response to an additional question, 14.0% thought (or were unsure) that HPV causes HIV/AIDS. The mean score on the HPV Triage and TOC knowledge questions was 7.4 out of 10, with only 9.1% scoring 100%. The mean score on the HPV vaccine knowledge questions was 6.0 out of 7 and 44.3% scored 100%. Only 62.6% of respondents agreed or strongly agreed that they were adequately informed about HPV, although 71.8% agreed or strongly agreed that they could confidently answer HPV-related questions asked by patients. Multivariate analyses revealed that knowledge in each domain predicted confidence in responding to patient questions. Furthermore, the number of years since training predicted both HPV knowledge and Triage and TOC knowledge.DiscussionAlthough overall level of knowledge was adequate, there were significant gaps in knowledge, particularly about the role of HPV testing in the New Zealand National Cervical Screening Programme. More education is required to ensure that misinformation and stigma do not inadvertently result from interactions between health practitioners and the public.

Background The pathophysiological changes occurring in the trabecular meshwork in primary open angle glaucoma are poorly understood, but are thought to include increased extracellular matrix deposition, trabecular meshwork cell apoptosis, inflammation, trabecular meshwork calcification and altered protein composition of the aqueous humor. Although many proteins are present in aqueous humor, relatively few have been studied extensively, and their potential roles in primary open angle glaucoma are unknown. Methods Analyte concentrations in aqueous humor from 19 primary open angle glaucoma and 18 cataract patients were measured using a multiplex immunoassay. Fisher’s exact test was used to assess statistical significance between groups, and correlations of analyte concentrations with age, intraocular pressure, pattern standard deviation, mean deviation, cup-to-disc ratio and disease duration since commencing treatment were tested by Spearman’s method. Results CHI3L1, FLRG, HGF, MIF, P-selectin and Uteroglobin were detected in more than 50% of samples of one or both patient groups, some of which have not previously been quantified in aqueous humor. In the glaucoma but not the cataract group, significant correlations were determined with age for Uteroglobin/SCGB1A1 (rs = 0.805, p

It has long been believed that benign prostatic hyperplasia (BPH) and prostate cancer involve reawakening of the prostate glands embryonic growth potential, and reactivation of its developmental programmes. An understanding of the molecular details of these processes, in normal development and in disease, requires robust developmental models for the human system. Towards that goal, we describe the step-wise differentiation of prostatic organoids from human embryonic stem cells (hESCs) in a three-dimensional system. To generate prostate organoids, hESCs are driven into FOXA2 + /Brachyury + definitive endoderm and Brachyury + mesoderm by treatment with activin A for three days. The differentiated cells are then cultured with WNT10B and FGF10 for four consecutive days to direct them into prostatic fate determination. During this step, three-dimensional bud-like structures arise from the monolayer attached to the culture dish. These three-dimensional structures are patterned into prostate organoids by further culture in Matrigel for 20–30 days, with prostate-specific medium. The organoids derived from hESCs exhibit comparable architecture and functional properties to those found in the human prostate. So far, this is the only method for the directed differentiation of hESCs into human prostate tissues in vitro.

Current vaccines against Streptococcus pneumoniae, a bacterial species that afflicts people by causing a wide spectrum of diseases, do not protect against all pneumococcal serotypes. Thus, alternative vaccines to fight pneumococcal infections that target common proteins are under investigation. One promising strategy is to take advantage of immune cross-reactivity between commensal and pathogenic microbes for cross-protection. In this study, we examined the antibody-mediated cross-reactivity between S. pneumoniae and Streptococcus mitis, a commensal species closely related to S. pneumoniae. Western blot analysis showed that rabbit antisera raised against S. mitis reacted with multiple proteins of virulent S. pneumoniae strains (6B, TIGR4, and D39). Rabbit anti-S. pneumoniae IgG antibodies also showed binding to S. mitis antigens. Incubation of rabbit antisera raised against S. mitis with heterologous or homologous bacterial lysates resulted in marked inhibition of the developments of bands in the Western blots. Furthermore, plasma IgG antibodies from adult human volunteers intranasally inoculated with S. pneumoniae 6B revealed enhanced S. mitis-specific IgG titers compared with the pre-inoculation samples. Using an on-chip protein microarray representing a number of selected membrane and extracellular S. pneumoniae proteins, we identified choline-binding protein D (CbpD), cell division protein (FtsH), and manganese ABC transporter or manganese-binding adhesion lipoprotein (PsaA) as common targets of the rabbit IgG antibodies raised against S. mitis or S. pneumoniae. Cumulatively, these findings provide evidence on the antibody-mediated cross-reactivity of proteins from S. mitis and S. pneumoniae, which may have implications for development of effective and wide-range pneumococcal vaccines.

Major advances during the past decade have permitted a clearer understanding of processes that regulate stem cell self-renewal and lineage commitment toward differentiated progeny that populate all tissues. Considerable evidence has also accumulated to indicate that aberrations in the stem and progenitor cell populations can lead to increased cancer risk in specific organs systems. It is long recognized that environmental factors play a major role in cancer etiology, and emerging data suggest that endocrine-disrupting chemicals (EDCs) may contribute to an increased cancer risk. Using the prostate gland as a model system, the present review highlights recent data that find that estrogens and EDCs can reprogram prostate stem and progenitor cell populations, leading to increased cancer susceptibility. We propose that stem cell programming during early development in hormone-regulated tissues may lead to heightened sensitivity to early-life EDC exposures and that aberrant stem cell reprogramming by EDCs may contribute to the developmental basis of adult cancer risk.

INTRODUCTION: Hand, foot, and mouth disease continues to cause seasonal epidemics in the Asia-Pacific Region. Since the current Enterovirus 71 vaccines do not provide cross-protection for all Enterovirus species that cause hand, foot, and mouth disease, there is an urgent need to identify appropriate detection tools and best practice to prevent its transmission and to effectively control its outbreaks. This systematic review aimed to identify characteristics of outbreak and assess the impact and effectiveness of detection tools and public health preventive measures to interrupt transmission. The findings will be used to recommend policy on the most effective responses and interventions in Hong Kong to effectively minimise and contain the spread of the disease within childcare facilities. METHODS: We searched the following databases for primary studies written in Chinese or English: MEDLINE, EMBASE, Global Health, WHO Western Pacific Region Index Medicus database, China National Knowledge Infrastructure Databases, and Chinese Scientific Journals Database. Studies conducted during or retrospective to outbreaks of hand, foot, and mouth disease caused by Enterovirus 71 from 1980 to 2012 within childcare facilities and with a study population of 0 to 6 years old were included. RESULTS: Sixteen studies conducted on outbreaks in China showed that hand, foot, and mouth disease spread rapidly within the facility, with an outbreak length of 4 to 46 days, especially in those with delayed notification (after 24 hours) of clustered outbreak (with five or more cases discovered within the facility) to the local Center for Disease Control and Prevention and delayed implementation of a control response. The number of classes affected ranged from 1 to 13, and the attack rate for children ranged from 0.97% to 28.18%. CONCLUSIONS: Communication between key stakeholders about outbreak confirmation, risk assessment, and surveillance should be improved. Effective communication facilitates timely notification (within 24 hours) of clustered outbreaks to a local Center for Disease Control and Prevention. Timely implementation of a control response is effective in minimising incidence and length of an outbreak in childcare facilities. The government should provide incentives for childcare facilities to train infection control specialists who can serve as the first contact, knowledge, and communication points, as well as facilitate exchange of information and provision of support across stakeholders during a communicable disease epidemic.

Biomarkers are important for accurate diagnosis of complex disorders such as traumatic brain injury (TBI). For a complex and multifaceted condition such as TBI, it is likely that a single biomarker will not reflect the full spectrum of the response of brain tissue to injury. Ubiquitin C-terminal hydrolase L1 (UCH-L1) and glial fibrillary acidic protein (GFAP) are among of the most widely studied biomarkers for TBI. Because UCH-L1 and GFAP measure distinct molecular events, we hypothesized that analysis of both biomarkers would be superior to analysis of each alone for the diagnosis and prognosis of TBI. Serum levels of UCH-L1 and GFAP were measured in a cohort of 206 patients with TBI enrolled in a multicenter observational study (Transforming Research and Clinical Knowledge in Traumatic Brain Injury [TRACK-TBI]). Levels of the two biomarkers were weakly correlated to each other (r=0.364). Each biomarker in isolation had good sensitivity and sensitivity for discriminating between TBI patients and healthy controls (area under the curve [AUC] 0.87 and 0.91 for UCH-L1 and GFAP, respectively). When biomarkers were combined, superior sensitivity and specificity for diagnosing TBI was obtained (AUC 0.94). Both biomarkers discriminated between TBI patients with intracranial lesions on CT scan and those without such lesions, but GFAP measures were significantly more sensitive and specific (AUC 0.88 vs. 0.71 for UCH-L1). For association with outcome 3 months after injury, neither biomarker had adequate sensitivity and specificity (AUC 0.65-0.74, for GFAP, and 0.59-0.80 for UCH-L1, depending upon Glasgow Outcome Scale Extended [GOS-E] threshold used). Our results support a role for multiple biomarker measurements in TBI research. (ClinicalTrials.gov Identifier NCT01565551)

Fracture healing in animal models has been shown to be altered in both ground based analogs of spaceflight and in those exposed to actual spaceflight. The molecular mechanisms behind altered fracture healing as a result of chronic exposure to microgravity remain to be elucidated. This study investigates temporal gene expression of multiple factors involved in secondary fracture healing, specifically those integral to the development of a soft tissue callus and the transition to that of hard tissue. Skeletally mature female rats were subjected to a 4 week period of simulated microgravity and then underwent a closed femoral fracture procedure. Thereafter, they were reintroduced to the microgravity and allowed to heal for a 1 or 2 week period. A synchronous group of weight bearing rats was used as a normal fracture healing control. Utilizing Real-Time quantitative PCR on mRNA from fracture callus tissue, we found significant reductions in the levels of transcripts associated with angiogenesis, chondrogenesis, and osteogenesis. These data suggest an altered fracture healing process in a simulated microgravity environment, and these alterations begin early in the healing process. These findings may provide mechanistic insight towards developing countermeasure protocols to mitigate these adaptations.

The functional consequences of nest size variation in cavity nesting birds have been thoroughly investigated in recent years. However, it is unknown whether recent nest-building experience influences subsequent nest architecture. By providing Eastern Bluebirds (Sialia sialis) with either deep or shallow nest boxes for their initial nest of the breeding season, we tested whether the height of their subsequent nest in the same breeding season reflected the height of their initial nest or whether they built nests to match their current nest cavity regardless of their previous nest height. We found that second nests built in deep boxes were significantly taller than first nests built in shallow boxes by the same females. This difference could be because of nest date, cavity depth, or experience. We tested each possible driver and found that cavity depth better explains variation in nest height than either nest date or prior experience. We conclude that, for this multi-brooded secondary cavity nesting...

We compared House Wren (Troglodytes aedon) nests in nest boxes with either small (28 mm) or large (38 mm) entrance holes. Spring clutch size did not differ between wrens using boxes with large and small holes. Nests in boxes with large holes had a significantly taller ‘berm’ of sticks between the box entrance and the nest cup than did nests in boxes with small holes. This was true even when the same wren pairs were given boxes with different-size holes for spring and summer nests. We compared the difficulty of touching the eggs when we removed the frontplate of the nest box as an alternative measure of nest vulnerability to predators and brood parasites. There was a significant association between hole size and accessibility: boxes without the protection of a small entrance hole were far more likely to be categorized as ‘inaccessible.’ We found a similar relationship between berm height and nest architecture: nests with tall berms were generally less accessible than were nests with shorter berms....

The corticotropin-releasing hormone (CRH) family regulates the endocrine stress response. Here, we examined the effect of immobilization stress (IMO) on gene expression of adrenomedullary CRH family members. Urocortin 2 (Ucn2) has the highest basal gene expression and is increased by > 30-fold in response to single IMO and about 10-fold after six daily repeated IMO. IMO also induced a smaller rise in CRH (six-fold) and CRH receptor type 1 (CRHR1; two-fold with single IMO). The influence of glucocorticoids was examined. Dexamethasone (DEX) or corticosterone greatly increased Ucn2 mRNA levels in PC12 cells in a dose-dependent manner. The DEX elicited rise in Ucn2 was abolished by actinomycin D pre-treatment, indicating a transcriptionally mediated response. DEX also triggered a rise in CRHR1 and lowered CRH mRNA levels. In CRH-knockout mice, where the IMO-induced rise in corticosterone was attenuated, the response of IMO on Ucn2, as well as CRHR2 mRNAs was absent. Overall, the results suggest that the stress-triggered rise in glucocorticoids is involved in the large induction of Ucn2 mRNA levels by IMO, which may allow Ucn2 to act in an autocrine/paracrine fashion to modulate adrenomedullary function, or act as an endocrine hormone.

Reliable diagnosis of traumatic brain injury (TBI) is a major public health need. Glial fibrillary acidic protein (GFAP) is expressed in the central nervous system, and breakdown products (GFAP-BDP) are released following parenchymal brain injury. Here, we evaluate the diagnostic accuracy of elevated levels of plasma GFAP-BDP in TBI. Participants were identified as part of the prospective Transforming Research And Clinical Knowledge in Traumatic Brain Injury (TRACK-TBI) Study. Acute plasma samples (

Background\ud The incorporation of Human papillomavirus (HPV) testing into the English cervical screening programme has been met with fear and anxiety. Healthcare professionals need to be adequately informed about HPV to help alleviate patient concerns.\ud \ud The aim of this study was to evaluate the HPV training provided to practice nurses (PNs) and determine their level of HPV knowledge.\ud \ud Method\ud A web-based survey was distributed to 147 General Practice surgeries in the Leicester, Leicestershire and Rutland regions, between May and July 2015. The survey explored four broad areas; demographics/level of experience, HPV knowledge, attitudes towards the HPV vaccine and self-perceived adequacy of HPV knowledge.\ud \ud Results\ud A total of 128 surveys were completed, with 94 complete responses. Overall awareness of basic HPV facts was adequate; however, detailed, and in some cases basic, knowledge was lacking. 9.6% failed to identify that HPV can cause cervical cancer and 62.8% believed that HPV requires treatment. Not all PNs felt adequately informed about HPV and a need to improve the provision of training was identified.\ud \ud Conclusion\ud PNs play a key role in increasing public awareness of HPV and implementing cervical cancer screening. The provision of education to PNs needs to be a priority and current methods of training need to be re-evaluated.

We described recently a subacute serum autoantibody response toward glial fibrillary acidic protein (GFAP) and its breakdown products 5-10 days after severe traumatic brain injury (TBI). Here, we expanded our anti-GFAP autoantibody (AutoAb[GFAP]) investigation to the multicenter observational study Transforming Research and Clinical Knowledge in TBI Pilot (TRACK-TBI Pilot) to cover the full spectrum of TBI (Glasgow Coma Scale 3-15) by using acute (

Brain-derived neurotrophic factor (BDNF) is important for neuronal survival and regeneration. We investigated the diagnostic and prognostic values of serum BDNF in traumatic brain injury (TBI). We examined serum BDNF in two independent cohorts of TBI cases presenting to the emergency departments (EDs) of the Johns Hopkins Hospital (JHH; n = 76) and San Francisco General Hospital (SFGH, n = 80), and a control group of JHH ED patients without TBI (n = 150). Findings were subsequently validated in the prospective, multi-center Transforming Research and Clinical Knowledge in TBI (TRACK-TBI) Pilot study (n = 159). We investigated the association between BDNF, glial fibrillary acidic protein (GFAP), and ubiquitin C-terminal hydrolase-L1 (UCH-L1) and recovery from TBI at 6 months in the TRACK-TBI Pilot cohort. Incomplete recovery was defined as having either post-concussive syndrome or a Glasgow Outcome Scale Extended score

The purpose of this study was to evaluate microarray technology of HNSCC cells in muscle tissue. 200 SCCVII tumor cells were injected intramuscularly into the right flank of ten C3H/Km mice each. One week later the animals were killed and the tissue taken out. Histology (H&E staining) and microarray of the tissue were performed. Histology showed a few tumor cells between the muscle fibers. Microarray technology showed different gene expression pattern of the muscle tissue with SCCVII cells in comparison with normal muscle tissue. Only those genes showing a fold change difference of 5 or higher were considered. Gene expression analysis revealed changes in the expression levels of SCCVII cells in muscle tissue in 220 genes. Significant gene expression differences between SCCVII cells in muscle tissue and pure muscle tissue could be seen.

Introduction. To investigate the indications for performing a second large loop excision of the transformation zone (LLETZ) biopsy and to compare the associated colposcopic and pathological findings and treatment morbidity compared with 1 LLETZ. Methods. This is a case-control study that reviewed case notes and histology reports on women who had undergone 1 LLETZ biopsy (control group) and women who had undergone 2 biopsies (index group). A comparison of referral cytology, colposcopic findings, and pathological and clinical outcomes was performed. Results. Of the women who went on to have 2 LLETZ biopsies, 88% had histologically proven high-grade cervical intraepithelial neoplasia (CIN) or invasion on their first biopsy. A significantly greater proportion of high-grade cytologic and histologic diagnoses were associated with the first LLETZ compared with the second LLETZ biopsy, 76.5% and 69.1% versus 39.5% and 30.9%, respectively. A significantly greater proportion of women in the control group were referred with low-grade cytology (28.0%) and were diagnosed with human papillomavirus/low-grade CIN on histology (31.7%) compared with the first cytologic and LLETZ results in the index group, 9.9% and 8.6%, respectively. Complications were low in both groups; the immediate complication rate was 4% after the first LLETZ compared with 1% after the second LLETZ. Conclusions. Most second LLETZ biopsies are performed in women with a history of biopsy-proven high-grade CIN and are not associated with an increased risk of immediate complications.

Experiments performed in vivo and in cell culture have demonstrated that oestradiol induces dopamine β-hydroxylase (DBH) gene transcription. In the present study, we examined oestrogen-responsive elements of the rat DBH gene promoter aiming to characterise the mechanisms of oestradiol-induced DBH transcription. Various mutations and deletions of DBH promoter reporter constructs were tested for responsiveness to 17β-oestradiol (E(2) ). Mutation of the half palindromic oestrogen response element (ERE) at position -759 reduced the response to E(2) in PC12 cells co-transfected with oestrogen receptor (ER) α, indicating a functional role for this motif. In cells co-transfected with ERβ, mutations at the -759 site were unresponsive to E(2) . To characterise the additional E(2) responsive elements, mediated by ERα, the DBH promoter was truncated to the proximal 249 or 200 nucleotides upstream of the transcription start site. Despite either truncation, 10 nm E(2) still elicited an approximately two-fold induction of DBH promoter activity. Mutation of a possible ERE-like sequence at -59 had no effect. The lack of a functional ERE in the proximal region of the rat DBH promoter despite E(2) -mediated DBH promoter activity, suggests regulation by a nonclassical mechanism, such as a membrane-initiated signalling pathway. Moreover, the induction of DBH promoter activity and the rise in DBH mRNA levels were observed within hours. To determine whether membrane-initiated E(2) signalling is involved in rat DBH gene transcription, a membrane impermeable E(2) conjugate, β-oestradiol-6-(O-carboxy-methyl) oxime-bovine serum albumin (E(2) BSA), was used. Incubation with E(2) -BSA induced luciferase activity and elicited a significant rise in DBH mRNA levels in the ERα transfected cells. The findings indicate two different mechanisms whereby DBH transcription is regulated by E(2) in the presence of ERα. The results implicate both genomic and membrane-initiated mechanisms, mediated by ERα, in E(2) -induced DBH gene transcription.

Vesicular monoamine transporters (VMATs) mediate transmitter uptake into neurosecretory vesicles. There are two VMAT isoforms, VMAT1 and VMAT2, encoded by separate genes and displaying different cellular distributions and pharmacological properties. We examined the effect of immobilization stress (IMO) on expression of VMATs in the rat adrenal medulla. Under basal conditions, VMAT1 is widely expressed in all adrenal chromaffin cells, while VMAT2 is co-localized with tyrosine hydroxylase (TH) but not phenylethanolamine N-methyltransferase (PNMT), indicating its expression in norepinephrine (NE)-, but not epinephrine (Epi)-synthesizing chromaffin cells. After exposure to IMO, there was no change in levels of VMAT1 mRNA. However, VMAT2 mRNA was elevated after exposure of rats to 2 h IMO once (1× IMO) or daily for 6 days (6× IMO). The changes in VMAT2 mRNA were reflected by increased VMAT2 protein after the repeated IMO. Immunofluorescence revealed an increased number of cells expressing VMAT2 following repeated IMO and its colocalization with PNMT in many chromaffin cells. The findings suggest an adaptive mechanism in chromaffin cells whereby enhanced catecholamine storage capacity facilitates more efficient utilization of the well-characterized heightened catecholamine biosynthesis with repeated IMO stress.

Objective Selection of an appropriate diet for rodent spaceflight experiments is critical and may have significant effects on mission results. The National Aeronautics and Space Administration (NASA) rodent food bar (RFB) was reformulated and designated as the nutrient-upgraded RFB (NuRFB). The objectives of this study were to determine whether the NuRFB nutrient formulation meets the 1995 National Research Council (NRC) nutrient recommendations and whether the NuRFB can be used for short-term (45-d) and long-term (90-d) spaceflight experiments. Methods Nutrient and moisture analyses of the NuRFB were performed. Young (age 13–14 wk) male Sprague-Dawley rats ( n = 16/group) were individually caged and fed a diet treatment consisting of 1) NuRFB, 2) RFB, or 3) modified AIN-93 G containing 4% instead of the 7% fat for 45- or 90-d. At the end of the study, organs were weighted, and serum clinical chemistry indicators of organ function and hematologic measurements were determined. Results Chemical analysis of the diet ingredients showed that the NuRFB met the 1995 NRC nutrient recommendations for rats. Subsequent animal feeding studies showed that NuRFB was comparable to RFB and modified AIN-93 G for supporting rat growth and body weight maintenance. In addition, the safety of the NuRFB for use as a spaceflight diet was indicated by the absence of changes in organ weight or function. Conclusion Based on the study results, the NuRFB performed similarly to the RFB and met the criteria necessary for short-term and long-term rodent spaceflight experiments.

The PAFAH 1b complex links phospholipid remodeling and membrane tubulation within the Golgi to dynein-dependent transport., We report that platelet-activating factor acetylhydrolase (PAFAH) Ib, comprised of two phospholipase A2 (PLA2) subunits, α1 and α2, and a third subunit, the dynein regulator lissencephaly 1 (LIS1), mediates the structure and function of the Golgi complex. Both α1 and α2 partially localize on Golgi membranes, and purified catalytically active, but not inactive α1 and α2 induce Golgi membrane tubule formation in a reconstitution system. Overexpression of wild-type or mutant α1 or α2 revealed that both PLA2 activity and LIS1 are important for maintaining Golgi structure. Knockdown of PAFAH Ib subunits fragments the Golgi complex, inhibits tubule-mediated reassembly of intact Golgi ribbons, and slows secretion of cargo. Our results demonstrate a cooperative interplay between the PLA2 activity of α1 and α2 with LIS1 to facilitate the functional organization of the Golgi complex, thereby suggesting a model that links phospholipid remodeling and membrane tubulation to dynein-dependent transport.

Corticotropin-releasing factor is well known activator of the hypothalamic-pituitary-adrenocortical axis, that represents crucial system participating on stress response of the organism. Urocortins are members of the corticotropin-releasing factor family of peptides with proposed effects on neuroendocrine and behavioral stress response mechanisms. Urocortin 2, one of three known urocortins, is present in central and peripheral stress response system and its expression can be augmented by glucocorticoids. In the present study we have examined how glucocorticoid withdrawal affects urocortin 2 gene expression after acute immobilization in the adrenal medulla and selected brain areas in rats. We used pharmacological adrenalectomy to block synthesis of corticosterone. Our results show that the immobilization-induced rise in urocortin 2 mRNA levels in rat adrenal medulla was not inhibited by glucocorticoid withdrawal. On the other hand, observed changes in the brain indicate that the effect of stress and pharmacological adrenalectomy on urocortin 2 gene expression is site-specific. While in the paraventricular nucleus and locus coeruleus the immobilization induced rise of urocortin 2 was not inhibited by pharmacological adrenalectomy in the arcuate nucleus and central amygdala it was. Moreover, we have seen a significant depletion of urocortin 2 plasma levels after immobilization. The immobilization induced rise of urocortin 2 gene expression in rat adrenal medulla and brain areas regulating stress response pathways and preservation of its induction after adrenalectomy suggests a role of urocortin 2 in the neuroendocrine stress response of an organism. This article is protected by copyright. All rights reserved.

The development of chemotherapy resistance by cancer cells is complex, using different mechanisms and pathways. The gene FAU (Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV)-associated ubiquitously expressed gene) was identified through functional expression cloning and previous data have shown that overexpression enhances apoptosis in several cell types. We demonstrate that the expression of FAU was reduced in the A2780cis (cisplatin resistant subclone of A2780) cell line compared with the A2780 ovarian cancer cell line, and was directly related to the cell line's sensitivity to carboplatin. Downregulation of FAU in the A2780 cell line by transfection with two predesigned short-interfering RNAs (siRNAs) to FAU resulted in a significant increase in resistance to carboplatin-induced cell death. Downregulation resulted in increased cell viability and reduced apoptosis after 72 hr of drug treatment compared with the negative controls (Kruskal-Wallis P = 0.0002). Transfection of the A2780cis cell line with the pcDNA3 plasmid containing FAU was associated with increased sensitivity to carboplatin-induced apoptosis, with decreased cell viability and increased apoptosis (Mann Whitney P < 0.0001). The expression of FAU was examined by quantitative real-time reverse transcriptase polymerase chain reaction in normal and malignant ovarian tissue. A significant reduction in the expression of FAU was seen in the malignant compared with normal ovarian samples (Kruskal-Wallis P = 0.0261). These data support a role for FAU in the regulation of platinum-resistance in ovarian cancer. Further research is needed into the apoptotic pathway containing FAU to investigate the potential for targeted therapies to increase or restore the platinum sensitivity of ovarian cancer.