Breasi-CRISPR: an efficient genome-editing method to interrogate protein localization and protein-protein interactions in the embryonic mouse cortex.

In developing tissues, knowing the localization and interactors of proteins of interest is key to understanding their function. Here, we describe the Breasi-CRISPR approach (Brain Easi-CRISPR), combining Easi-CRISPR with in utero electroporation to tag endogenous proteins within embryonic mouse brains. Breasi-CRISPR enables knock-in of both short and long epitope tag sequences with high efficiency. We visualized epitope-tagged proteins with varied expression levels, such as ACTB, LMNB1, EMD, FMRP, NOTCH1 and RPL22. Detection was possible by immunohistochemistry as soon as 1 day after electroporation and we observed efficient gene editing in up to 50% of electroporated cells. Moreover, tagged proteins could be detected by immunoblotting in lysates from individual cortices. Next, we demonstrated that Breasi-CRISPR enables the tagging of proteins with fluorophores, allowing visualization of endogenous proteins by live imaging in organotypic brain slices. Finally, we used Breasi-CRISPR to perform co-immunoprecipitation mass-spectrometry analyses of the autism-related protein FMRP to discover its interactome in the embryonic cortex. Together, these data demonstrate that Breasi-CRISPR is a powerful tool with diverse applications that will propel the understanding of protein function in neurodevelopment.

Cigarette smoking and misperceived norms among adults in rural Uganda: a population-based study.

BACKGROUND: Little is known about perceived norms about cigarette smoking in Uganda or the extent to which perceptions drive personal cigarette smoking behaviour. METHODS: We conducted a cross-sectional study in 2016-2018 that targeted all adults who resided within eight villages in Rwampara District, southwestern Uganda. Personal cigarette smoking frequency was elicited by self-report. We also asked participants what they believed to be the cigarette smoking frequency of most other adult men and women in their villages (i.e., perceived norms). Frequent cigarette smoking was defined as 4+ times/week. We compared perceived norms to cigarette smoking frequency reports aggregated at the village level. We used multivariable Poisson regression to estimate the association between perceived norms and personal cigarette smoking behaviour. RESULTS: Among 1626 participants (91% response rate), 92 of 719 men (13%) and 6 of 907 women (0.7%) reported frequent smoking. However, 1030 (63%) incorrectly believed most men in their villages smoked cigarettes frequently. Additionally, 116 (7%) incorrectly believed that most women in their villages smoked cigarettes frequently. These misperceptions were pervasive across social strata. Men who misperceived frequent cigarette smoking as the norm among other men in their villages were more likely to smoke frequently themselves (adjusted relative risk=1.49; 95% CI, 1.13 to 1.97). CONCLUSIONS: Most adults overestimated cigarette smoking frequency among village peers. Men who incorrectly believed that frequent smoking was the norm were more likely to engage in frequent smoking themselves. Applying a 'social norms approach' intervention by promoting existing healthy norms may prevent smoking initiation or motivate reductions in smoking among men in rural Uganda.

Correlates of attendance at community engagement meetings held in advance of bio-behavioral research studies: A longitudinal, sociocentric social network study in rural Uganda.

BACKGROUND: Community engagement is central to the conduct of health-related research studies as a way to determine priorities, inform study design and implementation, increase recruitment and retention, build relationships, and ensure that research meets the goals of the community. Community sensitization meetings, a form of community engagement, are often held prior to the initiation of research studies to provide information about upcoming study activities and resolve concerns in consultation with potential participants. This study estimated demographic, health, economic, and social network correlates of attendance at community sensitization meetings held in advance of a whole-population, combined behavioral, and biomedical research study in rural Uganda. METHODS AND FINDINGS: Research assistants collected survey data from 1,630 adults participating in an ongoing sociocentric social network cohort study conducted in a rural region of southwestern Uganda. These community survey data, collected between 2016 and 2018, were linked to attendance logs from community sensitization meetings held in 2018 and 2019 before the subsequent community survey and community health fair. Of all participants, 264 (16%) attended a community sensitization meeting before the community survey, 464 (28%) attended a meeting before the community health fair, 558 (34%) attended a meeting before either study activity (survey or health fair), and 170 (10%) attended a meeting before both study activities (survey and health fair). Using multivariable Poisson regression models, we estimated correlates of attendance at community sensitization meetings. Attendance was more likely among study participants who were women (adjusted relative risk [ARR]health fair = 1.71, 95% confidence interval [CI], 1.32 to 2.21, p < 0.001), older age (ARRsurvey = 1.02 per year, 95% CI, 1.01 to 1.02, p < 0.001; ARRhealth fair = 1.02 per year, 95% CI, 1.01 to 1.02, p < 0.001), married (ARRsurvey = 1.74, 95% CI, 1.29 to 2.35, p < 0.001; ARRhealth fair = 1.41, 95% CI, 1.13 to 1.76, p = 0.002), and members of more community groups (ARRsurvey = 1.26 per group, 95% CI, 1.10 to 1.44, p = 0.001; ARRhealth fair = 1.26 per group, 95% CI, 1.12 to 1.43, p < 0.001). Attendance was less likely among study participants who lived farther from meeting locations (ARRsurvey = 0.54 per kilometer, 95% CI, 0.30 to 0.97, p = 0.041; ARRhealth fair = 0.57 per kilometer, 95% CI, 0.38 to 0.86, p = 0.007). Leveraging the cohort's sociocentric design, social network analyses suggested that information conveyed during community sensitization meetings could reach a broader group of potential study participants through attendees' social network and household connections. Study limitations include lack of detailed data on reasons for attendance/nonattendance at community sensitization meetings; achieving a representative sample of community members was not an explicit aim of the study; and generalizability may not extend beyond this study setting. CONCLUSIONS: In this longitudinal, sociocentric social network study conducted in rural Uganda, we observed that older age, female sex, being married, membership in more community groups, and geographical proximity to meeting locations were correlated with attendance at community sensitization meetings held in advance of bio-behavioral research activities. Information conveyed during meetings could have reached a broader portion of the population through attendees' social network and household connections. To ensure broader input and potentially increase participation in health-related research studies, the dissemination of research-related information through community sensitization meetings may need to target members of underrepresented groups.

Association Between Treatments and Short-Term Biochemical Improvements and Clinical Outcomes in Post-Severe Acute Respiratory Syndrome Coronavirus-2 Inflammatory Syndrome.

OBJECTIVES: To 1) analyze the short-term biochemical improvements and clinical outcomes following treatment of children with post-severe acute respiratory syndrome coronavirus-2 inflammatory syndrome (multisystem inflammatory syndrome in children/pediatric inflammatory multisystem syndrome temporally associated with severe acute respiratory syndrome coronavirus-2) admitted to U.K. PICUs and 2) collate current treatment guidance from U.K. PICUs. DESIGN: Multicenter observational study. SETTING: Twenty-one U.K. PICUs. PATIENTS: Children (< 18 yr) admitted to U.K. PICUs between April 1, 2020, and May 10, 2020, fulfilling the U.K. case definition of pediatric inflammatory multisystem syndrome temporally associated with severe acute respiratory syndrome coronavirus-2. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Routinely collected, deidentified data were analyzed. Propensity score and linear mixed effects models were used to analyze the effect of steroids, IV immunoglobulin, and biologic agents on changes in C-reactive protein, platelet counts, and lymphocyte counts over the course of PICU stay. Treatment recommendations from U.K. clinical guidelines were analyzed. Over the 6-week study period, 59 of 78 children (76%) received IV immunoglobulin, 57 of 78 (73%) steroids, and 18 of 78 (24%) a biologic agent. We found no evidence of a difference in response in clinical markers of inflammation between patients with multisystem inflammatory syndrome in children/pediatric inflammatory multisystem syndrome temporally associated with severe acute respiratory syndrome coronavirus-2 who were treated with IV immunoglobulin, steroids, or biologics, compared with those who were not. By the end of the study period, most patients had received immunomodulation. The 12 patients who did not receive any immunomodulators had similar decrease in inflammatory markers as those treated. Of the 14 guidelines analyzed, the use of IV immunoglobulin, steroids, and biologics was universally recommended. CONCLUSIONS: We were unable to identify any short-term benefit from any of the treatments, or treatment combinations, administered. Despite a lack of evidence, treatment guidelines for multisystem inflammatory syndrome in children/pediatric inflammatory multisystem syndrome temporally associated with severe acute respiratory syndrome coronavirus-2 have become very similar in advising step-wise treatments. Retaining clinical equipoise regarding treatment will allow clinicians to enroll children in robust clinical trials to determine the optimal treatment for this novel important condition.

The Contribution of a Charitable Organization to Regional Cleft Lip and Palate Services in England and Scotland.

BACKGROUND: From diagnosis through to adulthood, a cleft lip and/or palate (CL/P) poses a number of challenges for those affected. Alongside the care provided by clinical teams, complementary information and support is offered by charitable organizations. In 2011, the Cleft Lip and Palate Association received funding to implement a new regional service across England and Scotland, with the aim of increasing support at a local level. The Centre for Appearance Research at the University of the West of England were commissioned to conduct an independent evaluation of the service over 7 years. METHODS: A pragmatic, mixed-methods approach was utilized to assess the impact of the service from the perspective of charity volunteers; children, young people, and adults with CL/P; caregivers; and clinicians. Feedback forms were distributed to stakeholders at a variety of events, and qualitative feedback was collected via focus groups and an online survey. RESULTS: The majority of participants indicated they had gained access to a local support network, felt more able to cope with CL/P-related challenges, and felt more confident in themselves. Qualitative investigation provided further support for these findings and highlighted additional benefits of the regional service for clinical teams. CONCLUSIONS: The evaluation provides encouraging evidence toward the contribution of a relatively small charitable organization in the context of cleft care. The importance of a pragmatic approach to community-based evaluation and the benefits of collaborative working between researchers and the charitable sector were also highlighted.

Evaluation and comparison of staining patterns of factor XIIIa (AC-1A1), adipophilin and GATA3 in sebaceous neoplasia.

BACKGROUND: Reliable nuclear immunohistochemical stains for sebaceous neoplasms have not been readily available. Positive nuclear staining has been reported for GATA3 and factor XIIIa (AC-1A1). We sought to determine the diagnostic utility of these nuclear stains by comparing their staining pattern to adipophilin, a consistently positive cytoplasmic stain. METHODS: Cases with the diagnosis of sebaceous hyperplasia, sebaceous adenoma, sebaceous epithelioma/sebaceoma, sebaceous carcinoma, and nonsebaceous neoplasms (basal cell carcinoma and squamous cell carcinoma) were examined. Intensity and extent of staining of the basal cells and mature sebocytes were evaluated for each stain. RESULTS: Factor XIIIa (AC-1A1) was 87.3% sensitive and 95.1% specific for all sebaceous neoplasms sand showed high inter-observer reliability. Adipophilin was 83.2% sensitive and 87.8% specific. GATA3 was the least sensitive (80.9%) and specific (75.6%) marker. When factor XIIIa was compared against composite staining of all three markers its staining was still uniquely significant (P = .0210). CONCLUSION: Factor XIIIa (AC-1A1) is a sensitive and specific nuclear marker for sebaceous differentiation. Its diagnostic utility exceeds that of adipophilin. Factor XIIIa should be included in the expanding group of immunohistochemical and special stains which can be utilized to aid in the diagnosis of sebaceous neoplasms.

Drivers of interannual variability in virioplankton abundance at the coastal western Antarctic peninsula and the potential effects of climate change.

An 8-year time-series in the Western Antarctic Peninsula (WAP) with an approximately weekly sampling frequency was used to elucidate changes in virioplankton abundance and their drivers in this climatically sensitive region. Virioplankton abundances at the coastal WAP show a pronounced seasonal cycle with interannual variability in the timing and magnitude of the summer maxima. Bacterioplankton abundance is the most influential driving factor of the virioplankton, and exhibit closely coupled dynamics. Sea ice cover and duration predetermine levels of phytoplankton stock and thus, influence virioplankton by dictating the substrates available to the bacterioplankton. However, variations in the composition of the phytoplankton community and particularly the prominence of Diatoms inferred from silicate drawdown, drive interannual differences in the magnitude of the virioplankton bloom; likely again mediated through changes in the bacterioplankton. Their findings suggest that future warming within the WAP will cause changes in sea ice that will influence viruses and their microbial hosts through changes in the timing, magnitude and composition of the phytoplankton bloom. Thus, the flow of matter and energy through the viral shunt may be decreased with consequences for the Antarctic food web and element cycling.

No influence of ischemic preconditioning on running economy.

PURPOSE: Many of the potential performance-enhancing properties of ischemic preconditioning suggest that the oxygen cost for a given endurance exercise workload will be reduced, thereby improving the economy of locomotion. The aim of this study was to identify whether ischemic preconditioning improves exercise economy in recreational runners. METHODS: A randomized sham-controlled crossover study was employed in which 18 adults (age 27 ± 7 years; BMI 24.6 ± 3 kg/m2) completed two, incremental submaximal (65-85% VO2max) treadmill running protocols (3 × 5 min stages from 7.2-14.5 km/h) coupled with indirect calorimetry to assess running economy following ischemic preconditioning (3 × 5 min bilateral upper thigh ischemia) and sham control. Running economy was expressed as mlO2/kg/km and as the energy in kilocalories required to cover 1 km of horizontal distance (kcal/kg/km). RESULTS: Ischemic preconditioning did not influence steady-state heart rate, oxygen consumption, minute ventilation, respiratory exchange ratio, energy expenditure, and blood lactate. Likewise, running economy was similar (P = 0.647) between the sham (from 201.6 ± 17.7 to 204.0 ± 16.1 mlO2/kg/km) and ischemic preconditioning trials (from 202.8 ± 16.2 to 203.1 ± 15.6 mlO2/kg/km). There was no influence (P = 0.21) of ischemic preconditioning on running economy expressed as the caloric unit cost (from 0.96 ± 0.12 to 1.01 ± 0.11 kcal/kg/km) compared with sham (from 1.00 ± 0.10 to 1.00 ± 0.08 kcal/kg/km). CONCLUSIONS: The properties of ischemic preconditioning thought to affect exercise performance at vigorous to severe exercise intensities, which generate more extensive physiological challenge, are ineffective at submaximal workloads and, therefore, do not change running economy.

Alteration of Mammary Gland Development and Gene Expression by In Utero Exposure to Cadmium.

Environmental exposure to estrogens and estrogen like contaminants during early development is thought to contribute to the risk of developing breast cancer primarily due to an early onset of puberty; however, exposure during key developing windows may also influence the risk of developing the disease. The goal of this study was to ask whether in utero exposure to the metalloestrogen cadmium alters mammary gland development due to acceleration of puberty onset or to an effect on early development of the mammary gland. The results show that, in addition to advancing the onset of puberty, in utero exposure to the metalloestrogen cadmium altered mammary gland development prior to its effect on puberty onset. In utero exposure resulted in an expansion of the number of mammosphere-forming cells in the neonatal mammary gland and an increase in branching, epithelial cells, and density in the prepubertal mammary gland. In the postpubertal mammary gland, there was a further expansion of the mammary stem/progenitor cell population and overexpression of estrogen receptor-alpha (ERα) that was due to the overexpression and altered regulation of the ERα transcripts derived from exons O and OT in response to estradiol. These results suggest that in utero exposure to cadmium increases stem/progenitor cells, cell density, and expression of estrogen receptor-alpha that may contribute to the risk of developing breast cancer.

Epitope-based DNA vaccine for Alzheimer's disease: translational study in macaques.

BACKGROUND: Clinical trials with passive and active Alzheimer's disease (AD) vaccines suggest that early interventions are needed for improvement of cognitive and/or functional performance in patients, providing impetus for the development of safe and immunologically potent active vaccines targeting amyloid ß (Aß). The AN-1792 trial has indicated that Aß-specific T cells may be unsafe for humans; therefore, other vaccines based on small Aß epitopes are undergoing preclinical and clinical testing. METHODS: Humoral and cellular immune responses elicited in response to a novel DNA epitope-based vaccine (AV-1955) delivered to rhesus macaques using the TriGrid electroporation device were evaluated. Functional activities of anti-Aß antibodies generated in response to vaccination were assessed in vitro. RESULTS: AV-1955 generates long-term, potent anti-Aß antibodies and cellular immune responses specific to foreign T-helper epitopes but not to self-Aß. CONCLUSIONS: This translational study demonstrates that a DNA-based epitope vaccine for AD could be appropriate for human clinical testing.

Clinical effectiveness and cost-effectiveness of a brief accessible cognitive behavioural therapy programme for stress in school-aged adolescents (BESST): a cluster randomised controlled trial in the UK.

BACKGROUND: Depression and anxiety are increasingly prevalent in adolescents. The Brief Educational Workshops in Secondary Schools Trial investigated the effectiveness of a brief accessible stress workshop programme for 16-18-year-olds. We aimed to investigate the clinical effectiveness and cost-effectiveness of the DISCOVER cognitive behavioural therapy (CBT) workshop on symptoms of depression in 16-18-year-olds at 6 months compared with treatment-as-usual. METHODS: We conducted a multicentre, cluster randomised controlled trial in UK schools or colleges with sixth forms to evaluate clinical effectiveness and cost-effectiveness of a brief CBT workshop (DISCOVER) compared with treatment-as-usual. We planned to enrol 60 schools and 900 adolescents, using a self-referral system to recruit participants. Schools were randomised in a 1:1 ratio for participants to receive either the DISCOVER workshop or treatment-as-usual, stratified by site and balanced on school size and index of multiple deprivation. Participants were included if they were 16-18 years old, attending for the full school year, seeking help for stress, and fluent in English and able to provide written informed consent. The outcome assessors, senior health economist, senior statistician, and chief investigator were masked. People with lived experience were involved in the study. The primary outcome was depression symptoms measured with the Mood and Feelings Questionnaire (MFQ) at 6-month follow-up, in the intention-to-treat population of all participants with full covariate data. The trial was registered with the ISRCTN registry (ISRCTN90912799). FINDINGS: 111 schools were invited to participate in the study, seven were deemed ineligible, and 47 did not provide consent. Between Oct 4, 2021, and Nov 10, 2022, 933 students at 57 schools were screened for eligibility, seven were not eligible for inclusion, and 26 did not attend the baseline meeting and assessment, resulting in 900 adolescents participating in the study. The DISCOVER group included 443 participants (295 [67%] female and 136 [31%] male) and the treatment-as-usual group included 457 participants (346 [76%] female and 92 [20%] male). 468 (52%) of the 900 participants were White, and the overall age of the participants was 17·2 years (SD 0·6). 873 (97%) adolescents were followed up in the intention-to-treat population. The primary intention-to-treat analysis (n=854) found an adjusted mean difference in MFQ of -2·06 (95% CI -3·35 to -0·76; Cohen's d=-0·17; p=0·0019) at the 6-month follow-up, indicating a clinical improvement in the DISCOVER group. The probability that DISCOVER is cost- effective compared with treatment-as-usual ranged from 61% to 78% at a £20 000 to £30 000 per quality-adjusted life-year threshold. Nine adverse events (two of which were classified as serious) were reported in the DISCOVER group and 14 (two of which were classified as serious) were reported in the treatment-as-usual group. INTERPRETATION: Our findings indicate that the DISCOVER intervention is modestly clinically effective and economically viable and could be a promising early intervention in schools. Given the importance of addressing mental health needs early in this adolescent population, additional research is warranted to explore this intervention. FUNDING: National Institute for Health and Care Research Health Technology Assessment Programme.

Brief Educational Workshops in Secondary Schools Trial (BESST trial), a school-based cluster randomised controlled trial of the DISCOVER workshop for 16-18-year-olds: recruitment and baseline characteristics.

BACKGROUND: The Brief Educational Workshops in Secondary Schools Trial (BESST) is an England-wide school-based cluster randomised controlled trial assessing the clinical and cost-effectiveness of an open-access psychological workshop programme (DISCOVER) for 16-18-year-olds. This baseline paper describes the self-referral and other recruitment processes used in this study and the baseline characteristics of the enrolled schools and participants. METHOD: We enrolled 900 participants from 57 Secondary schools across England from 4th October 2021 to 10th November 2022. Schools were randomised to receive either the DISCOVER day-long Stress workshop or treatment as usual which included signposting information. Participants will be followed up for 6 months with outcome data collection at baseline, 3-month, and 6-month post randomisation. RESULTS: Schools were recruited from a geographically and ethnically diverse sample across England. To reduce stigma, students were invited to self-refer into the study if they wanted help for stress. Their mean age was 17.2 (SD = 0.6), 641 (71%) were female and 411 (45.6%) were from ethnic minority groups. The general wellbeing of our sample measured using the Mood and Feelings Questionnaire (MFQ) found 314 (35%) of students exhibited symptoms of depression at baseline. Eighty percent of students reported low wellbeing on the Warwick Edinburgh Mental Wellbeing Scale (WEMWBS) suggesting that although the overall sample mean is below the cut-off for depression, the self-referral approach used in this study supports distressed students in coming forward. CONCLUSION: The BESST study will continue to follow up participants to collect outcome data and results will be analysed once all the data have been collected. TRIAL REGISTRATION: ISRCTN registry ISRCTN90912799. Registered on 28 May 2020.

Electroporation enhances immunogenicity of a DNA vaccine expressing woodchuck hepatitis virus surface antigen in woodchucks.

The development of therapeutic vaccines for chronic hepatitis B virus (HBV) infection has been hampered by host immune tolerance and the generally low magnitude and inconsistent immune responses to conventional vaccines and proposed new delivery methods. Electroporation (EP) for plasmid DNA (pDNA) vaccine delivery has demonstrated the enhanced immunogenicity of HBV antigens in various animal models. In the present study, the efficiency of the EP-based delivery of pDNA expressing various reporter genes first was evaluated in normal woodchucks, and then the immunogenicity of an analog woodchuck hepatitis virus (WHV) surface antigen (WHsAg) pDNA vaccine was studied in this model. The expression of reporter genes was greatly increased when the cellular uptake of pDNA was facilitated by EP. The EP of WHsAg-pDNA resulted in enhanced, dose-dependent antibody and T-cell responses to WHsAg compared to those of the conventional hypodermic needle injection of WHsAg-pDNA. Although subunit WHsAg protein vaccine elicited higher antibody titers than the DNA vaccine delivered with EP, T-cell response rates were comparable. However, in WHsAg-stimulated mononuclear cell cultures, the mRNA expression of CD4 and CD8 leukocyte surface markers and Th1 cytokines was more frequent and was skewed following DNA vaccination compared to that of protein immunization. Thus, the EP-based vaccination of normal woodchucks with pDNA-WHsAg induced a skew in the Th1/Th2 balance toward Th1 immune responses, which may be considered more appropriate for approaches involving therapeutic vaccines to treat chronic HBV infection.

Regional variation in lytic and lysogenic viral infection in the Southern Ocean and its contribution to biogeochemical cycling.

Lytic and lysogenic viral infection was investigated throughout the Southern Ocean at sites spanning the sub-Antarctic zone, the Antarctic Circumpolar Current, and an Antarctic continental sea. Higher lytic virus activity was recorded in the more productive sub-Antarctic zone than in the iron-limited waters of the Antarctic Circumpolar Current during two transects. Reduced lytic viral activity in the Antarctic Circumpolar Current was combined with a shift toward lysogenic infection, probably resulting from the lower concentration of potential prokaryotic hosts. Superimposed on this variation, lytic viral production was lower in a transect completed in the Drake Passage in autumn (1.8 × 10(8) to 1.5 × 10(9) liter(-1) day(-1)) than over the Greenwich Meridian during summer (5.1 × 10(8) to 2.0 × 10(10) cells liter(-1) day(-1)), indicating that viral activity is linked to the overall seasonal fluctuations in biotic activity. Interestingly, while prokaryotic abundance was lowest in the coastal Weddell Sea, levels of bacterial and lytic viral production (4.3 × 10(8) to 1.7 × 10(10) cells liter(-1) day(-1)) in this area were similar to those of the other zones. This may explain the weak relationship between the distribution of prokaryotes and chlorophyll in the Weddell Sea, as a high turnover of prokaryotic biomass may have been stimulated by the availability of substrates in the form of viral lysate. With estimated carbon and iron releases of 0.02 to 7.5 µg liter(-1) day(-1) and 1.5 to 175.7 pg liter(-1) day(-1), respectively, viral activity in the Southern Ocean is shown to be a major contributor to satisfying the elemental requirements of microbes, notably prokaryotes in the Weddell Sea and phytoplankton in the sub-Antarctic zone.

Delivery of a DNA vaccine for Alzheimer's disease by electroporation versus gene gun generates potent and similar immune responses.

BACKGROUND: Induction of a humoral response against amyloid-ß peptide may be beneficial for Alzheimer's disease (AD) patients and may alleviate the onset and progression of AD. DNA-based vaccination provides a unique alternative method of immunization for treatment and prevention of AD. Currently, the two major delivery methods used for enhancing DNA uptake and immune responses to DNA vaccines in humans are electroporation (EP) and gene gun (GG). OBJECTIVE: The goal of this translational study was to evaluate the efficacy of an AD DNA epitope vaccine (DepVac) delivered intramuscularly by EP or intradermally by GG. METHODS: Humoral and cellular immune responses to immunization with DepVac were evaluated by ELISA and ELISPOT, respectively. Functional activity of the antibodies was also assessed. RESULTS: EP- and GG-mediated immunizations with DepVac induced similar anti-amyloid-ß (Aß) antibody and T cell responses. Anti-Aß antibodies bound to amyloid plaques in AD brain tissue and to toxic forms of Aß(42) peptide. CONCLUSION: Both delivery methods are effective at promoting potent antibodies specific for Aß.

Perceived and misperceived norms about khat and/or cannabis use among adults in southwest Uganda.

BACKGROUND: Studies from high-income contexts have found evidence that norms about substance use are misperceived. The accuracy of perceived norms about khat and cannabis use in Uganda have not previously been described. METHODS: We conducted a population-based study targeting all resident adults across eight villages in southwestern Uganda. Personal khat and/or cannabis use frequency was based on self-report. We measured perceived norms about substance use by eliciting individuals' perceptions about how often most other adult men and most other adult women in their villages used these substances. We compared perceived norms to aggregated village rates of use to assess the extent to which norms were misperceived. We used multivariable Poisson regression to estimate correlates of misperceived norms. RESULTS: Among 1626 participants (91% response rate), only 29 men (4%) and 9 women (1%) reported any lifetime use of khat and/or cannabis. However, 695 participants (43%) did not think lifetime abstinence was the norm among men in their villages, and 256 participants (16%) did not think lifetime abstinence was the norm among women. Moreover, 219 participants (13%) incorrectly believed most men in their village regularly used khat and/or cannabis (≥4 times per week). Misperceived norms were present across subgroups and were correlated with larger social networks, symptoms of depression, loneliness, and younger age. CONCLUSION: In this study of all adults across 8 villages in rural Uganda, many participants misperceived norms about khat and/or cannabis use. Providing accurate information about prevailing norms in the local population may help prevent initiation of khat and/or cannabis use among adults in this context.

Brief Educational Workshops in Secondary Schools Trial (BESST): protocol for a school-based cluster randomised controlled trial of open-access psychological workshop programme for 16-18-year-olds.

Anxiety and depression are increasingly prevalent in adolescents, often causing daily distress and negative long-term outcomes. Despite the significant and growing burden, less than 25% of those with probable diagnosis of anxiety and depression are receiving help in England. Significant barriers to help-seeking exist in this population, with a scarcity of easily accessible, effective, and cost-effective interventions tailored specially for this age group. One intervention that has been shown to be feasible to deliver and with the promise of reducing stress in this age group is a school-based stress workshop programme for 16-18-year-olds (herein called DISCOVER). The next step is to rigorously assess the effectiveness, and cost-effectiveness, of the DISCOVER intervention in a fully powered cluster randomised controlled trial (cRCT). If found to be clinically and cost-effective, DISCOVER could be scaled up as a service model UK-wide and have a meaningful impact on the mental health of adolescents across the country.Trial registration: ISRCTN registry ISRCTN90912799. Registered with ISRCTN 28 May 2020.

Primary demyelination in transgenic mice expressing interferon-gamma.

Ever since the use of interferon-gamma to treat patients with multiple sclerosis resulted in enhanced disease, the role of IFN-gamma in demyelination has been under question. To address this issue directly, transgenic mice were generated that expressed the cDNA of murine IFN-gamma in the central nervous system by using an oligodendrocyte-specific promoter. Expression of the transgene occurred after 8 weeks of age, at which time the murine immune and central nervous systems are both fully developed. Directly associated with transgene expression, primary demyelination occurred and was accompanied by clinical abnormalities consistent with CNS disorders. Additionally, multiple hallmarks of immune-mediated CNS disease were observed including upregulation of MHC molecules, gliosis and lymphocytic infiltration. These results demonstrate a direct role for IFN-gamma as an inducer of CNS demyelination leading to disease and provide new opportunities for dissecting the mechanism of demyelination.

SARS-CoV-2 paediatric inflammatory syndrome.

The novel post SARS-COV2 Paediatric Inflammatory Syndrome, first recognized in April 2020, took worldwide clinicians by surprise. There rapidly followed a plethora of case definitions, nomenclatures, descriptive papers, and guidelines on treatment. There has been controversy around this condition. Is it really new? Is it an atypical form of a known disease? How should we communicate and report such cases? What is the pathogenesis? Which treatments are given, and which are effective? What are the short and long-term outcomes? We are all learning fast. The clinical and immunological patterns seen are unique. There are significant differences in both presentation and pathogenesis to any known condition, including Kawasaki's disease. This implies that treatments are not necessarily transferrable: and indeed it is unknown which treatments are effective at all. Outcomes, as far as are known, are good, but long term data is lacking. The international cooperation has been an example of how today's connected medicine can be a force for good, however calm assessment of evidence remains necessary to ensure the best outcomes for our patients. This short article identifies what we have learnt from the first surge of COVID-19 cases about paediatric inflammatory syndrome and how it affects children.

A Therapeutic Hepatitis B Virus DNA Vaccine Induces Specific Immune Responses in Mice and Non-Human Primates.

Despite the availability of an effective prophylactic vaccine for more than 30 years, nearly 300 million people worldwide are chronically infected with the hepatitis B virus (HBV), leading to 1 death every 30 s mainly from viral hepatitis-related cirrhosis and liver cancer. Chronic HBV patients exhibit weak, transient, or dysfunctional CD8+ T-cell responses to HBV, which contrasts with high CD8+ T-cell responses seen for resolvers of acute HBV infection. Therefore, a therapeutic DNA vaccine was designed, expressing both HBV core and polymerase proteins, and was sequence optimized to ensure high protein expression and secretion. Although the vaccine, administered intramuscularly via electroporation, had no effect on plasma viral parameters in a mouse model of persistent HBV infection, it did induce robust HBV-specific immune responses in healthy and adeno-associated hepatitis B virus (AAV-HBV) infected mice as well as in healthy non-human primates.