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Research suggests that there is a high prevalence of interpersonal violence and victimisation within the autistic population, and that this accounts for poor mental health outcomes. This study aimed to examine the impact of interpersonal violence and victimisation on autistic adults from their own perspective and explore what helps or hinders their recovery. In total, 102 autistic adults completed either an online survey or a spoken interview about their experiences of interpersonal violence and victimisation. We analysed the data using a thematic analysis, and found four themes: (1) The usual for autism (expectations of victimisation, experiences of othering), (2) Personhood revoked: The cost of living (being part of a neurominority, trauma, masking and burnout), (3) Unpacking the baggage (impact of hermeneutical injustice) and (4) ‘ If you want to make an apple pie from scratch, you have to invent the universe first’ (structural inequality/power dynamics, support and community). Findings highlight the importance of considering the relationship between stigma and victimisation, and the relationship between trauma, masking, and burnout in autistic people. Reducing barriers to support and recovery are contingent on reducing structural inequality and providing better training about autistic people to frontline professionals. Lay abstract Research suggests that autistic people are more likely to be hurt by someone they know (e.g. a friend or a family member) compared to non-autistic people. In this study, we wanted to know how being hurt by someone you know affects autistic people, and what might help them recover. In total, 102 autistic people took part in an interview, where we asked questions like how being hurt by people they know had made them feel and whether anyone they had asked for help had done a good job of supporting them. We analysed what they had said using thematic analysis, which involved reading what everyone said and looking for common themes. Our findings showed that a lot of autistic people think it is normal to be hurt by people you know because it has happened to them so much. This makes many autistic people feel like they need to mask parts of themselves to stay safe, but this also makes them really exhausted. It took some people a long time to realise what had happened to them was wrong, and it was hard to ask for help. People who did ask for help often had bad experiences with professionals (e.g. police) who did not know much about autistic people. They said better support would come from creating more autistic-run support groups and educating people about autism. These findings are important for working out how to help people who have been hurt by people they know.

Large epidemiological studies often require sample transportation and storage, presenting unique considerations when applying advanced lipidomics techniques. The goal of this study was to acquire lipidomics data on plasma and serum samples stored at potential preanalytical conditions (e.g., thawing, extracting, evaporating), systematically monitoring lipid species for a period of one month. Split aliquots of 10 plasma samples and 10 serum samples from healthy individuals were kept in three temperature-related environments: refrigerator, laboratory benchtop, or heated incubator. Samples were analyzed at six different time points over 28 days using a Bligh & Dyer lipid extraction protocol followed by direct infusion into a lipidomics platform using differential mobility with tandem mass spectrometry. The observed concentration changes over time were evaluated relative to method and inter-individual biological variability. In addition, to evaluate the effect of lipase enzyme levels on concentration changes during storage, we compared corresponding fasting and post-prandial plasma samples collected from 5 individuals. Based on our data, a series of low abundance free fatty acid (FFA), diacylglycerol (DAG), and cholesteryl ester (CE) species were identified as potential analytical markers for degradation. These FFA and DAG species are typically produced by endogenous lipases from numerous triacylglycerols (TAGs), and certain high abundance phosphatidylcholines (PCs). The low concentration CEs, which appeared to increase several fold, were likely mass-isobars from oxidation of other high concentration CEs. Although the concentration changes of the high abundant TAG, PC, and CE precursors remained within method variability, the concentration trends of FFA, DAG, and oxidized CE products should be systematically monitored over time to inform analysts about possible pre-analytical biases due to degradation in the study sample sets.

ImportanceUnderstanding Adverse Events (AEs) associated with SARS-CoV-2 vaccination has public health implications, especially with regards to vaccine hesitancy.ObjectiveTo establish whether individuals with prior history of COVID-19 were more likely to experience AEs after BNT162b2/Pfizer vaccination, than those without previous COVID-19, and whether COVID-19-vaccination interval influenced AE severity.DesignAn observational study explored AEs after vaccination. Participants were invited to complete an electronic survey, capturing self-reported COVID-19 symptoms, PCR/antibody results, and AEs following first dose of BNT162b2/Pfizer vaccine. In a subset where PCR/antibody results could be verified, a sensitivity analysis was conducted.SettingThree North-East England hospital Trusts in the United Kingdom.ParticipantsHealthcare workers formed an opportunistic sample – 265 of 974 reported prior positive SARS-CoV-2 PCR and/or antibody.ExposureAll participants had received their first dose of BNT162b2/Pfizer vaccine.Main Outcomes and MeasuresNature, severity, duration, and onset of self-reported AEs (reported via a modified version of the FDA Toxicity Grading Scale for vaccine-associated AEs), was compared between those with and without a prior history of COVID-19, using 2-way ANCOVA and logistic regression. Effects of age, gender, illness-vaccine interval, and ongoing symptoms (‘Long-COVID’) on AEs, were also explored.ResultsOf 974 respondents (81% female, mean age 48), 265 (27%) reported previous COVID-19 infection. Within this group (symptoms median 8.9 months pre-vaccination), 30 (11%) complained of Long-COVID. The proportion reporting one moderate/severe symptom was higher in the previous COVID-19 group (56% v 47%, OR=1.5 [95%CI, 1.1–2.0], p=.009), with fever, fatigue, myalgia-arthralgia and lymphadenopathy significantly more common. There was no significant relationship between illness-vaccine interval and symptom composite score (rs=0.09, p=.44). Long-COVID was not associated with worse AEs in comparison to the group without previous COVID-19. In the smaller sensitivity analysis cohort (412 people) similar findings were obtained although only myalgia and arthralgia remained significant.Conclusions and RelevancePrior COVID-19 infection but not ongoing Long-COVID symptoms were associated with an increase in the risk of self-reported adverse events following BNT162b2/Pfizer vaccination. COVID-19 illness-vaccination interval did not significantly influence AEs. This data can support education around vaccine-associated AEs and, through improved understanding, help to combat vaccine hesitancy.Key PointsQuestionDoes previous COVID-19 infection or ‘Long-COVID’ increase the frequency of Adverse Events (AEs) following first dose of BNT162b2/Pfizer vaccination?FindingsIn a survey-based observational study, healthcare workers in the United Kingdom reported AEs experienced after their first dose of BNT162b2/Pfizer vaccine. Prior COVID-19 infection, but not Long-COVID, were associated with increased risk of self-reported AEs including lymphadenopathy post-vaccination. Duration since COVID-19 infection did not affect severity of AEs.MeaningOur study can inform education and understanding of AEs associated with COVID-19 vaccination and help to combat vaccine hesitancy.

Prior studies suggest that adverse events (AEs) following doses one and two of BNT162b2/Pfizer vaccine are worse in those with a prior history of COVID-19. To establish whether this outcome applies to a third/booster dose, we conducted a survey with 534 healthcare workers (HCW) in Northeast England, who reported AEs following all three doses of BNT162b2/Pfizer vaccine. We also explored AEs associated with concurrent seasonal influenza immunisation. For all doses of BNT162b2/Pfizer vaccine there was a cluster of systemic AEs that were consistently worse in HCWs with a prior history of COVID-19. AEs were no worse in HCWs who received their third/booster dose within 7 days of the influenza jab, rather than further apart. Gender and the presence of ongoing COVID-19 symptoms (OCS) had no effect on AEs associated with COVID-19 or influenza vaccination, though younger HCWs experienced more AEs overall. Our findings have implications for vaccine hesitancy and immunisation protocols.

Purpose: We hypothesised that cleaning the internal limiting membrane (ILM) with a flexible nitinol loop following diabetic vitrectomy without peeling may reduce the common occurrence of postoperative epiretinal membrane (ERM) formation. Methods: Consecutive patients undergoing vitrectomy for proliferative diabetic retinopathy by one surgeon from 2015 to 2019 were studied and divided into 2 cohorts: the control group underwent standard surgery, and the ILM clean group underwent additional cleaning of the macular retina using a flexible nitinol loop after vitrectomy. Masked comparison of ERM on optical coherence tomography was performed at 3 months, and visual acuity (VA) was measured until 12 months postoperatively. Results: Baseline demographics, clinical features, and protein levels were similar between cohorts. The ILM clean group (n = 56) had fewer clinically significant ERM than the control group (n = 50; 4 vs. 20%; p = 0.01), and a significantly lower proportion of the ILM clean group required revision surgery (2 vs. 14%; p = 0.02). VA in the ILM clean group was significantly better than in the control group at 3 months (0.35 vs. 0.50 logMAR; p = 0.02) but not at 12 months (0.34 vs. 0.43 logMAR; p = 0.17). Conclusion: ILM cleaning with a flexible nitinol loop following diabetic vitrectomy resulted in significant reduction in ERM formation and reduced necessity for revision surgery. There was significant improvement in VA at 3 months but not over a longer follow-up.

Interpersonal violence and victimisation (IPV) is violence and abuse that occurs within personal relationships (e.g. from a friend, or family member).Figures suggest that between 50-89% of autistic people have been victimised by someone they know1,2, and that this high prevalence is related to poor mental health outcomes for autistic people3. We conducted a qualitative exploration of autistic adult’s experiences of IPV, focussing on how it impacts their identity, and their experiences of seeking help and support.

Muller cells seem to be important in maintaining foveal morphology through connections between their foot processes and the internal limiting membrane (ILM). Internal limiting membrane peeling causes Muller cell trauma. We hypothesized that leaving a rim of unpeeled ILM around idiopathic macular holes undergoing vitrectomy surgery would improve postoperative foveal morphology and vision.Prospective pilot study of fovea-sparing ILM peeling in a consecutive cohort of patients with macular holes over a 12-month period. Spectral-domain optical coherence tomography and Early Treatment Diabetic Retinopathy Study letters best-corrected visual acuity were assessed preoperatively and postoperatively, and foveal morphology and metamorphopsia postoperatively. The foveal sparing group was compared with a second consecutive cohort who received standard ILM peeling (control group).Thirty-four eyes of 34 patients were included in each group. Groups showed no significant preoperative differences. 34/34 holes were successfully closed with surgery in the foveal sparing group and 32/34 in the control group. The foveal sparing group showed better postoperative best-corrected visual acuity (67.7 vs. 63.8, P = 0.003) and best-corrected visual acuity improvement (25.1 vs. 20.2, P = 0.03). The foveal sparing group demonstrated thicker minimum foveal thickness (211 vs. 173 μm, P = 0.002) and less steep foveal depression (158 vs. 149, P = 0.002).Preserving nonpeeled ILM around macular holes resulted in a high closure rate, improved foveal morphology, and better postoperative best-corrected visual acuity. An appropriately powered randomized controlled study is warranted.

The BNT162b2/Pfizer SARS-CoV-2 vaccine has been widely used in the UK, particularly amongst healthcare workers (HCWs). To establish whether previous COVID-19 influenced vaccine-associated Adverse Events (AEs), we conducted a survey-based study of HCWs in Northeast England. Out of 1238 HCWs, 32% self-reported prior positive PCR and/or antibody test for SARS-CoV-2. Post-dose AEs were worse in those with prior COVID-19 after the first, but not the second dose of vaccine. Second dose AEs were greater in frequency/severity, regardless of COVID-19 history, and they were more systemic in nature. Women and younger HCW were more likely to report AEs after both doses, while dosing interval had no effect on AEs. Ongoing Symptomatic COVID-19 was associated with greater frequency/severity of AEs after dose 2, but not dose one. Overall, AEs were self-limiting and short-lived (i.e.

BACKGROUND: Autistic masking is an emerging research area, and so far, research has suggested that masking has a negative effect on autistic people. Masking relates to general social practices (such as identity management) and is often driven by stigma avoidance. Many nonautistic people also experience stigma that might drive them to suppress aspects of their identity. In this study, we investigate similarities and differences in experiences of masking in autistic and nonautistic people. METHODS: We conducted an online survey about experiences and views of masking in autistic people (n = 144), neurodivergent people without an autism diagnosis (n = 49), and neurotypical people (n = 45) recruited via social media. We used thematic analysis to analyze responses to open-ended items about masking, using an inductive approach, at a semantic level, with a critical realist paradigm. RESULTS: Thematic analysis revealed that some aspects of masking are shared across autistic and nonautistic people, such as utilizing mimicry of others as a social strategy or feeling exhausted from masking. All groups reported that masking made them feel disconnected from their true sense of identity and had a negative effect on them. Other aspects of masking seemed more specific to autistic people, such as sensory suppression, and masking leading to suicidal ideation. CONCLUSIONS: Our findings suggest that many aspects of masking are experienced across different neurotypes and are likely related to outside perceptions of difference and stigma. It is likely that what we call “autistic masking” is similar to other forms of stigma management previously theorized. Some aspects of masking do seem more specific to the autistic neurotype (e.g., suppression of stimming) and should be explored further to provide support for autistic people recovering from the negative impact of masking. LAY SUMMARY: WHY WAS THIS STUDY DONE? Masking is the process of intentionally, or unintentionally, hiding aspects of yourself to avoid harm. Recent research has suggested that autistic people might mask a lot and that this can lead to long-term problems in mental health. At the moment, we do not know which parts of masking are specific to autistic people, and which parts might be experienced by others, especially other neurodivergent people who are not autistic, for example, people with attention-deficit hyperactivity disorder (ADHD). WHAT WAS THE PURPOSE OF THIS STUDY? The purpose of this study was to find out about masking in autistic and nonautistic people, to find out whether some parts of masking are specific to autistic people, or experienced by others. WHAT DID THE RESEARCHERS DO? We conducted an online survey about masking with three groups of people: autistic people, nonautistic people who have another neurodevelopmental or mental health diagnosis, such as dyslexia, ADHD, or depression, and nonautistic people who do not have any neurodevelopmental or mental health diagnoses. The survey included an open text box for people to write about their experiences of masking and their opinions about masking. We analyzed these responses using a method called “thematic analysis.” This method helps us look for ideas that people commonly talk about in their answers. WHAT WERE THE RESULTS OF THIS STUDY? We found out that autistic and nonautistic people think masking is very complicated and has a negative effect on them. Both autistic and nonautistic people said that masking made them exhausted and really unhappy and that it made them feel like people did not know the “real them.” Only autistic people mentioned that masking sometimes makes them feel suicidal and that masking includes things that other groups did not mention (such as trying to hide being upset by sensory things such as loud noise). WHAT DO THESE FINDINGS ADD TO WHAT WAS ALREADY KNOWN? Our findings suggest that some aspects of masking do not just affect autistic people (such as feeling like people do not know the real you), but other parts might be more unique to autistic people (such as hiding stims from other people). WHAT ARE POTENTIAL WEAKNESSES IN THIS STUDY? More women than men and nonbinary people took part, which means that we know more about women than other people. We also do not really know whether everyone in the nonautistic groups were really “not autistic.” They could be autistic people who do not have a diagnosis, which might affect our results. HOW WILL THESE FINDINGS HELP AUTISTIC PEOPLE NOW OR IN THE FUTURE? We hope our findings will help people to understand how masking affects autistic and nonautistic people. We also hope that people designing new studies will look at important things such as whether someone is autistic and nonbinary, or autistic and ADHD as these things might affect their experiences.

Background Face transplantation is a surgical innovation to manage people with severely interrupted facial function and form. How the public perceive face transplantation and its potential implications for the recipient, donor, and society is unclear. The aim of this study was to understand the public perception of face transplantation, including when it is appropriate, what information is required to feel adequately informed, and which factors influence a person’s willingness to donate their face. Methods This was a nationwide survey of participants representative of the GB public. A quantitative analysis was performed. Free-text qualitative responses were coded with thematic content analysis and a narrative analysis was constructed. Results The survey included 2122 participants. Face transplantation was considered worth the potential risks if it improved an individual’s quality of life, gave them a ‘normal life’, and/or increased their confidence and social interaction. Respondents were worried about the impact face transplantation might have on donor families, especially recipient families adapting to the identity of the donor. Respondents most concerned about the concept of face transplantation were aged at least 55 years (χ2(4) = 38.9, P Conclusion The public perceive emotional and psychological outcomes as equally as important as, or more important than, surgical outcomes when determining the appropriateness of face transplantation. Future research should focus on measuring and describing emotional and psychological outcomes after face transplantation.

The gold standard method of tonsillectomy is "cold steel" dissection (DT). A dissector is used to separate the tonsil and capsule from the underlying muscle layer, with diathermy / surgical ties to achieve hemostasis. It causes significant postoperative pain and risk of bleeding (return to theatre rate 1.6%). [Abstract copyright: This article is protected by copyright. All rights reserved.]

The practice of histopathological assessment of the uterus following hysterectomy for benign indications including pelvic organ prolapse (POP) surgery is common and often routine. While pathology is not anticipated, the finding of pathology requiring further action is always a concern, in particular CIN (cervical intraepithelial neoplasia) or cervical/uterine malignancy. We aimed to perform a systematic review to understand the prevalence of actionable uterine and cervical pathology in hysterectomy specimens performed for POP. A literature search was performed in January 2020 of MEDLINE, Embase and CINAHL using the Healthcare Databases Advanced Search platform. Included studies reported CIN and/or uterine/cervical malignancy in histological assessment of hysterectomy specimens performed purely for POP. Meta-analysis of prevalence was performed using the MetaXL ( www.epigear.com ) add-in for Microsoft Excel. Six hundred seventy-seven records were identified, out of which 34 studies were eligible. Overall prevalence (95% confidence interval [CI]) of endometrial cancer in 33 studies was 0.004 (0.003–0.006), I2 = 41%, number needed to treat (NNT) 1:250. Total actionable uterine pathology was 0.005 (0.003–0.006) in 33 studies, I2 = 35%, NNT = 1:200. Overall prevalence of cervical cancer in 19 papers was 0.001 (0.000–0.002), I2 = 18%, NNT = 1:1000. In 16 studies the overall prevalence of CIN was 0.013 (0.001–0.033), I2 = 95%, NNT = 1:77. Prevalence of total actionable pathology was 0.013 (0.006–0.0023), I2 = 86%, NNT = 1:77. The risk of actionable pathology is low, but not negligible. The variation between populations is wide. The prevalence of finding such pathology supports the routine practice of sending all hysterectomy specimens performed for POP for histological assessment.

Objectives\ud This is a protocol for a Cochrane Review (intervention). The objectives are as follows:\ud \ud To assess the effect of surgical interventions on postoperative visual and anatomical outcomes in people with a confirmed degenerative lamellar macular hole.

Background: Autistic masking is an emerging research area, and so far, research has suggested that masking has a negative effect on autistic people. Masking relates to general social practices (such as identity management) and is often driven by stigma avoidance. Many non-autistic people also experience stigma that might drive them to suppress aspects of their identity. In this study we investigate similarities and difference in experiences of masking in autistic and non-autistic people. Method: We conducted an online survey about experiences and views of masking in autistic people (n=144), neurodivergent people without an autism diagnosis (n=49), and neurotypical people (n=45) recruited via social media. We used thematic analysis to analyse responses to open-ended items about masking, using an inductive approach, at a semantic level, with a critical realist paradigm. Results: Thematic analysis revealed that some aspects of masking are shared across autistic and non-autistic people, such as utilising mimicry of others as a social strategy or feeling exhausted from masking. All groups reported that masking made them feel disconnected from their true sense of identity and had a negative effect on them. Other aspects of masking seemed more specific to autistic people, such as sensory suppression, and masking leading to suicidal ideation. Conclusions: Our findings suggest that many aspects of masking are experienced across different neurotypes and are likely related to outside perceptions of difference and stigma. It is likely that what we call ‘autistic masking’ is similar to other forms of stigma management previously theorised. Some aspects of masking do seem more specific to the autistic neurotype (e.g. suppression of stimming), and should be explored further in order to provide support for autistic people recovering from the negative impact of masking.

Purpose Determining which factors influence idiopathic macular hole (MH) size is important because it is a major prognostic indicator of treatment success. Foveal pit morphologic features are highly symmetrical within individuals and may influence idiopathic MH size. Using a series of patients with unilateral idiopathic MHs, we examined the foveal floor size of the fellow eye to evaluate its relationship with idiopathic MH size and postoperative outcomes. Design Retrospective observational study. Participants Two hundred forty-one participants with a unilateral idiopathic MH treated with surgery and a fellow eye with no ocular pathologic features. Methods Both eyes underwent spectral-domain (SD) OCT imaging at the time of surgery. Minimum linear diameter (MLD) and base diameter (BD) defined idiopathic MH size. Foveal floor width (FFW) and minimal foveal thickness defined foveal pit morphologic features of the fellow eye. Main Outcome Measures Baseline characteristics, SD OCT measurements, and preoperative variables were compared to determine their relationship with idiopathic MH size and postoperative visual acuity (VA) in logarithm of the minimum angle of resolution units. Results Foveal floor width was correlated with MLD (r = 0.36; P ≤ 0.001) and BD (r = 0.30; P ≤ 0.001), but not postoperative VA. Minimum linear diameter correlated with preoperative VA (r = 0.49; P ≤ 0.0001) and postoperative VA (r = 0.54; P ≤ 0.0001). A 2-stage regression model was developed to predict postoperative VA (r2 = 0.28): preoperative VA (β = 0.36; P = 0.002) explained 13% of variability and MLD (β = 0.29; P = 0.002), and idiopathic MH duration (β = 0.23; P = 0.004) explained a further 16%. Conclusions Foveal floor width of the fellow eye in patients with a unilateral idiopathic MH was correlated significantly with idiopathic MH size and may explain some of the variability in idiopathic MH size observed between individuals. However, FFW could not predict postoperative vision.

Background As of January 7, 2020, a total of 2558 hospitalized patients with nonfatal cases and 60 patients with fatal cases of e-cigarette, or vaping, product use-associated lung injury (EVALI) had been reported to the Centers for Disease Control and Prevention (CDC). Methods In a national study, we compared the characteristics of patients with fatal cases of EVALI with those of patients with nonfatal cases to improve the ability of clinicians to identify patients at increased risk for death from the condition. Health departments reported cases of EVALI to the CDC and included, when available, data from medical-record abstractions and patient interviews. Analyses included all the patients with fatal or nonfatal cases of EVALI that were reported to the CDC as of January 7, 2020. We also present three case reports of patients who died from EVALI to illustrate the clinical characteristics common among such patients. Results Most of the patients with fatal or nonfatal cases of EVALI were male (32 of 60 [53%] and 1666 of 2498 [67%], respectively). The proportion of patients with fatal or nonfatal cases was higher among those who were non-Hispanic white (39 of 49 [80%] and 1104 of 1818 [61%], respectively) than among those in other race or ethnic groups. The proportion of patients with fatal cases was higher among those 35 years of age or older (44 of 60 [73%]) than among those younger than 35 years, but the proportion with nonfatal cases was lower among those 35 years of age or older (551 of 2514 [22%]). Among the patients who had an available medical history, a higher proportion of those with fatal cases than those with nonfatal cases had a history of asthma (13 of 57 [23%] vs. 102 of 1297 [8%]), cardiac disease (26 of 55 [47%] vs. 115 of 1169 [10%]), or a mental health condition (32 of 49 [65%] vs. 575 of 1398 [41%]). A total of 26 of 50 patients (52%) with fatal cases had obesity. Half the patients with fatal cases (25 of 54 [46%]) were seen in an outpatient setting before hospitalization or death. Conclusions Chronic conditions, including cardiac and respiratory diseases and mental health conditions, were common among hospitalized patients with EVALI.

Background: The victimisation of autistic people by familiar others (interpersonal victimisation) is an understudied phenomenon despite suggestions that prevalence rates may be disproportionately high. We know very little about the way autistic people perceive these experiences, and how to support them. The aim of the current study was to explore experiences of interpersonal victimisation among autistic adults from their own perspective. Method: We recruited 43 autistic adults to take part in a qualitative online study, and asked about their experiences of being victimised or taken advantage of by people they know in the past. We analysed their comments at the semantic level using inductive thematic analysis, from a critical realist perspective. Results: We identified two key themes in the data. The first theme, ‘cycles of victimisation’ highlighted the occurrence of polyvictimisation in the sample. The second (‘perceptions of victimisation’) focussed on how these experiences were related to difficulties with trust (of both self and others), the recognition of victimisation, and heightened compliance. The participants expressed difficulty with saying no to people, and found it difficult to identify when someone had negative or manipulative intentions. Conclusions: Our findings suggest that autistic adults experience victimisation from a range of close others, and may find it difficult to recognise when someone is acting in an abusive manner. Many participants had experienced heightened compliance in response to unreasonable requests from others, however reasons for this were varied (e.g. fear, desire to avoid confrontation) and require further investigation. These findings have implications for developing supports which enable autistic adults to recognise their own boundaries and advocate for themselves, in addition to helping them to recognise what a healthy relationship looks like.

Objectives\ud To explore the impact of implementation of Community‐Acquired Pneumonia (CAP) quality care bundle.\ud \ud Setting\ud Eight acute hospitals in the North East of England and North Cumbria.\ud \ud Participants\ud ICD‐10 coded CAP aged >18 were identified. A total of 16 201 CAP patients were discharged 2016/2017 (15 707; 2015/2016 and 10 733; 2014/2015).\ud \ud Outcome measures\ud Secondary User Service (SUS) data were collected monthly from April 2014 to 2017. Data were pseudonymised and data flows governed by Data Sharing Agreements. CAP measures were based on British Thoracic Society guidance and agreed following clinician consultation. CAP admissions and individual organisational compliance with and impact of, CAP quality bundle measures was explored.\ud \ud Results\ud Average length of stay (LOS) was 10.4 days (median 6) 25% >13 days. Crude in‐hospital mortality rate was 17.6%, significantly lower (95% CI) than 19.1% in 2015/2016 and 19.3% in 2014/2015. Emergency readmissions within 28 days were 19.7% (19.2%; 2015/2016, 17.9%; 2014/2015). A total of 39.5% of patients received all appropriate care measures. Compliance has improved over time, although not for all hospitals. Most quality measures have higher mortality for those passing measures compared to those failing (P < .05 95% CI). Giving oxygen, had a significantly higher emergency readmission rate, 3.3% higher (95% CI 1.1% to 5.5%). Appropriate antibiotics and recording CURB‐65 scores reduced the emergency readmission rates (−2.7% (95% CI −4.5% to −0.8%) −2.6% (95% CI −3.8% to −1.4%), respectively, (P = ns)).\ud \ud Conclusion\ud CAP accounts for significant bed days, mortality and readmissions. Although mortality was lower, LOS and readmission rates were not, despite improvements in compliance after implementation of the care bundle. Care bundle use remained sub‐optimal.

Apolipoproteins measured in plasma or serum are potential biomarkers for assessing metabolic irregularities that are associated with the development of cardiovascular disease (CVD). LC-MS/MS allows quantitative measurement of multiple apolipoproteins in the same sample run. However, the accuracy and precision of the LC-MS/MS measurement depends on the reproducibility of the enzymatic protein digestion step. With the application of an immobilized enzyme reactor (IMER), the reproducibility of the trypsin digestion can be controlled with high precision via flow rate, column volume and temperature. In this report, we demonstrate the application of an integrated IMER-LC-MS/MS platform for the simultaneous quantitative analysis of eight apolipoproteins. Using a dilution series of a characterized serum pool as calibrator, the method was validated by repeated analysis of pooled sera and individual serum samples with a wide range of lipid profiles, all showing intra-assay CV < 4.4% and inter-assay CV < 8%. In addition, the method was compared with traditional homogeneous digestion coupled LC-MS/MS for the quantification of apoA-I and apoB-100. Applied in large scale human population studies, this method can serve the translation of a wider panel of apolipoprotein biomarkers from research to clinical application. SIGNIFICANCE: Currently, the translation of apolipoprotein biomarkers to clinical application is impaired because of the high cost of large cohort studies using traditional single-analyte immunoassays. The application of on-line tryptic digestion coupled with LC-MS/MS analysis is an effective way to address this problem. In this work we demonstrate a high throughput, multiplexed, automated proteomics workflow for the simultaneous analysis of multiple proteins.

As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950-Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each laboratory using a different lipidomics workflow. A total of 1,527 unique lipids were measured across all laboratories and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra- and interlaboratory quality control and method validation. These analyses were performed using nonstandardized laboratory-independent workflows. The consensus locations were also compared with a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement.

Apolipoprotein L1 (ApoL1) is an intriguing protein and was shown to be involved in numerous diseases states such as chronic kidney disease (CKD), cardiovascular diseases(CVD), Human Immunodeficiency Virus associated Nephropathy (HIVAN), systemic lupus erythematosus (SLE) (associated collapsing glomerulopathy), and type 2 diabetes(especially for people of African descent). People of African ancestry were shown to have either one or two alleles which contribute to their susceptibility of diseases related to CKD. ApoL1 is characterized as a HDL binding protein. In this work we studied ApoL1 binding to HDL by using a gentle size fractionation technique, asymmetric flow field-flow fractionation (AF4), and liquid chromatography (LC) with quantitative tandem mass spectrometry (MS/MS) detection. We injected 50 uL of whole plasma onto the AF4 system, where smaller HDL particles eluted first followed by the larger HDL, and then by LDL, IDL and VLDL particles. The lipoproteins were separated into 40 fractions and quantitatively analyzed using on-line trypsin digestion coupled with LC-MS/MS analysis. We analyzed plasma samples both with and without purification by UC. When HDL particles were separated by UC, apoL1 was found in the higher density HDL fractions, as was also found by previous studies. When HDL separated using AF4 with or without UC, apoL1 eluted in the larger HDL size region (12-13 nm). With UC vs. without UC, the apoL1 peptide signal intensities were reduced by more than 50%, indicating significant loss of apoL1 from the surface of HDL particles during UC. Our data on apoL1 provides further evidence for the fact that size and density are not directly interconvertible physical characteristics of lipoproteins. Furthermore, our data shows that apoL1 is also an example of exchangeable apolipoproteins whose binding can be significantly diminished due to the effect of intense shear forces during UC separation. This work also demonstrates the advantages of utilizing AF4-IMER-UPLC-MS/MS methodology to separate lipoprotein particles and study of their apolipoprotein composition.

To identify the incidence, presenting features, treatment, and clinical course of optic disc pit maculopathy (ODPM) in the United Kingdom (UK).A 2-year nationwide prospective population-based study.All new incident cases of ODPM presenting to UK ophthalmologists using the British Ophthalmic Surveillance Unit monthly reporting system.All reporting ophthalmologists were sent an initial questionnaire requesting data on previous medical and ophthalmic history, presentation details, investigation findings, and management. A further questionnaire was sent at 12 months post diagnosis to ascertain further outcome data.Visual acuity at initial presentation, at 1 year, and after any intervention. Foveal involvement and optical coherence tomography (OCT) findings, including retinal layers affected, and the location and size of the optic disc pit. Management, including observation, vitrectomy, and associated procedures.There were 74 confirmed new cases, giving an annual incidence of approximately 1 per 2 million. Complete data were available on 70 patients (70 eyes) at baseline and 68 after 1 year. There were 35 (50%) female patients with a mean age of 35 years (range, 3-82 years). Visual acuity at baseline ranged from 6/5 to hand movements. In 43 patients (61%) subretinal fluid (SRF) was present, whereas 27 (39%) had intraretinal fluid only. The presence of SRF was associated with worse vision and foveal involvement. Of the 53 eyes initially observed with 1-year follow-up, 10 (19%) deteriorated and 9 (16%) improved on OCT; eyes with SRF were more likely to worsen and those without SRF were more likely to improve. Fifteen of the 70 patients (21%) at baseline had primary surgery and a further 10 had deferred surgery within 1 year of presentation; 19 of these 25 eyes (76%) showed anatomic success with a dry fovea at 1 year of follow-up, and 15 (60%) had a greater than 0.1 logMAR improvement in visual acuity.The incidence and presenting features of ODPM were defined. Patients with SRF had worse vision and were more likely to deteriorate than patients with intraretinal fluid only. Surgery was anatomically successful in 75% of cases. Patients without SRF tended to remain stable with observation.

The number of caesarean sections at maternal request without medical indication is increasing. We aimed to explore the views of pregnant women, midwives and doctors using six hypothetical clinical scenarios and compare group views on: (a) perceived appropriateness of requests for caesarean section and (b) the reasons underlying these requests.A questionnaire was distributed to 166 pregnant women, 31 midwives and 52 doctors within maternity units at two hospitals in the North East region of England. Six hypothetical clinical scenarios for maternal requests were used: (1) uncomplicated first pregnancy, (2) one previous normal delivery, (3) one previous instrumental delivery, (4) one previous caesarean section, (5) one previous caesarean section with vaginal delivery since and (6) uncomplicated twin pregnancy. To highlight the differences in group responses, two main questions were asked for each scenario: 1. Should women be able to request a caesarean section? 2. What do you feel are the reasons for requesting a caesarean section? Data was analysed using Chi-squared or likelihood ratio as appropriate.In scenarios 1-3, professional groups were 'less likely' than pregnant women to always support a request (2.4% vs. 19.4%), (2.6% vs. 15.6%), (4.6% vs. 22%), (p0.001). No significant differences were shown between doctors and midwives except for scenario 6 (twins), where midwives more often felt maternal requests should be declined (26.1% vs. 1.9%) (p=0.001). Multiparous women (n=95) were more likely to agree 'sometimes' to maternal requests in scenarios 1, compared to nulliparous women (n=71) (21.1% vs. 4.2%) (p=0.04). 'Safety of the baby' was ranked highly with pregnant women in scenarios 1-3 (mean 24.4%, range [15.8-38%]) compared with healthcare professionals (7.6% [3.4-12.8%]). However in scenario 3, healthcare professionals attributed 'fear of injury to self' (29.6%) as the most likely reason compared to 14.6% of pregnant women.Healthcare professionals and pregnant women's views differ significantly. Multiparous patients' views differ from those who have not had children before. We should provide clearer information on risks and benefits which encompass areas that concern women most.

To improve awareness and recognition of chronic bacterial prostatitis (CBP) and chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) among non-specialists and patients. To provide guidance to healthcare professionals treating patients with CBP and CP/CPPS, in both non-specialist and specialist settings. To promote efficient referral of care between non-specialists and specialists and the involvement of the multidisciplinary team (MDT).The guideline population were men with CBP or CP/CPPS (persistent or recurrent symptoms and no other urogenital pathology for ≥3 of the previous 6 months). Consensus recommendations for the guidelines were based on a search to identify literature on the diagnosis and management of CBP and CP/CPPS (published between 1999 and February 2014). A Delphi panel process was used where high-quality, published evidence was lacking.CBP and CP/CPPS can present with a wide range of clinical manifestations. The four main symptom domains are urogenital pain, lower urinary tract symptoms (LUTS - voiding or storage symptoms), psychological issues and sexual dysfunction. Patients should be managed according to their individual symptom pattern. Options for first-line treatment include antibiotics, α-adrenergic antagonists (if voiding LUTS are present) and simple analgesics. Repeated use of antibiotics, such as quinolones, should be avoided if there is no obvious symptomatic benefit from infection control or cultures do not support an infectious cause. Early use of treatments targeting neuropathic pain and/or referral to specialist services should be considered for patients who do not respond to initial measures. An MDT approach (urologists, pain specialists, nurse specialists, specialist physiotherapists, general practitioners, cognitive behavioural therapists/psychologists, and sexual health specialists) is recommended. Patients should be fully informed about the possible underlying causes and treatment options, including an explanation of the chronic pain cycle.Chronic prostatitis can present with a wide variety of signs and symptoms. Identification of individual symptom patterns and a symptom-based treatment approach are recommended. Further research is required to evaluate management options for CBP and CP/CPPS.

The continued advance of antibiotic resistance in clinically relevant bacterial strains necessitates the development and refinement of assays that can rapidly and cost-effectively identify bacteria and determine their susceptibility to a panel of antibiotics. A methodology is described herein that exploits the specificity and physiology of the Staphylococci bacteriophage K to identify Staphylococcus aureus (S. aureus) and determine its susceptibility to clindamycin and cefoxitin. The method uses liquid chromatography-mass spectrometry to monitor the replication of bacteriophage after it is used to infect samples thought to contain S. aureus. Amplification of bacteriophage K indicates the sample contains S. aureus, for it is only in the presence of a suitable host that bacteriophage K can amplify. If bacteriophage amplification is detected in samples containing the antibiotics clindamycin or cefoxitin, the sample is deemed to be resistant to these antibiotics, respectively, for bacteriophage can only amplify in a viable host. Thus, with a single work flow, S. aureus can be detected in an unknown sample and susceptibility to clindamycin and cefoxitin can be ascertained. This Article discusses implications for the use of bacteriophage amplification in the clinical laboratory.

Lipoproteins are complex molecular assemblies that are key participants in the intricate cascade of extracellular lipid metabolism with important consequences in the formation of atherosclerotic lesions and the development of cardiovascular disease. Multiplexed mass spectrometry (MS) techniques have substantially improved the ability to characterize the composition of lipoproteins. However, these advanced MS techniques are limited by traditional pre-analytical fractionation techniques that compromise the structural integrity of lipoprotein particles during separation from serum or plasma. In this work, we applied a highly effective and gentle hydrodynamic size based fractionation technique, asymmetric flow field-flow fractionation (AF4), and integrated it into a comprehensive tandem mass spectrometry based workflow that was used for the measurement of apolipoproteins (apos A-I, A-II, A-IV, B, C-I, C-II, C-III and E), free cholesterol (FC), cholesterol esters (CE), triglycerides (TG), and phospholipids (PL) (phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and lysophosphatidylcholine (LPC)). Hydrodynamic size in each of 40 size fractions separated by AF4 was measured by dynamic light scattering. Measuring all major lipids and apolipoproteins in each size fraction and in the whole serum, using total of 0.1 ml, allowed the volumetric calculation of lipoprotein particle numbers and expression of composition in molar analyte per particle number ratios. Measurements in 110 serum samples showed substantive differences between size fractions of HDL and LDL. Lipoprotein composition within size fractions was expressed in molar ratios of analytes (A-I/A-II, C-II/C-I, C-II/C-III. E/C-III, FC/PL, SM/PL, PE/PL, and PI/PL), showing differences in sample categories with combinations of normal and high levels of Total-C and/or Total-TG. The agreement with previous studies indirectly validates the AF4-LC-MS/MS approach and demonstrates the potential of this workflow for characterization of lipoprotein composition in clinical studies using small volumes of archived frozen samples.

In this report we demonstrate a practical multivariate design of experiment (DoE) approach for asymmetric flow field-flow fractionation (AF4) method optimization using separation of lipoprotein subclasses as an example. First, with the aid of commercially available software, we built a full factorial screening design where the theoretical outcomes were calculated by applying established formulas that govern AF4 channel performance for a 5–35 nm particle size range of interest for lipid particles. Second, using the desirable ranges of instrumental parameters established from theoretical optimization, we performed fractional factorial DoE for AF4 separation of pure albumin and ferritin with UV detection to narrow the range of instrumental parameters and allow optimum size resolution while minimizing losses from membrane immobilization. Third, the optimal range of conditions were tested using response surface DoE for sub-fractionation of high and low density lipoproteins (HDL and LDL) in human serum, where the recovery of the analytes were monitored by fraction collection and isotope-dilution LC-MS/MS analysis of each individual fraction for cholesterol and apolipoproteins (ApoA-1 and ApoB-100). Our results show that DoE is an effective tool in combining AF4 theoretical knowledge and experimental data in finding the most optimal set of AF4 instrumental parameters for quantitative coupling with LC-MS/MS measurements.

The bacterial surfaceome, comprising outer membrane-sorted and/or associated (i.e., cell transporters), cell surface-exposed (i.e., adhesins) and extracellularly secreted proteins (i.e., toxins), has been characterized in bacterial pathogens, such as Bordetella pertussis (Bp) to provide information for use in development of diagnostic and prevention strategies. This protein subset has clinical significance, as these bacterial proteins are often associated with attachment to host cells, microbial pathogenesis and antibody-mediated immunity. Here we describe classical surface membrane protein enrichment techniques, followed by proteomic methodologies, such as gel-free protein separation and antibody-affinity capture technologies in combination with nano-liquid chromatography mass spectrometry, for the identification and characterization of Bp surfaceome proteins.

The bacterial surfaceome, comprising outer membrane-sorted and/or associated (i.e., cell transporters), cell surface-exposed (i.e., adhesins) and extracellularly secreted proteins (i.e., toxins), has been characterized in bacterial pathogens, such as Bordetella pertussis (Bp) to provide information for use in development of diagnostic and prevention strategies. This protein subset has clinical significance, as these bacterial proteins are often associated with attachment to host cells, microbial pathogenesis and antibody-mediated immunity. Here we describe classical surface membrane protein enrichment techniques, followed by proteomic methodologies, such as gel-free protein separation and antibody-affinity capture technologies in combination with nano-liquid chromatography mass spectrometry, for the identification and characterization of Bp surfaceome proteins.

Purpose\ud To evaluate the effect of a high-vacuum setting versus a low-vacuum setting on the efficiency of phacoemulsification.\ud \ud Setting\ud Sunderland Eye Infirmary, Sunderland, United Kingdom.\ud \ud Design\ud Prospective clinical trial.\ud \ud Methods\ud Consecutive patients having cataract surgery in 2014 were recruited. Cataract surgery was performed by 2 experienced surgeons using a phacoemulsification machine with monitored forced infusion. The cataractous lens was split into 2 heminuclei using the stop-and-chop technique; in 1 heminucleus, phacoemulsification and aspiration used a high-vacuum setting (600 mm Hg; treatment group) and in the other heminucleus, a low-vacuum setting (350 mm Hg; control group). The high and low settings were alternated by case per the operating list to reduce surgeon bias. The main outcome measures were cumulative dissipated energy (CDE) and active heminucleus removal time.\ud \ud Results\ud One hundred sixty patients (160 eyes) were enrolled in the study, and 158 were included in the analysis. The CDE per heminucleus was significantly lower with the high-vacuum setting than with the low-vacuum setting (mean 2.81 percent-seconds; 95% confidence interval (CI), 2.44-3.21 versus 3.81 percent-seconds; 95% CI, 3.38-4.20; P < .001). The active heminucleus removal time was significantly shorter in the high-vacuum group than the low-vacuum group (mean 27.77 seconds; 95% CI, 25.26-30.19 versus 33.59 seconds; 95% CI, 31.07-35.92; P < .001). The observed differences were independent of the surgeon, patient age and sex, incision size, and nucleus density. No intraoperative complications were observed in either group.\ud \ud Conclusion\ud A high-vacuum setting improved phacoemulsification efficiency using an active fluidics system and torsional phacoemulsification

A multiplexed, quantitative analytical workflow was applied to 120 fasting human serum samples collected from 30 normolipidemic, and 90 dyslipidemic donors. The analysis included separation by asymmetric flow field-flow fractionation while collecting fractions with ~1 nm increments of 7-15 nm (HDL), 20-30 nm (LDL) and greater than 30 nm lipoproteins. Size separation was followed by concentration measurements in each size fraction for non-polar lipids (FC, CE and TG), polar lipids (PC, SM, PE and PI), and apolipoproteins (apos A-I, A-II, A-IV, B, C-I, C-II, C-III and E); using three parallel, high throughput, quantitative liquid chromatography-tandem mass spectrometry methods developed in our laboratory. The average hydrodynamic size in each size fraction was also determined by dynamic light scattering. Measuring all major lipid and protein components and the size in all fractions allowed volumetric estimation of lipoprotein particle numbers (Lp-P). In the LDL size range of 22-26 nm, the calculated mean (N=120 samples) of the average (N=5 fraction/sample) apoB/LDL-P was 1.00 (Stdev=0.31, N=120). The resulting highly comprehensive data set allowed the evaluation of correlations between cardiovascular disease (CVD) risk factors and metabolic pathway specific indicators. For example, multivariate modelling of Total-TG/HDL-C ratios (1.5-17.5) showed negative correlation with HDL-apoC-III/LDL-apoC-III ratios.

Hypertriglyceridemia is frequently observed with diabetes mellitus (DM) and is associated with increased cardiovascular disease (CVD) risk. In conditions of insulin deficiency or resistance, increased levels of free fatty acids are seen by the liver, which trigger increased lipogenesis and secretion of triglyceride (TG) rich lipoprotein (TRL) particles. However, a high number of specific ApoC-III on TRL particles suppress their return to the liver leading to a continuous circulation of highly atherogenic TRL remnants. To better understand the effect of intra-individual temporal variations on the differentiation of lipid and apolipoprotein profiles in DM vs. normal individuals, a small time course study over a 4 week period was performed on 3 normolipidemic, and 3 type II DM patients. Our laboratory has developed a method to separate lipoproteins based on hydrodynamic size coupled with mass spectrometry based analysis. Whole serum and lipoprotein size fractions were analyzed for 8 apolipoproteins (ApoA-I, ApoA-II, ApoA-IV, B-100, ApoC-I, ApoC-II, ApoC-III and E) and non-polar lipids (FC, CE, TG) while monitoring other proteins (ApoD, ApoM, CETP, LCAT, PLTP, PON1 and SAA4). In whole serum over the course of 4 weeks, ApoA-IV levels of DM vs. normal subjects differed the most, 3.2 (0.7) vs. 1.8 (0.4) μM, respectively, more significantly than intra-group or intra-individual variations. On the contrary, significantly lower whole serum levels were found for FC, CE, ApoM and PON1 (Prob

The presence of diabetes mellitus in patients significantly increases the risk of cardiovascular disease, among many negative health outcomes. Several research groups have reported lipid species anomalies in subjects showing insulin resistance, including higher concentrations of sphingomyelin species relative to normal controls. Advances in lipidomic methodologies has allowed for the profiling of numerous lipid species in a single extraction and analytical run. We used the SCIEX Lipidyzer platform to determine the absolute concentration of 770 distinct lipid species from 52 subjects categorized into hyperglycemic (n=30) and normal subjects (n=22). Lipid species were determined from the following classes: cholesterol esters (CE), ceramides (CER), diacylglycerols (DAG), dihydroceramide (DCER), free fatty acids (FFA), hexosylceramides (HCER), lactosylceramides (LCER), lysophosphatidylcholines (LPC), lysophosphatidylethanolamines (LPE), phosphatidylcholines (PC), phosphatidylethanolamines (LPE), sphingomyelins (SM), and triacylglycerols (TAG). After normalizing by the sum of total apolipoprotein A and apolipoprotein B (to account for the possibility of reduced particle number due to medicinal intervention), lipids both by class and species were compared using one way analysis of variance. On a class level, relative to normal subjects, hyperglycemic patients showed increased levels of CER and FFA. Individual lipid species of these classes that were higher in hyperglycemic patients include CER(18:0), CER(20:0), CER(24:1), FFA(16:0), FFA(16:1), FFA(18:0), FFA(18:1), FFA(18:2), FFA(18:3), and FFA(20:3) Differences were also found in SM concentrations between hyperglycemic and normal patients resulting in a higher the SM/PC ratio, indicating changes in the lipid fluidity of the shell in lipoprotein particles of hyperglycemic patients. On a species level, SM(16:0), SM(18:1), and SM(24.1), were elevated in hyperglycemic subjects relative to normal subjects. This finding of elevated unsaturated SM species in hyperglycemic patients is at odds with other studies where it was found that the saturated SM moieties were elevated in insulin resistant subjects.

Clearance of elevated triglycerides (TG) level in response to fat challenge is a clinically significant indicator of CVD risk. HDL can play a key role in facilitating TG metabolism explaining the reverse correlation of HDL levels with CVD risk. In this study 6 individuals, low BMI (21-25) and 3 high BMI (30-49) were challenged with a meal containing ~90 g of total fat. Blood samples were drawn in fasting state before meal and 3 times after meal in 2, 4 and 6 hours. Serum was analyzed by a quantitative, multiplexed analytical workflow which included analysis of whole serum and the separation of lipoproteins by asymmetric flow field-flow fractionation where fractions were collected with 1 nm increments of 7-15 nm (HDL), 20-30 nm (LDL) and >30 nm lipoprotein classes. Dynamic light scattering was used to determine hydrodynamic size in each size fraction. Whole serum and individual fractions were analyzed to determine concentrations of apolipoproteins (apo A-I, A-II, A-IV, B, C-I, C-II, C-III and E), and non-polar lipids (FC, CE and TG), using two parallel liquid chromatography tandem mass spectrometry methods developed in our laboratory. In whole serum, the total TG levels for low and high BMI donors, relative to pre-meal levels (57-137 mg/dL and 111-183 md/dL), peaked at between the 2 and 4 hours post meal (103-214 mg/dL and 210-309 mg/dL). LCAT were lower (20%) and CETP levels were higher (30%), in low vs high BMI groups, but did not show change in repose to the fat challenge. In both low and high BMI groups, the most significant change between pre-meal and post-meal after 2 hours occurred in HDL-ApoC-I, HDL-ApoC-II, and HDL-ApoC-III levels mainly in the HDL size range of 9-12 nm (medium HDL) and at 13-16 nm (large HDL), with concurrent TG increase observed in LDL and remnant fractions (Prob

Bacterial fatty acid profiling is a well-established technique for bacterial identification. Ten bacteria were analyzed using both positive- and negative-ion modes with a modified matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) approach using CaO as a matrix replacement (metal oxide laser ionization MS (MOLI MS)). The results show that reproducible lipid cleavage similar to thermal in situ tetramethyl ammonium hydroxide saponification/derivatization had occurred. Principal component analysis showed that replicates from each organism grouped in a unique space. Cross validation (CV) of spectra from both ionization modes resulted in greater than 94% validation of the data. When CV results were compared for the two ionization modes, negative-ion data produced a superior outcome. MOLI MS provides clinicians a rapid, reproducible and cost-effective bacterial diagnostic tool.

Serum Institute of India is among the world's largest vaccine producers. Here, we report the complete genome sequences for four Bordetella pertussis strains used by Serum Institute of India in the production of whole-cell pertussis vaccines.

Antibiotic resistance continues to contribute significantly to morbidity and mortality across the world. Developing new tests for antibiotic-resistant bacteria is a core action to combat resistant infections. We describe a method that uses phage amplification detection (PAD) combined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to rapidly identify Staphylococcus aureus and determine phenotypic susceptibility to cefoxitin. Samples tested for S. aureus are incubated together with bacteriophage in the presence and absence of cefoxitin and subjected to rapid trypsin digestion followed by MALDI-MS analysis. Tryptic peptides derived from amplified phage proteins can be detected by MALDI-MS, as validated by time-of-flight (TOF)/TOF analysis of each peptide combined with database searching. Methicillin-resistant S. aureus show significant phage amplification in the presence of cefoxitin, while methicillin-sensitive S. aureus show no phage amplification relative to a no-antibiotic control. We also show that PAD methodology can be implemented on an FDA-approved commercial MALDI-MS bacterial identification system to identify S. aureus and determine antibiotic susceptibility. The novelty of this assay includes the use of phage-derived tryptic peptides as detected by MALDI-MS to monitor the results of PAD on an instrument common to many modern microbiology laboratories. Electronic supplementary material The online version of this article (doi:10.1007/s00216-016-0070-3) contains supplementary material, which is available to authorized users.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a valuable tool for rapid bacterial detection and identification but is limited by the need for relatively high cell count samples, which have been grown under strictly controlled conditions. These requirements can be eliminated by the natural infection of a viable bacterial species of interest with a host-specific phage. This produces a rapid increase in phage protein concentrations in comparison to bacterial concentrations, which can in turn be exploited as a method for signal amplification during MALDI-TOF MS. One drawback to this approach is the requirement for repetitive, time-consuming sample preparation and analysis applied over the course of a phage infection to monitor phage concentrations as a function of time to determine the MALDI-TOF MS detection limit. To reduce the requirement for repeated preparation and analysis, a modified phage therapy model was investigated as a means for predicting the time during a given phage infection when a detectable signal would occur. The modified model used a series of three differential equations composed of predetermined experimental parameters including phage burst size and burst time to predict progeny phage concentrations as a function of time. Using Yersinia pestis with plague diagnostic phage φA1122 and Escherichia coli with phage MS2 as two separate, well-characterized model phage-host pairs, we conducted in silico modeling of the infection process and compared it with experimental infections monitored in real time by MALDI-TOF MS. Significant agreement between mathematically calculated phage growth curves and those experimentally obtained by MALDI-TOF MS was observed, thus verifying this method's utility for significant time and labor reduction.

Mass spectrometry (MS) coupled with 1-D and 2-D electrophoresis can be utilized to detect and identify immunogenic proteins, but these methods are laborious and time-consuming. We describe an alternative, simple, rapid gel-free strategy to identify multiple immunogenic proteins from Bordetella pertussis (Bp). It couples immunoprecipitation to nano liquid chromatography- tandem mass spectrometry (IP-nLC-MS/MS) and is significantly both time- and labor-saving. We developed a gel-free magnetic bead-based immunoprecipitation (IP) method using different NP-40/PBS concentrations in which solubilized proteins of Bp Tohama I membrane fractions were precipitated with polyclonal rabbit anti-Bp whole cell immune sera. Immune complexes were analyzed by MS and Scaffold analysis (>95% protein identification probability). Total immunoproteins identified were 50, 63 and 49 for 0.90%, 0.45% and 0.22% NP-40/PBS buffer concentrations respectively. Known Bp proteins identified included pertactin, serotype 2 fimbrial subunit and filamentous hemagglutinin. As proof of concept that this gel-free protein immunoprecipitation method enabled the capture of multiple immunogenic proteins, IP samples were also analyzed by SDS-PAGE and immunoblotting. Bypassing gels and subjecting immunoprecipitated proteins directly to MS is a simple and rapid antigen identification method with relatively high throughput. IP-nLC-MS/MS provides a novel alternative approach for current methods used for the identification of immunogenic proteins.

In late February 2008, law enforcement officials in Las Vegas, Nevada, discovered in a hotel room, a copy of The Anarchist Cookbook, suspected castor beans and a "white powder" thought to be a preparation of ricin. Ricin is a deadly toxin from the seed of the castor bean plant (Ricinus communis). The United States regulates the possession, use, and transfer of ricin and it is the only substance considered a warfare agent in both the Chemical and the Biological Weapons Conventions. Six samples obtained from the hotel room were analyzed by laboratories at the Centers for Disease Control and Prevention using a panel of biological and mass spectrometric assays. The biological assays (real time-PCR, time resolved fluorescence and cytotoxicity) provided presumptive evidence of active ricin in each of the samples. This initial screen was followed by an in-depth analysis using a novel, state-of-the-art mass spectrometry-based ricin functional assay and high sensitivity tandem mass spectrometry for protein identification. Mass spectrometric analysis positively identified ricin and confirmed that in each of the samples it was enzymatically active. The tandem mass spectrometry analysis used here is the most selective method available to detect ricin toxin. In each sample, ricin was unequivocally identified along with other R. communis plant proteins, including the highly homologous protein RCA120. Although database searches using tandem mass spectra acquired from the samples indicated that additional controlled substances were not present in these samples, the mass spectrometric results did provide extensive detail about the sample contents. To the best of our knowledge following a review of the available literature, this report describes the most detailed analysis of a white powder for a public health or forensic investigation involving ricin.