Different electrophysiological profiles of genetically labelled dopaminergic neurons in the mouse midbrain and olfactory bulb.

Dopaminergic (DA) neurons play pivotal roles in diverse brain functions, spanning movement, reward processing and sensory perception. DA neurons are most abundant in the midbrain (Substantia Nigra pars compacta [SNC] and Ventral Tegmental Area [VTA]) and the olfactory bulb (OB) in the forebrain. Interestingly, a subtype of OB DA neurons is capable of regenerating throughout life, while a second class is exclusively born during embryonic development. Compelling evidence in SNC and VTA also indicates substantial heterogeneity in terms of morphology, connectivity and function. To further investigate this heterogeneity and directly compare form and function of midbrain and forebrain bulbar DA neurons, we performed immunohistochemistry and whole-cell patch-clamp recordings in ex vivo brain slices from juvenile DAT-tdTomato mice. After confirming the penetrance and specificity of the dopamine transporter (DAT) Cre line, we compared soma shape, passive membrane properties, voltage sags and action potential (AP) firing across midbrain and forebrain bulbar DA subtypes. We found that each DA subgroup within midbrain and OB was highly heterogeneous, and that DA neurons across the two brain areas are also substantially different. These findings complement previous work in rats as well as gene expression and in vivo datasets, further questioning the existence of a single "dopaminergic" neuronal phenotype.

Barriers and facilitators for people with severe mental illness accessing cancer screening: A systematic review.

OBJECTIVE: Evidence suggests that people with severe mental illness (PwSMI) are 2.1 times more likely to die from cancer before the age of 75, compared to people without Severe mental illness (SMI). Yet, cancer screening uptake is low among PwSMI. This mixed-methods systematic review aimed to identify the barriers and facilitators for PwSMI deciding to access and attend primary cancer screening of the cervix, breast and colon. METHODS: Six electronic databases and two grey literature sources were searched, with 1017 records screened against inclusion criteria. Included papers were appraised and data synthesised using the constructs of Normalisation Process Theory. RESULTS: Twenty papers met the inclusion criteria. Factors that impact upon uptake of PwSMI accessing cancer screening were found to include age, gender, race, and income. Common barriers to attending screening included poor communication from healthcare staff, stigmatising attitudes, and accessibility problems such as no access to transportation. While, facilitators included social support from friends, family, and healthcare providers. CONCLUSIONS: Due to ease and privacy, colorectal screening was found to have fewer barriers when compared to cervical and breast screening. The review identified multiple barriers that can be addressed and targeted to support decision-making for cancer screening among PwSMI. The protocol was registered with PROSPERO (CRD42022331781).

Normalisation and equity of referral to the NHS Low Calorie Diet programme pilot; a qualitative evaluation of the experiences of health care staff.

BACKGROUND: Health and wellbeing can be profoundly impacted by both obesity and type 2 diabetes, while the normalisation and equity of care for people living with these non-communicable diseases remain as challenges for local health systems. The National Health Service Low Calorie Diet programme in England, aims to support people to achieve type 2 diabetes remission, while also reducing health inequalities. We have explored the experiences of health care staff who have made a referral to the LCD programme, while identifying effective and equitable delivery of programme referrals, and their normalisation into routine care. METHODS: Nineteen individual semi-structured interviews were completed health care staff in the first year of the Low Calorie Diet programme. Interviewees were purposively sampled from the ten localities who undertook the Low Calorie Diet programme pilot. Each interview explored a number of topics of interest including communication and training, referrals, equity, and demands on primary care, before being subjected to a thematic analysis. RESULTS: From the data, five core themes were identified: Covid-19 and the demands on primary care, the expertise and knowledge of referrers, patient identification and the referral process, barriers to referrals and who gets referred to the NHS LCD programme. Our findings demonstrate the variation in the real world settings of a national diabetes programme. It highlights the challenge of COVID-19 for health care staff, whereby the increased workload of referrals occurred at a time when capacity was curtailed. We have also identified several barriers to referral and have shown that referrals had not yet been normalised into routine care at the point of data collection. We also raise issues of equity in the referral process, as not all eligible people are informed about the programme. CONCLUSIONS: Referral generation had not yet been consistently normalised into routine care, yet our findings suggest that the LCD programme runs the risk of normalising an inequitable referral process. Inequalities remain a significant challenge, and the adoption of an equitable referral process, normalised at a service delivery level, has the capacity to contribute to the improvement of health inequalities.

Commercial provider staff experiences of the NHS low calorie diet programme pilot: a qualitative exploration of key barriers and facilitators.

BACKGROUND: The National Health Service Type 2 Diabetes Path to Remission programme in England (known as the NHS Low Calorie Diet programme when piloted) was established to support people living with excess weight and Type 2 Diabetes to lose weight and improve their glycaemic control. A mixed method evaluation was commissioned to provide an enhanced understanding of the long-term cost effectiveness of the pilot programme, its implementation, equity and transferability across broad and diverse populations. This study provided key insights on implementation and equity from the service providers' perspective. METHODS: Thirteen focus groups were conducted with commercial providers of the programme, during the initial pilot rollout. Participants were purposively sampled across all provider organisations and staff roles involved in implementing and delivering the programme. Normalisation Process Theory (NPT) was used to design the topic schedule, with the addition of topics on equity and person-centredness. Data were thematically analysed using NPT constructs with additional inductively created codes. Codes were summarised, and analytical themes generated. RESULTS: The programme was found to fulfil the requirements for normalisation from the providers' perspective. However, barriers were identified in engaging GP practices and receiving sufficient referrals, as well as supporting service users through challenges to remain compliant. There was variation in communication and training between provider sites. Areas for learning and improvement included adapting systems and processes and closing the gap where needs of service users are not fully met. CONCLUSIONS: The evaluation of the pilot programme demonstrated that it was workable when supported by effective primary care engagement, comprehensive training, and effective internal and external communication. However, limitations were identified in relation to programme specifications e.g. eligibility criteria, service specification and local commissioning decisions e.g. pattern of roll out, incentivisation of general practice. A person-centred approach to care is fundamental and should include cultural adaptation(s), and the assessment and signposting to additional support and services where required.

Age and an obesogenic diet affect mouse behaviour in a sex-dependent manner.

Obesity is rising globally and is associated with neurodevelopmental and psychiatric disorders among children, adolescents and young adults. Whether obesity is the cause or the consequence of these disorders remains unclear. To examine the behavioural effects of obesity systematically, locomotion, anxiety and social behaviour were assessed in male and female C57Bl/6J mice using the open field, elevated plus maze and social preference task. First, the effects of age and sex were examined in control mice, before investigating post-weaning consumption of a high fat-high sugar diet commonly consumed in human populations with high rates of obesity. In the open field and elevated plus maze, locomotor activity and anxiety-related behaviours reduced with aging in both sexes, but with different sex-specific profiles. The high fat-high sugar diet reduced food and calorie intake and increased body mass and fat deposition in both sexes. In the open field, both male and female mice on the obesogenic diet showed reduced locomotion; whereas, in the elevated plus maze, only females fed with the obesogenic diet displayed reduced anxiety-related behaviours. Both male and female mice on the obesogenic diet had a significantly higher social preference index than the control group. In conclusion, the findings demonstrate that the behavioural effects of age and diet-induced obesity all depend on the sex of the mouse. This emphasises the importance of considering the age of the animal and including both sexes when assessing behavioural phenotypes arising from dietary manipulations.

Emollients for preventing atopic eczema: Cost-effectiveness analysis of the BEEP trial.

BACKGROUND: Recent discoveries have led to the suggestion that enhancing skin barrier from birth might prevent eczema and food allergy. OBJECTIVE: To determine the cost-effectiveness of daily all-over-body application of emollient during the first year of life for preventing atopic eczema in high-risk children at 2 years from a health service perspective. We also considered a 5-year time horizon as a sensitivity analysis. METHODS: A within-trial economic evaluation using data on health resource use and quality of life captured as part of the BEEP trial alongside the trial data. Parents/carers of 1394 infants born to families at high risk of atopic disease were randomised 1:1 to the emollient group, which were advised to apply emollient (Doublebase Gel or Diprobase Cream) to their child at least once daily to the whole body during the first year of life or usual care. Both groups received advice on general skin care. The main economic outcomes were incremental cost-effectiveness ratio (ICER), defined as incremental cost per percentage decrease in risk of eczema in the primary cost-effectiveness analysis. Secondary analysis, undertaken as a cost-utility analysis, reports incremental cost per Quality-Adjusted Life Year (QALY) where child utility was elicited using the proxy CHU-9D at 2 years. RESULTS: At 2 years, the adjusted incremental cost was £87.45 (95% CI -54.31, 229.27) per participant, whilst the adjusted proportion without eczema was 0.0164 (95% CI -0.0329, 0.0656). The ICER was £5337 per percentage decrease in risk of eczema. Adjusted incremental QALYs were very slightly improved in the emollient group, 0.0010 (95% CI -0.0069, 0.0089). At 5 years, adjusted incremental costs were lower for the emollient group, -£106.89 (95% CI -354.66, 140.88) and the proportion without eczema was -0.0329 (95% CI -0.0659, 0.0002). The 5-year ICER was £3201 per percentage decrease in risk of eczema. However, when inpatient costs due to wheezing were excluded, incremental costs were lower and incremental effects greater in the usual care group. CONCLUSIONS: In line with effectiveness endpoints, advice given in the BEEP trial to apply daily emollient during infancy for eczema prevention in high-risk children does not appear cost-effective.

Emollients for prevention of atopic dermatitis: 5-year findings from the BEEP randomized trial.

BACKGROUND: The effectiveness of emollients for preventing atopic dermatitis/eczema is controversial. The Barrier Enhancement for Eczema Prevention trial evaluated the effects of daily emollients during the first year of life on atopic dermatitis and atopic conditions to age 5 years. METHODS: 1394 term infants with a family history of atopic disease were randomized (1:1) to daily emollient plus standard skin-care advice (693 emollient group) or standard skin-care advice alone (701 controls). Long-term follow-up at ages 3, 4 and 5 years was via parental questionnaires. Main outcomes were parental report of a clinical diagnosis of atopic dermatitis and food allergy. RESULTS: Parents reported more frequent moisturizer application in the emollient group through to 5 years. A clinical diagnosis of atopic dermatitis between 12 and 60 months was reported for 188/608 (31%) in the emollient group and 178/631 (28%) in the control group (adjusted relative risk 1.10, 95% confidence interval 0.93 to 1.30). Although more parents in the emollient group reported food reactions in the previous year at 3 and 4 years, cumulative incidence of doctor-diagnosed food allergy by 5 years was similar between groups (92/609 [15%] emollients and 87/632 [14%] controls, adjusted relative risk 1.11, 95% confidence interval 0.84 to 1.45). Findings were similar for cumulative incidence of asthma and hay fever. CONCLUSIONS: Daily emollient application during the first year of life does not prevent atopic dermatitis, food allergy, asthma or hay fever.

Polygenic risk scores and the need for pharmacotherapy in neonatal abstinence syndrome.

BACKGROUND: The aim of this study was to identify genetic variants associated with NAS through a genome-wide association study (GWAS) and estimate a Polygenic Risk Score (PRS) model for NAS. METHODS: A prospective case-control study included 476 in utero opioid-exposed term neonates. A GWAS of 1000 genomes-imputed genotypes was performed to identify variants associated with need for pharmacotherapy for NAS. PRS models for estimating genetic predisposition were generated via a nested cross-validation approach using 382 neonates of European ancestry. PRS predictive ability, discrimination, and calibration were assessed. RESULTS: Cross-ancestry GWAS identified one intergenic locus on chromosome 7 downstream of SNX13 exhibiting genome-wide association with need for pharmacotherapy. PRS models derived from the GWAS for a subset of the European ancestry neonates reliably discriminated between need for pharmacotherapy using cis variant effect sizes within validation sets of European and African American ancestry neonates. PRS were less effective when applying variant effect sizes across datasets and in calibration analyses. CONCLUSIONS: GWAS has the potential to identify genetic loci associated with need for pharmacotherapy for NAS and enable development of clinically predictive PRS models. Larger GWAS with additional ancestries are needed to confirm the observed SNX13 association and the accuracy of PRS in NAS risk prediction models. IMPACT: Genetic associations appear to be important in neonatal abstinence syndrome. This is the first genome-wide association in neonates with neonatal abstinence syndrome. Polygenic risk scores can be developed examining single-nucleotide polymorphisms across the entire genome. Polygenic risk scores were higher in neonates receiving pharmacotherapy for treatment of their neonatal abstinence syndrome. Future studies with larger cohorts are needed to better delineate these genetic associations.

Acute Kidney Injury and Abnormalities on Brain Magnetic Resonance Imaging or Death in Infants with Hypoxic-Ischemic Encephalopathy: A Case-Control Study.

OBJECTIVE: This study aimed to analyze the association between acute kidney injury (AKI) and abnormalities on brain magnetic resonance imaging (MRI) or death in neonates treated with therapeutic hypothermia for hypoxic-ischemic encephalopathy (HIE). STUDY DESIGN: This is a retrospective case-control analysis of 380 neonates born at ≥35 weeks' gestation treated with therapeutic hypothermia for HIE. Death or abnormal brain MRI using the basal ganglia watershed scoring system was compared between neonates with and without AKI. RESULTS: A total of 51 (13.4%) neonates had AKI. Infants with AKI had higher rates of the composite of death or abnormal brain MRI (74.5 vs. 38.3%; p < 0.001). Rate of death (21.6 vs. 5.5%; p < 0.001) and severe abnormalities on MRI or death (43.1 vs. 19.1%; p < 0.001) were also higher in neonates with AKI. CONCLUSION: AKI is strongly associated with abnormalities on brain MRI or death in neonates with HIE. Identification of AKI in this patient population may be helpful in guiding clinical management and predicting potential neurodevelopmental impairment. KEY POINTS: · Neonates with HIE are at increased risk for AKI.. · AKI is associated with hypoxic-ischemic injury on brain MRI or death among neonates with HIE.. · Identification of AKI in infants with HIE may help predict neurodevelopmental impairment..

Living with plague: Lessons from the Soviet Union's antiplague system.

Zoonoses, such as plague, are primarily animal diseases that spill over into human populations. While the goal of eradicating such diseases is enticing, historical experience validates abandoning eradication in favor of ecologically based control strategies (which reduce morbidity and mortality to a locally accepted risk level). During the 20th century, one of the most extensive plague-eradication efforts in recorded history was undertaken to enable large-scale changes in land use in the former Soviet Union (including vast areas of central Asia). Despite expending tremendous resources in its attempt to eradicate plague, the Soviet antiplague response gradually abandoned the goal of eradication in favor of plague control linked with developing basic knowledge of plague ecology. Drawing from this experience, we combine new gray-literature sources, historical and recent research, and fieldwork to outline best practices for the control of spillover from zoonoses while minimally disrupting wildlife ecosystems, and we briefly compare the Soviet case with that of endemic plague in the western United States. We argue for the allocation of sufficient resources to maintain ongoing local surveillance, education, and targeted control measures; to incorporate novel technologies selectively; and to use ecological research to inform developing landscape-based models for transmission interruption. We conclude that living with emergent and reemergent zoonotic diseases-switching to control-opens wider possibilities for interrupting spillover while preserving natural ecosystems, encouraging adaptation to local conditions, and using technological tools judiciously and in a cost-effective way.

Is Maternal Methadone Dose Associated with the Severity of Neonatal Abstinence Syndrome?

OBJECTIVE: The aim of the study is to assess the correlation between maternal methadone dose and severity of neonatal abstinence syndrome (NAS) in infants that required pharmacological treatment for NAS. STUDY DESIGN: This is a retrospective analysis of 574 infants ≥35 weeks' gestation exposed to methadone in utero, born between August 2006 and May 2018, and who required pharmacological therapy for NAS. Indicators of NAS severity (duration of morphine treatment, maximum morphine dose, use of phenobarbital, and length of hospitalization) were compared between infants exposed to high (≥200 mg), intermediate (100-199 mg), and low doses (<100 mg) of methadone. Logistic and linear regression models were used to adjust for the covariates. RESULTS: Median (interquartile range) duration of medical treatment with morphine was higher in infants exposed to higher doses of methadone (low dose 23 [14-37] days, intermediate dose 31 [18-45] days, and high dose 35 [20-48] days, p < 0.001). Higher methadone doses were also predictive of longer duration of hospitalization, higher maximum morphine dose, and increased likelihood of treatment with phenobarbital. The association between maternal methadone dose and the severity of NAS persisted in multivariable regression models. CONCLUSION: Infants exposed to higher methadone doses displayed more severe NAS, as indicated by longer durations of treatment, higher maximum morphine dose, longer duration of hospitalization, and increased likelihood of phenobarbital use. KEY POINTS: · Methadone maintenance therapy is used during pregnancy to control maternal withdrawal symptoms.. · Relationship between maternal methadone dose and severity of NAS is not adequately investigated.. · Increased doses of methadone during pregnancy correlate with increased severity of NAS..

Daily emollient during infancy for prevention of eczema: the BEEP randomised controlled trial.

BACKGROUND: Skin barrier dysfunction precedes eczema development. We tested whether daily use of emollient in the first year could prevent eczema in high-risk children. METHODS: We did a multicentre, pragmatic, parallel-group, randomised controlled trial in 12 hospitals and four primary care sites across the UK. Families were approached via antenatal or postnatal services for recruitment of term infants (at least 37 weeks' gestation) at high risk of developing eczema (ie, at least one first-degree relative with parent-reported eczema, allergic rhinitis, or asthma, diagnosed by a doctor). Term newborns with a family history of atopic disease were randomly assigned (1:1) to application of emollient daily (either Diprobase cream or DoubleBase gel) for the first year plus standard skin-care advice (emollient group) or standard skin-care advice only (control group). The randomisation schedule was created using computer-generated code (stratified by recruiting centre and number of first-degree relatives with atopic disease) and participants were assigned to groups using an internet-based randomisation system. The primary outcome was eczema at age 2 years (defined by UK working party criteria) with analysis as randomised regardless of adherence to allocation for participants with outcome data collected, and adjusting for stratification variables. This trial is registered with ISRCTN, ISRCTN21528841. Data collection for long-term follow-up is ongoing, but the trial is closed to recruitment. FINDINGS: 1394 newborns were randomly assigned to study groups between Nov 19, 2014, and Nov 18, 2016; 693 were assigned to the emollient group and 701 to the control group. Adherence in the emollient group was 88% (466 of 532) at 3 months, 82% (427 of 519) at 6 months, and 74% (375 of 506) at 12 months in those with complete questionnaire data. At age 2 years, eczema was present in 139 (23%) of 598 infants with outcome data collected in the emollient group and 150 (25%) of 612 infants in the control group (adjusted relative risk 0·95 [95% CI 0·78 to 1·16], p=0·61; adjusted risk difference -1·2% [-5·9 to 3·6]). Other eczema definitions supported the results of the primary analysis. Mean number of skin infections per child in year 1 was 0·23 (SD 0·68) in the emollient group versus 0·15 (0·46) in the control group; adjusted incidence rate ratio 1·55 (95% CI 1·15 to 2·09). INTERPRETATION: We found no evidence that daily emollient during the first year of life prevents eczema in high-risk children and some evidence to suggest an increased risk of skin infections. Our study shows that families with eczema, asthma, or allergic rhinitis should not use daily emollients to try and prevent eczema in their newborn. FUNDING: National Institute for Health Research Health Technology Assessment.

An Investigation of Early Syphilis Among Men Who have Sex with Men: Alaska, 2018: Findings from a 2018 Rapid Ethnographic Assessment.

The state of Alaska had a sharp increase in cases of primary and secondary syphilis among gay, bisexual, and other men who have sex with men (GBMSM) in 2018, centered in Anchorage. A rapid ethnographic assessment was conducted in October 2018 to examine contextual factors contributing to local increases in syphilis. The assessment team conducted qualitative interviews with 64 (N=49 interviews) key informants in Anchorage and Matanuska-Susitna Valley identified through the STD/HIV program at the Alaska Department of Health and Social Services, Division of Public Health (ADPH):  ADPH staff (n = 11; 22%) Medical Providers (n = 18; 37%), Community-Based Organizations/Partners (n = 9; 18%), and GBMSM Community Members (n = 11; 22%). This project was deemed exempt from IRB review. Primary factors affecting syphilis transmission, care, and treatment among GBMSM were: (1) Low awareness about the current syphilis outbreak and ambivalence about syphilis and other STIs; (2) Aspects of sexual partnering such as travel, tourism, and the use of online sites and apps to facilitate anonymous sex and multiple (both sequential/concurrent) partnering; (3) The synergistic effects of substance use, homelessness, and transactional sex; (4) Choosing condomless sex; and (5) Challenges accessing healthcare, including the ability to find appropriate and culturally competent care. Syphilis increases may have been influenced by factors which spanned multiple sectors of the Anchorage community, including individual behavior, community-level risk and protective factors, and use of and interactions with resources offered by ADPH, community-based organizations, and medical providers.

Buprenorphine for the Treatment of the Neonatal Abstinence Syndrome.

BACKGROUND: Current pharmacologic treatment of the neonatal abstinence syndrome with morphine is associated with a lengthy duration of therapy and hospitalization. Buprenorphine may be more effective than morphine for this indication. METHODS: In this single-site, double-blind, double-dummy clinical trial, we randomly assigned 63 term infants (≥37 weeks of gestation) who had been exposed to opioids in utero and who had signs of the neonatal abstinence syndrome to receive either sublingual buprenorphine or oral morphine. Infants with symptoms that were not controlled with the maximum dose of opioid were treated with adjunctive phenobarbital. The primary end point was the duration of treatment for symptoms of neonatal opioid withdrawal. Secondary clinical end points were the length of hospital stay, the percentage of infants who required supplemental treatment with phenobarbital, and safety. RESULTS: The median duration of treatment was significantly shorter with buprenorphine than with morphine (15 days vs. 28 days), as was the median length of hospital stay (21 days vs. 33 days) (P<0.001 for both comparisons). Adjunctive phenobarbital was administered in 5 of 33 infants (15%) in the buprenorphine group and in 7 of 30 infants (23%) in the morphine group (P=0.36). Rates of adverse events were similar in the two groups. CONCLUSIONS: Among infants with the neonatal abstinence syndrome, treatment with sublingual buprenorphine resulted in a shorter duration of treatment and shorter length of hospital stay than treatment with oral morphine, with similar rates of adverse events. (Funded by the National Institute on Drug Abuse; BBORN ClinicalTrials.gov number, NCT01452789 .).

Acceptability of a cessation intervention for pregnant smokers: a qualitative study guided by Normalization Process Theory.

BACKGROUND: Smoking during pregnancy has serious consequences for maternal and child health. An intervention package to embed National Institute for Health and Care Excellence guidance (babyClear©) was delivered across maternity and stop smoking services (SSS) within an English region, to support pregnant women to stop smoking. We aimed to ascertain acceptability among pregnant smokers receiving the intervention. METHODS: Pregnant smokers who received the intervention and participated in the study were interviewed, first at around 16 weeks of pregnancy (n = 17) and again several weeks later (n = 8) or postpartum (n = 3). Interview schedules were informed by Normalization Process Theory (NPT) and Theoretical Domains Framework; interviews were audio-recorded, transcribed and analysed thematically, using the Framework method and NPT. Findings are grouped according to the four NPT concepts. RESULTS: Coherence: Carbon monoxide monitoring appeared to make sense; women were motivated to quit by being monitored. Cognitive participation: When linked to a professional discourse of caring and concern, some women were prompted to engage with the SS message. Women were more guarded in their reaction to initial contact from the SSS; reporting attending appointments successfully, or in some cases, experiencing problems that decreased engagement and made quitting harder. Collective action: Where women continued to smoke or failed to attend SSS appointments, an extra intervention was delivered, the Risk Perception Tool (RPT), which often prompted pregnant women to act. Reflexive monitoring: Most women accepted the need for a hard-hitting approach (RPT) and, while it distressed them at the time, they claimed they were subsequently grateful for it. SSS intervention post-RPT was seen as supportive, partly because it often involved home visits. Aspects of family inclusion in babyClear© were reported as beneficial. In Trusts where women experienced services as less focused on prioritising the stop smoking message, less well integrated or reported maternity staff as less adept at delivering the RPT, women found babyClear© less acceptable overall. CONCLUSIONS: The babyClear© package was acceptable to pregnant smokers interviewed during and shortly after pregnancy and, in some cases, to promote quitting. However, some contexts were more optimal than others, leading to variation in acceptability overall.

Hepatic FcRn regulates albumin homeostasis and susceptibility to liver injury.

The neonatal crystallizable fragment receptor (FcRn) is responsible for maintaining the long half-life and high levels of the two most abundant circulating proteins, albumin and IgG. In the latter case, the protective mechanism derives from FcRn binding to IgG in the weakly acidic environment contained within endosomes of hematopoietic and parenchymal cells, whereupon IgG is diverted from degradation in lysosomes and is recycled. The cellular location and mechanism by which FcRn protects albumin are partially understood. Here we demonstrate that mice with global or liver-specific FcRn deletion exhibit hypoalbuminemia, albumin loss into the bile, and increased albumin levels in the hepatocyte. In vitro models with polarized cells illustrate that FcRn mediates basal recycling and bidirectional transcytosis of albumin and uniquely determines the physiologic release of newly synthesized albumin into the basal milieu. These properties allow hepatic FcRn to mediate albumin delivery and maintenance in the circulation, but they also enhance sensitivity to the albumin-bound hepatotoxin, acetaminophen (APAP). As such, global or liver-specific deletion of FcRn results in resistance to APAP-induced liver injury through increased albumin loss into the bile and increased intracellular albumin scavenging of reactive oxygen species. Further, protection from injury is achieved by pharmacologic blockade of FcRn-albumin interactions with monoclonal antibodies or peptide mimetics, which cause hypoalbuminemia, biliary loss of albumin, and increased intracellular accumulation of albumin in the hepatocyte. Together, these studies demonstrate that the main function of hepatic FcRn is to direct albumin into the circulation, thereby also increasing hepatocyte sensitivity to toxicity.

Kodoja: A workflow for virus detection in plants using k-mer analysis of RNA-sequencing data.

RNA-sequencing of plant material allows for hypothesis-free detection of multiple viruses simultaneously. This methodology relies on bioinformatics workflows for virus identification. Most workflows are designed for human clinical data, and few go beyond sequence mapping for virus identification. We present a new workflow (Kodoja) for the detection of plant virus sequences in RNA-sequence data. Kodoja uses k-mer profiling at the nucleotide level and sequence mapping at the protein level by integrating two existing tools Kraken and Kaiju. Kodoja was tested on three existing RNA-seq datasets from grapevine, and two new RNA-seq datasets from raspberry. For grapevine, Kodoja was shown to be more sensitive than a method based on contig building and blast alignments (27 viruses detected compared to 19). The application of Kodoja to raspberry, showed that field-grown raspberries were infected by multiple viruses, and that RNA-seq can identify lower amounts of virus material than reverse transcriptase PCR. This work enabled the design of new PCR-primers for detection of Raspberry yellow net virus and Beet ringspot virus. Kodoja is a sensitive method for plant virus discovery in field samples and enables the design of more accurate primers for detection. Kodoja is available to install through Bioconda and as a tool within Galaxy.

A conformational switch high-throughput screening assay and allosteric inhibition of the flavivirus NS2B-NS3 protease.

The flavivirus genome encodes a single polyprotein precursor requiring multiple cleavages by host and viral proteases in order to produce the individual proteins that constitute an infectious virion. Previous studies have revealed that the NS2B cofactor of the viral NS2B-NS3 heterocomplex protease displays a conformational dynamic between active and inactive states. Here, we developed a conformational switch assay based on split luciferase complementation (SLC) to monitor the conformational change of NS2B and to characterize candidate allosteric inhibitors. Binding of an active-site inhibitor to the protease resulted in a conformational change of NS2B and led to significant SLC enhancement. Mutagenesis of key residues at an allosteric site abolished this induced conformational change and SLC enhancement. We also performed a virtual screen of NCI library compounds to identify allosteric inhibitors, followed by in vitro biochemical screening of the resultant candidates. Only three of these compounds, NSC135618, 260594, and 146771, significantly inhibited the protease of Dengue virus 2 (DENV2) in vitro, with IC50 values of 1.8 µM, 11.4 µM, and 4.8 µM, respectively. Among the three compounds, only NSC135618 significantly suppressed the SLC enhancement triggered by binding of active-site inhibitor in a dose-dependent manner, indicating that it inhibits the conformational change of NS2B. Results from virus titer reduction assays revealed that NSC135618 is a broad spectrum flavivirus protease inhibitor, and can significantly reduce titers of DENV2, Zika virus (ZIKV), West Nile virus (WNV), and Yellow fever virus (YFV) on A549 cells in vivo, with EC50 values in low micromolar range. In contrast, the cytotoxicity of NSC135618 is only moderate with CC50 of 48.8 µM on A549 cells. Moreover, NSC135618 inhibited ZIKV in human placental and neural progenitor cells relevant to ZIKV pathogenesis. Results from binding, kinetics, Western blot, mass spectrometry and mutagenesis experiments unambiguously demonstrated an allosteric mechanism for inhibition of the viral protease by NSC135618.

Potato Mop-Top Virus Co-Opts the Stress Sensor HIPP26 for Long-Distance Movement.

Virus movement proteins facilitate virus entry into the vascular system to initiate systemic infection. The potato mop-top virus (PMTV) movement protein, TGB1, is involved in long-distance movement of both viral ribonucleoprotein complexes and virions. Here, our analysis of TGB1 interactions with host Nicotiana benthamiana proteins revealed an interaction with a member of the heavy metal-associated isoprenylated plant protein family, HIPP26, which acts as a plasma membrane-to-nucleus signal during abiotic stress. We found that knockdown of NbHIPP26 expression inhibited virus long-distance movement but did not affect cell-to-cell movement. Drought and PMTV infection up-regulated NbHIPP26 gene expression, and PMTV infection protected plants from drought. In addition, NbHIPP26 promoter-reporter fusions revealed vascular tissue-specific expression. Mutational and biochemical analyses indicated that NbHIPP26 subcellular localization at the plasma membrane and plasmodesmata was mediated by lipidation (S-acylation and prenylation), as nonlipidated NbHIPP26 was predominantly in the nucleus. Notably, coexpression of NbHIPP26 with TGB1 resulted in a similar nuclear accumulation of NbHIPP26. TGB1 interacted with the carboxyl-terminal CVVM (prenyl) domain of NbHIPP26, and bimolecular fluorescence complementation revealed that the TGB1-HIPP26 complex localized to microtubules and accumulated in the nucleolus, with little signal at the plasma membrane or plasmodesmata. These data support a mechanism where interaction with TGB1 negates or reverses NbHIPP26 lipidation, thus releasing membrane-associated NbHIPP26 and redirecting it via microtubules to the nucleus, thereby activating the drought stress response and facilitating virus long-distance movement.

What helped and hindered implementation of an intervention package to reduce smoking in pregnancy: process evaluation guided by normalization process theory.

BACKGROUND: Smoking in pregnancy causes harm to mother and baby. Despite evidence from trials of what helps women quit, implementation in the real world has been hard to achieve. An evidence-based intervention, babyClear©, involving staff training, universal carbon monoxide monitoring, opt-out referral to smoking cessation services, enhanced follow-up protocols and a risk perception tool was introduced across North East England. This paper presents the results of the qualitative analyses, reporting acceptability of the system changes to staff, as well as aids and hindrances to implementation and normalization of this complex intervention. METHODS: Process evaluation was used to complement an effectiveness study. Interviews with maternity and smoking cessation services staff and observations of training were undertaken. Normalization Process Theory (NPT) was used to frame the interview guides and analysis. NPT is an empirically-derived theory, developed by sociologists, that uses four concepts to understand the process of routinising new practices. RESULTS: Staff interviews took place across eight National Health Service trusts at a time of widespread restructuring in smoking cessation services. Principally interviewees worked in maternity (n = 63) and smoking cessation services (n = 35). Five main themes, identified inductively, influenced the implementation: 1) initial preparedness of the organisations; 2) staff training; 3) managing partnership working; 4) resources; 5) review and planning for sustainability. CONCLUSIONS: NPT was used to show that the babyClear© package was acceptable to staff in a range of organisations. Illustrated in Themes 1, 2 & 3, staff welcomed ways to approach pregnant women about their smoking, without damaging their professional relationship with them. Predicated on producing individual behaviour change in women, the intervention does this largely through reorganising and standardising healthcare systems that are required to implement best practice guidelines. Changing organisational systems requires belief and commitment from staff, so that they set up and maintain practical adjustments to their practice and are reflective about adapting themselves and the work context as new challenges are encountered. The ongoing challenge is to identify and maintain the elements of the intervention package which are essential for its effectiveness and how to tailor them to local circumstances and resources without compromising its core ingredients.