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Washing red blood cell (RBC) units prior to transfusion is indicated for certain patients. In the United States, units stored at 1°C-6°C or transported at 1°C-10°C are available for issue up to 24 h, if not used immediately. The washing procedure commonly utilizes room temperature saline resulting in units starting out above the allowed temperature range. This leads to wastage if units are issued and returned too quickly before having a chance to equilibrate in a transport cooler.Here we performed an experimental study of washed RBC quality comparing "ideal" storage conditions in a blood bank refrigerator to a "real-world" simulation of unit transport, including holding in a transport cooler. Twelve RBC units were washed and allocated evenly into either condition.Measurements at 0, 1, 3, 6, 12, and 24 h post-washing revealed that placement in a transport cooler was associated with higher unit temperature prior to 12 h (p = .013) with a maximum difference of 9.3°C. Despite this difference, several measures of unit quality including extracellular potassium, pH, lactate, and free hemoglobin were indistinguishable between conditions (p = .382, .224, .286, .691, respectively). We selected half of the tested units from our irradiated inventory and confirmed increased potassium leak (p .001) and accumulation of free hemoglobin (p = .012) in irradiated units.Washed units stored under approved transport conditions are acceptable to return to inventory up to 24 h after washing and we provide a prediction interval-based temperature threshold for rejecting these units, permitting reduced waste.

Despite the enormous advancements in nanomedicine research, a limited number of nanoformulations are available on the market, and few have been translated to clinics. An easily scalable, sustainable, and cost-effective manufacturing strategy and long-term stability for storage are crucial for successful translation. Here, we report a system and method to instantly formulate NF achieved with a nanoscale polyelectrolyte coacervate-like system, consisting of anionic pseudopeptide poly(l-lysine isophthalamide) derivatives, polyethylenimine, and doxorubicin (Dox) via simple "mix-and-go" addition of precursor solutions in seconds. The coacervate-like nanosystem shows enhanced intracellular delivery of Dox to patient-derived multidrug-resistant (MDR) cells in 3D tumor spheroids. The results demonstrate the feasibility of an instant drug formulation using a coacervate-like nanosystem. We envisage that this technique can be widely utilized in the nanomedicine field to bypass the special requirement of large-scale production and elongated shelf life of nanomaterials.

Synchronisation of Plasmodium cultures is essential to investigate the complexities of time-dependent events associated with the asexual blood stage of the malaria parasite life cycle. Here we describe a procedure using ML10, a highly specific inhibitor of the parasite cyclic GMP-dependent protein kinase (PKG), to attain high synchronicity of Plasmodium falciparum and P. knowlesi asexual blood-stage cultures and to obtain high levels of arrested mature schizonts as well as viable released merozoites. Additionally, we describe how to use ML10 to improve the transfection efficiency of P. falciparum parasites and also how to derive the half maximal effective concentration (EC

SummaryCommon variable immunodeficiency disorders (CVID) are multi-system disorders where target organ damage is mediated by infective, autoimmune and inflammatory processes. Bronchiectasis is probably the most common disabling complication of CVID. The risk factors for bronchiectasis in CVID patients are incompletely understood. The New Zealand CVID study (NZCS) is a nationwide longitudinal observational study of adults, which commenced in 2006. In this analysis, the prevalence and risk factors for bronchiectasis were examined in the NZCS. After informed consent, clinical and demographic data were obtained with an interviewer-assisted questionnaire. Linked electronic clinical records and laboratory results were also reviewed. Statistical methods were applied to determine if variables such as early-onset disease, delay in diagnosis and increased numbers of infections were associated with greater risk of bronchiectasis. One hundred and seven adult patients with a diagnosis of CVID are currently enrolled in the NZCS, comprising approximately 70% of patients known to have CVID in New Zealand. Fifty patients (46·7%) had radiologically proven bronchiectasis. This study has shown that patients with compared to those without bronchiectasis have an increased mortality at a younger age. CVID patients with bronchiectasis had a greater number of severe infections consequent to early-onset disease and delayed diagnosis. Indigenous Māori have a high prevalence of CVID and a much greater burden of bronchiectasis compared to New Zealand Europeans. Diagnostic latency has not improved during the study period. Exposure to large numbers of infections because of early-onset disease and delayed diagnosis was associated with an increased risk of bronchiectasis. Earlier diagnosis and treatment of CVID may reduce the risk of bronchiectasis and premature death in some patients.

OBJECTIVES/HYPOTHESIS The objective of this meta-analysis was to look at the pooled prevalence of symptoms, laboratory tests, and imaging of all COVID-19 infected patients. This will allow better identification of potential COVID-19 patients and take appropriate precautions. STUDY DESIGN Meta analysis. METHODS We searched three databases, PubMed, EMBASE, and Ovid to identify studies published between Dec-2019 and May-2020. All studies reporting upper-aerodigestive symptoms of COVID-19 infection were included. The meta-analysis was conducted following meta-analyses of observational studies in epidemiology (MOOSE) guidelines, which have evaluated the pooled prevalence of 14 symptoms and nine laboratory investigations. RESULTS Based on inclusion criteria, 67 publications consisting of 8302 patients were included. Among adults, the pooled proportion of hypertensive and diabetic patients was 18% and 7%. Cough (53% [0.46-0.61]), anosmia (38% [0.19-0.58]), loss/distortion of taste (31% [0.17-0.45]), and nasal obstruction (26% [0.12-0.39]) were the most common ear, nose & throat (ENT) symptoms. Fever (69% [0.62-0.76]) and fatigue (31% [0.26-0.37]) were the commonest generalized symptoms. C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) were raised in 56% (0.41-0.71) and 49% (0.21-0.77), respectively. Interestingly, lymphopenia (41% [0.30-0.53]) and leucopenia (22% [0.16-0.29]) were more common than lymphocytosis (33% [0.02-0.64]) and leucocytosis (12% [0.09-0.16]). Fever (69% vs. 44%), cough (53% vs. 33%), and dyspnea (20% vs. 4%) were more common in adults as compared to the pediatric population. Diarrhea was more common among the pediatric cases (12%) versus (9%). The pooled estimate of fatality was 4%. CONCLUSIONS The most commonly experienced ENT symptom was cough followed by anosmia and dysguesia. Raised ESR and CRP with leukopenia and lymphopenia are common laboratory findings. Majority of the infected patients had abnormal computed tomography findings. COVID infection is less severe in pediatric patients. Laryngoscope, 131:1254-1265, 2021.

Purpose: The 3 Wishes Project (3WP) promotes holistic end-of-life care in the intensive care unit (ICU) to honor dying patients, support families, and encourage clinician compassion. Organ donation is a wish that is sometimes made by, or on behalf of, critically ill patients. Our objective was to describe the interface between the 3WP and organ donation as experienced by families, clinicians, and organ donation coordinators. Methods: In a multicenter evaluation of the 3WP in 4 Canadian ICUs, we conducted a thematic analysis of transcripts from interviews and focus groups with clinicians, organ donation coordinators, and families of dying or died patients for whom donation was considered. Results: We analyzed transcripts from 26 interviews and 2 focus groups with 18 family members, 17 clinicians, and 6 organ donation coordinators. The central theme describes the mutual goals of the 3WP and organ donation—emphasizing personhood and agency across the temporal continuum of care. During family decision-making, conversations encouraged by the 3WP can facilitate preliminary discussions about donation. During preparation for donation, memory-making activities supported by the 3WP redirect focus toward personhood. During postmortem family care, the 3WP supports families, including when donation is unsuccessful, and highlights aspirational pursuits of donation while encouraging reflections on other fulfilled wishes. Conclusions: Organ donation and the 3WP provide complementary opportunities to engage in value-based conversations during the dying process. The shared values of these programs may help to incorporate organ donation and death into a person’s life narrative and incorporate new life into a person’s death narrative.

A dual-channel optical coherence tomography system with wavelengths in the visible and near-infrared light ranges can provide both structural and functional information for retinal microvasculature simultaneously. We applied this integrated system in an ongoing clinical study of patients with various retinal pathologies. Here, we present case study results of patients with diabetic retinopathy, central retinal vein occlusion, and sickle cell retinopathy compared to a healthy subject. For the first time, this comparison validates the system’s ability to detect structural anomalies in both en face and B-scan images with simultaneous retinal optical coherence tomography angiography and measurement of sO2 in parafoveal vessels that are around 20–30 µm in diameter. This integrated system represents a powerful instrument with potentially far-reaching clinical implications for the early detection and diagnosis of retinal vascular diseases.

The stroma in pancreatic ductal adenocarcinoma (PDAC) contributes to its immunosuppressive nature and therapeutic resistance. Herein we sought to modify signaling and enhance immunotherapy efficacy by targeting multiple stromal components through both intracellular and extracellular mechanisms.A murine liver metastasis syngeneic model of PDAC was treated with focal adhesion kinase inhibitor (FAKi), anti-programmed cell death protein 1 (PD-1) antibody, and stromal hyaluronan (HA) degradation by PEGylated recombinant human hyaluronidase (PEGPH20) to assess immune and stromal modulating effects of these agents and their combinations.The results showed that HA degradation by PEGPH20 and reduction in phosphorylated FAK expression by FAKi leads to improved survival in PDAC-bearing mice treated with anti-PD-1 antibody. HA degradation in combination with FAKi and anti-PD-1 antibody increases T-cell infiltration and alters T-cell phenotype toward effector memory T cells. FAKi alters the expression of T-cell modulating cytokines and leads to changes in T-cell metabolism and increases in effector T-cell signatures. HA degradation in combination with anti-PD-1 antibody and FAKi treatments reduces granulocytes, including granulocytic- myeloid-derived suppressor cells and decreases C-X-C chemokine receptor type 4 (CXCR4)-expressing myeloid cells, particularly the CXCR4-expressing granulocytes. Anti-CXCR4 antibody combined with FAKi and anti-PD-1 antibody significantly decreases metastatic rates in the PDAC liver metastasis model.This represents the first preclinical study to identify synergistic effects of targeting both intracellular and extracellular components within the PDAC stroma and supports testing anti-CXCR4 antibody in combination with FAKi as a PDAC treatment strategy.

The literature is divided with regards to contralateral tonsillectomy in a known/suspected case of ipsilateral tonsillar malignancy. In this study, we evaluate the incidence of indolent synchronous contralateral tonsillar malignancy (SCTC) in patients with known ipsilateral tonsillar malignancy.All patients diagnosed with ipsilateral tonsillar carcinoma (TC) at a tertiary teaching center between January 2016 and December 2019 were screened. None of the patients were suspected to have bilateral TC. All patients underwent appropriate imaging in the form of Magnetic resonance imaging and computed tomography of head and neck region and then underwent bilateral tonsillectomy. The prevalence of bilateral tonsillar malignancy and the factors predicting them were analyzed.In all 59 patients were included in the study. The mean and median age of the patient population was 60.8 and 59 years, respectively, with a male to female ratio of 3.2:1. The incidence of bilateral tonsillar malignancy in carcinoma of unknown primary (CUP) was 3/10 (33.3%). Among the remaining 49 patients, incidence of synchronous contralateral tonsillar carcinoma (SCTC) was 2/49 (4.08%). Overall, 5/59 (8.5%) patients had synchronous bilateral tonsillar malignancy. Furthermore, dysplasia was found in the contralateral tonsil in 4/10 (40%) CUP patients. Among the remaining 49 patients, dysplasia was seen in the contralateral tonsil in 20/49 (40.8%) patients. The absence of p16 expression predicted higher probability of SCTC. Factors like gender, T stage, nodal status or smoking did not predict SCTC.We recommend bilateral tonsillectomy in all patients with suspected or proven TC (unilateral or bilateral) and CUP as it helps identify indolent SCTC and it does not add any significant morbidity to the patients.III.

To evaluate the utility of positron-emission tomography (FDG PET) in initial staging and management of head and neck cancers. This is a retrospective study of 99 treatment naive head and neck cancer patients treated between January 2017 and December 2020 at a tertiary teaching centre. Change in initial staging and management was noted based on PET scan compared to cross-sectional imaging (CT and MRI). There were 73 (73.7%) males and 26 (26.2%) females with male-to-female ratio of 2.8:1.Overall, change in management was seen in 36/99 (36.4%) patients due to PET scan. With regards to initial staging, T, N and M stage was changed in 14/99 (14.1%), 19/99 (19.1%) and 3/99 (3%) patients, respectively. These changes were significantly higher in patients with unknown primary (63.3%, p value −0.001) and N3 (41%, p −0.045) nodal disease. PET-CT plays an important role in appropriate initial staging and subsequent treatment planning of head and neck cancers. Initial staging PETCT changes management in 36.4% cases. Accuracy of various different imaging modalities have been compared.

To evaluate the feasibility of a clinical trial involving participants with concussion randomized to treatments designed to address fear avoidance or endurance coping, which are risk factors for disability. A secondary objective was to evaluate whether each treatment could affect selective change on targeted coping outcomes.Randomized controlled trial.Outpatient concussion clinics.Adults (N=73, mean age=42.5y) who had persistent postconcussion symptoms and high avoidance or endurance behavior were enrolled at a mean of 12.9 weeks post injury. Ten participants did not complete treatment.Participants were randomized to an interdisciplinary rehabilitation program delivered via videoconferencing and tailored to avoidance coping (graded exposure therapy [GET]) or endurance coping (operant condition-based pacing strategies plus mindfulness training [Pacing+]).Feasibility outcomes included screening efficiency, accrual, credibility, treatment fidelity, adherence, and retention. Avoidance was measured with the Fear Avoidance Behavior after Traumatic Brain Injury Questionnaire and endurance behavior with the Behavioral Response to Illness Questionnaire.Screening efficiency, or the proportion of clinic patients who were assessed for eligibility, was 44.5% (275 of 618). A total of 65.8% (73 of 111) of eligible patients were randomized (37 to GET, 36 to Pacing+), meeting accrual targets; 91.7% (55 of 60) of participants perceived treatment as credible. Therapists covered a mean of 96.8% of essential prescribed elements, indicating excellent fidelity. The majority (71.2%; 47 of 66) of participants consistently attended treatment sessions and completed between-session homework. Retention was strong, with 65 of 73 (89%) randomized participants completing the outcome assessment. GET was associated with greater posttreatment reductions in avoidance behavior compared with Pacing+ (Cohen's dThese findings support a future efficacy-focused clinical trial. GET has the potential to selectively reduce fear avoidance behavior after concussion, and, via this mechanism, to prevent or reduce disability.

Preoperative carbohydrate beverages have been shown to be beneficial in improving patient outcomes. There have been several investigations into the safety of maltodextrin as a preoperative carbohydrate. Although alternative preoperative carbohydrate sources have been proposed, there have been few investigations into the safety and gastric emptying of novel carbohydrate beverages. The present study aimed to compare the gastric emptying of phytoglycogen and maltodextrin to evaluate safety for use as presurgical carbohydrate beverages. In a quasi-experimental design, ten healthy participants orally consumed either a 12.5% maltodextrin or a 12.5% phytoglycogen solution. Gamma scintigraphy was used to evaluate gastric emptying at baseline at 45, 90, and 120 min. Serum insulin and serum glucose were measured at baseline at 15, 30, 45, 60, 90, and 120 min. Gastric volume was significantly lower in the phytoglycogen group at 45 min (p = 0.01) and 90 min (p = 0.01), but this difference lost significance at 120 min (p = 0.17). There were no significant differences between treatments for serum insulin or serum glucose at any time point. This study indicates that the gastric emptying of phytoglycogen is comparable to maltodextrin at 120 min after ingestion, opening the opportunity for the study of alternative carbohydrates for utilization as preoperative carbohydrates.

DNMDP and related compounds, or velcrins, induce complex formation between the phosphodiesterase PDE3A and the SLFN12 protein, leading to a cytotoxic response in cancer cells that express elevated levels of both proteins. The mechanisms by which velcrins induce complex formation, and how the PDE3A-SLFN12 complex causes cancer cell death, are not fully understood. Here, we show that PDE3A and SLFN12 form a heterotetramer stabilized by binding of DNMDP. Interactions between the C-terminal alpha helix of SLFN12 and residues near the active site of PDE3A are required for complex formation, and are further stabilized by interactions between SLFN12 and DNMDP. Moreover, we demonstrate that SLFN12 is an RNase, that PDE3A binding increases SLFN12 RNase activity, and that SLFN12 RNase activity is required for DNMDP response. This new mechanistic understanding will facilitate development of velcrin compounds into new cancer therapies., The small molecule DNMDP acts as a velcrin by inducing complex formation between phosphodiesterase PDE3A and SLFN12, which kills cancer cells that express sufficient levels of both proteins. Here, the authors present the cryo-EM structure of the DNMDP-stabilized PDE3A-SLFN12 complex and show that SLFN12 is an RNase. PDE3A binding increases SLFN12 RNase activity, and SLFN12 RNase activity is required for DNMDP-mediated cancer cell killing.

The recent eighth edition of the American Joint Committee on Cancer (AJCC) staging system has introduced depth of invasion (DOI) as one of its important components. DOI is also important for deciding neck management in superficial tongue cancers. Magnetic resonance imaging (MRI) is mainly used to assess DOI, and in this study we have evaluated the efficacy of MRI to detect it. This is a retrospective study consisting of 60 treatment-naive tongue cancer patients operated on between July 2017 and June 2019. Patients underwent MR imaging on an Optima MR450W 1.5T unit, and MRI was reported by two experienced ead and eck adiologistss. Postoperative histological DOI was considered the gold standard. The correlation coefficient was derived for postoperative DOI and MRI-detected DOI. A subgroup analysis of superficial tongue cancer was also done. The mean MRI DOI was 13.7 mm and the mean histological DOI 12.45 mm. The shrinkage factor was 0.6 mm. Pearson's correlation coefficient was 0.80 (p=

Objective The objective was to assess the diagnostic efficacy of combined computed tomography (CT) and magnetic resonance imaging (MRI) in detecting metastases of oral cancers to lymph nodes in the neck. Study Design In total, 105 patients with previously untreated oral squamous cell carcinoma underwent both computed tomography (CT) and magnetic resonance imaging (MRI) within 6 weeks before tumor excision and selective neck dissection. The diagnostic efficacy of combined CT and MRI was compared to published data on the efficacy of CT and MRI alone. Results The sensitivity, specificity, positive and negative predictive values, and accuracy of diagnosis based on CT and MRI were 80%, 90%, 80%, 90%, and 87%, respectively. These values were similar to or higher than those reported in a meta-analysis for CT or MRI alone. The highest and lowest sensitivities were found for metastatic nodes at levels I (75%) and III (33%). The highest and lowest specificities were discovered for nodes at levels IV (99%) and I (88%). Conclusion Combined CT and MRI may be superior to a single modality alone in detecting metastatic neck nodes. This may have an advantage in both pre-operative planning and neck surveillance in superficial oral cancers.

Background The minimum duration of pulselessness required before organ donation after circulatory determination of death has not been well studied. Methods We conducted a prospective observational study of the incidence and timing of resumption of cardiac electrical and pulsatile activity in adults who died after planned withdrawal of life-sustaining measures in 20 intensive care units in three countries. Patients were intended to be monitored for 30 minutes after determination of death. Clinicians at the bedside reported resumption of cardiac activity prospectively. Continuous blood-pressure and electrocardiographic (ECG) waveforms were recorded and reviewed retrospectively to confirm bedside observations and to determine whether there were additional instances of resumption of cardiac activity. Results A total of 1999 patients were screened, and 631 were included in the study. Clinically reported resumption of cardiac activity, respiratory movement, or both that was confirmed by waveform analysis occurred in 5 patients (1%). Retrospective analysis of ECG and blood-pressure waveforms from 480 patients identified 67 instances (14%) with resumption of cardiac activity after a period of pulselessness, including the 5 reported by bedside clinicians. The longest duration after pulselessness before resumption of cardiac activity was 4 minutes 20 seconds. The last QRS complex coincided with the last arterial pulse in 19% of the patients. Conclusions After withdrawal of life-sustaining measures, transient resumption of at least one cycle of cardiac activity after pulselessness occurred in 14% of patients according to retrospective analysis of waveforms; only 1% of such resumptions were identified at the bedside. These events occurred within 4 minutes 20 seconds after a period of pulselessness. (Funded by the Canadian Institutes for Health Research and others.).

STRUCTURED ABSTRACTBackgroundEndothelial cell (EC) activation, endotheliitis, vascular permeability, and thrombosis have been observed in patients with severe COVID-19, indicating that the vasculature is affected during the acute stages of SARS-CoV-2 infection. It remains unknown whether circulating vascular markers are sufficient to predict clinical outcomes, are unique to COVID-19, and if vascular permeability can be therapeutically targeted.MethodsEvaluating the prevalence of circulating inflammatory, cardiac and EC activation markers, and the development of a microRNA atlas in 241 patients with suspected SARS-CoV-2 infection, allowed their prognostic value to be assessed by a Random Forest model machine learning approach. Subsequent ex vivo experiments assessed EC permeability responses to patient plasma and were used to uncover modulated gene regulatory networks from which rational therapeutic design was inferred.FindingsMultiple inflammatory and EC activation biomarkers were associated with mortality in COVID-19 patients and in severity-matched SARS-CoV-2-negative patients, while dysregulation of specific microRNAs at presentation was specific for poor COVID-19-related outcomes and revealed disease-relevant pathways. Integrating the datasets using a machine learning approach further enhanced clinical risk prediction for in-hospital mortality. Exposure of ECs to COVID-19 patient plasma resulted in severity-specific gene expression responses and EC barrier dysfunction which was ameliorated using angiopoietin-1 mimetic or recombinant Slit2-N.InterpretationIntegration of multi-omics data identified microRNA and vascular biomarkers prognostic of in-hospital mortality in COVID-19 patients and revealed that vascular stabilizing therapies should be explored as a treatment for endothelial dysfunction in COVID-19, and other severe diseases where endothelial dysfunction has a central role in pathogenesis.RESEARCH IN CONTEXTEvidence before this studyWhile diagnostic testing has allowed for the rapid identification of COVID-19 cases, the lack of post-diagnosis risk assessment metrics, especially among the highest-risk subgroups, thereby undermined the cascade and allocation of care. To date, the integration of clinical data with broad omics technologies has opened up new avenues for efficiently delineating complex patient phenotypes and their associations with clinical outcomes, with circulating profiles of plasma microRNAs (miRNA), in particular, having been shown to be tightly associated with disease, and capable of providing not only detailed prognostic information but also mechanistic insight.Added value of this studyMarkers of endothelial dysfunction at presentation, while indicative of poor outcomes in COVID-19-positive patients, likely reflect systemic vascular dysfunction in critically ill patients and are not specific to SARS-CoV-2 infection. More so, the generation of a plasma microRNA atlas uncovers COVID-19-specific prognostic markers and multiple disease-specific pathways of interest, including endothelial barrier dysfunction. Furthermore, synthesis of electronic health record data with clinically relevant multi-omic datasets using a machine learning approach provides substantially better metrics by which mortality can be estimated in patients with severe COVID-19. Finally, targeted stabilization of the endothelial barrier with Q-Peptide and Slit2-N are novel therapeutic avenues that should be explored in COVID-19 patients.Implications of all the available evidenceTogether, our work provides biological insight into the role of the endothelium in SARS-CoV-2 infection, the importance of miRNA as disease- and pathway-specific biomarkers, and the exciting possibility that endothelial barrier stabilizing treatments might hold promise in COVID-19.

Mortality due to Covid-19 is highly associated with advanced age, owing in large part to severe lower respiratory tract infection. SARS-CoV-2 utilizes the host ACE2 receptor for infection. Whether ACE2 abundance in the lung contributes to age-associated vulnerability is currently unknown. We set out to characterize the RNA and protein expression profiles of ACE2 in aging human lung in the context of phenotypic parameters likely to affect lung physiology. Examining publicly available RNA sequencing data, we discovered that mechanical ventilation is a critical variable affecting lung ACE2 levels. Therefore, we investigated ACE2 protein abundance in patients either requiring mechanical ventilation or spontaneously breathing. ACE2 distribution and expression were determined in archival lung samples by immunohistochemistry (IHC). Tissues were selected from the specimen inventory at a large teaching hospital collected between 2010-2020. Twelve samples were chosen from patients receiving mechanical ventilation for acute hypoxic respiratory failure (AHRF). Twenty samples were selected from patients not requiring ventilation. We compared samples across age, ranging from 40-83 years old in the ventilated cohort and 14-80 years old in the non-ventilated cohort. Within the alveolated parenchyma, ACE2 expression is predominantly observed in type II pneumocytes (or alveolar type II / AT2 cells) and alveolar macrophages. All 12 samples from our ventilated cohort showed histologic features of diffuse alveolar damage including reactive, proliferating AT2 cells. In these cases, ACE2 was strongly upregulated with age when normalized to lung area (p = 0.004) or cellularity (p = 0.003), associated with prominent expression in AT2 cells. In non-ventilated individuals, AT2 cell reactive changes were not observed and ACE2 expression did not change with age when normalized to lung area (p = 0.231) or cellularity (p = 0.349). Additionally, we observed prominent pulmonary endothelial ACE2 expression in 2 patients on either an angiotensin-converting enzyme inhibitor (ACEI) or angiotensin receptor blocker (ARB). In summary, ACE2 expression increases with age in the setting of alveolar damage observed in patients on mechanical ventilation, providing a potential mechanism for higher Covid-19 mortality in the elderly.

In the United Kingdom (UK) the estimated prevalence of dental infection involving the supporting bone is 2%, and from 2014-2015 there were 2281 admissions in England alone due to dental abscess. We undertook an analysis of 184 dental abscesses that required surgical drainage, as there is surprisingly little in the literature on the subject. This was a retrospective study of 184 consecutive patients with dental abscesses who were admitted between January 2016 and September 2019. On admission, all patients had orthopantomograms (OPG) and baseline blood tests. Surgical drainage was performed under a general or local anaesthetic and a pus swab sent for culture and sensitivity. The submandibular space was the most commonly involved site and paediatric patients most often presented with buccal space abscesses. A lower molar tooth was the cause in 132 patients. White blood cells (WBC) and C-reactive protein (CRP) were both raised in 63.6% (n=117), but were normal in 4.9% (n=9). The remaining patients had either raised WBC (2.7%) or CRP (28.8%). Streptococcus milleri was the most common organism isolated in 66.6% (n=42). There was no association between CRP or WBC values and duration of hospital stay. Paediatric patients had a shorter duration of admission (1.96 days vs 2.81 days) and significantly lower CRP values (120.9 vs 45.7; p=0.001). The submandibular space was the commonest site involved and mandibular molars the most frequent source of infection. An elevated CRP value appeared to be a more sensitive indicator of infection in this study population. Adult and paediatric patients present in a different manner.

In this report, we explored the benefits of cyclic ion mobility (cIM) mass spectrometry in the analysis of isomeric post-transcriptional modifications of RNA. Standard methyl-cytidine samples were initially utilized to test the ability to correctly distinguish different structures sharing the same elemental composition, and thus molecular mass. Analyzed individually, the analytes displayed characteristic arrival times (t(D)) determined by the different positions of the modifying methyl groups onto the common cytidine scaffold. Analyzed in mixture, the widths of the respective signals resulted in significant overlap that initially prevented their resolution on the t(D) scale. The separation of the four isomers was achieved by increasing the number of passes through the cIM device, which enabled to fully differentiate the characteristic ion mobility behaviors associated with very subtle structural variations. The placement of the cIM device between the mass-selective quadrupole and the time-of-flight analyzer allowed us to perform gas-phase activation of each of these ion populations, which had been first isolated according to a common mass-to-charge ratio, and then separated on the basis of different ion mobility behaviors. The observed fragmentation patterns confirmed the structures of the various isomers, thus substantiating the benefits of complementing unique t(D) information with specific fragmentation data to reach more stringent analyte identification. These capabilities were further tested by analyzing natural mono-nucleotide mixtures obtained by exonuclease digestion of total RNA extracts. In particular, the combination of cIM separation and post-mobility dissociation allowed us to establish the composition of methyl-cytidine and methyl-adenine components present in the entire transcriptome of HeLa cells. For this reason, we expect that this technique will benefit not only epitranscriptomics studies requiring the determination of identity and expression levels of RNA modifications, but also metabolomics investigations involving the analysis of natural extracts that may possibly contain subsets of isomeric/isobaric species.

Background Inter-individual variability during sepsis limits appropriate triage of patients. Identifying, at first clini-cal presentation, gene expression signatures that predict subsequent severity will allow clinicians to identify the most at-risk groups of patients and enable appropriate antibiotic use.& nbsp;Methods Blood RNA-Seq and clinical data were collected from 348 patients in four emergency rooms (ER) and one intensive-care-unit (ICU), and 44 healthy controls. Gene expression profiles were analyzed using machine learning and data mining to identify clinically relevant gene signatures reflecting disease severity, organ dysfunction, mortal-ity, and specific endotypes/mechanisms.& nbsp;Findings Gene expression signatures were obtained that predicted severity/organ dysfunction and mortality in both ER and ICU patients with accuracy/AUC of 77-80%. Network analysis revealed these signatures formed a coherent biological program, with specific but overlapping mechanisms/pathways. Given the heterogeneity of sepsis, we asked if patients could be assorted into discrete groups with distinct mechanisms (endotypes) and varying severity. Patients with early sepsis could be stratified into five distinct and novel mechanistic endotypes, named Neutrophilic-Suppressive/NPS, Inflammatory/INF, Innate-Host-Defense/IHD, Interferon/IFN, and Adaptive/ADA, each based on ~& nbsp;200 unique gene expression differences, and distinct pathways/mechanisms (e.g., IL6/STAT3 in NPS). Endo-types had varying overall severity with two severe (NPS/INF) and one relatively benign (ADA) groupings, consistent with reanalysis of previous endotype studies. A 40 gene-classification tool (accuracy=96%) and several gene-pairs (accuracy=89-97%) accurately predicted endotype status in both ER and ICU validation cohorts.& nbsp;Interpretation The severity and endotype signatures indicate that distinct immune signatures precede the onset of severe sepsis and lethality, providing a method to triage early sepsis patients.& nbsp;Copyright (C) 2021 The Authors. Published by Elsevier B.V.

Objectives To curtail inappropriate plasma transfusions, we instituted clinical decision support as an alert upon order entry if the patient's recent international normalized ratio (INR) was 1.7 or less. Methods The alert was suppressed for massive transfusion and within operative or apheresis settings. The plasma order was automatically removed upon alert acceptance while clinical exception reasons allowed for continued transfusion. Alert impact was studied comparing a 7-month control period with a 4-month intervention period. Results Monthly plasma utilization decreased 17.4%, from a mean ± SD of 3.40 ± 0.48 to 2.82 ± 0.6 plasma units per hundred patient days (95% confidence interval [CI] of difference, -0.1 to 1.3). Plasma transfused below an INR of 1.7 or less decreased from 47.6% to 41.6% (P = .0002; odds ratio, 0.78; 95% CI, 0.69-0.89). The alert recommendation was accepted 33% of the time while clinical exceptions were chosen in the remaining cases (active bleeding, 31%; other clinical indication, 33%; and apheresis, 2%). Alert acceptance rate varied significantly among different provider specialties. Conclusions Clinical decision support can help curtail inappropriate plasma use but needs to be part of a comprehensive strategy including audit and feedback for comprehensive, long-term changes.

Objectives The diagnosis of heparin-induced thrombocytopenia (HIT) ideally requires a functional assay to confirm. 14C-serotonin release assay (SRA) as "gold standard" is technically challenging and unsuitable for routine use. We conducted a study to assess the performance of whole-blood impedance aggregometry (WBIA) as a simple and rapid HIT functional assay. Methods Platelet factor 4 (PF4)/immunoglobulin G (IgG) antibody, WBIA, and SRA were tested on 70 patients suspected of having HIT. Patients with a 4Ts score of 4 or more, positive PF4/IgG, and positive SRA were considered HIT positive; others were designated HIT negative. Results WBIA had 85.7% (6/7) sensitivity and 98.4% (61/62) specificity, which were not statistically different compared with SRA. Sixty-two of 70 patients had concordant results (five positive and 57 negative) by both WBIA and SRA. Eight discordant cases revealed the importance of recognizing donor effect, interferences, and the presence of heparin-independent or non-heparin-dependent antibodies in functional assays. Conclusions Implementation of WBIA could facilitate timely diagnosis and management of HIT.

Objective: To perform a systematic review and meta-analysis to compare the diagnostic accuracy of CT and initial reverse transcriptase polymerase chain reaction (RT-PCR) for detecting COVID-19 infection. Methods: We searched three databases, PubMed, EMBASE, and EMCARE, to identify studies reporting diagnostic accuracy of both CT and RT-PCR in detecting COVID-19 infection between December 2019 and May 2020. For accurate comparison, only those studies that had patients undergoing both CT and RT-PCR were included. Pooled diagnostic accuracy of both the tests was calculated by using a bivariate random effects model. Results: Based on inclusion criteria, only 11 studies consisting of 1834 patients were included in the final analysis that reported diagnostic accuracy of both CT and RT-PCR, in the same set of patients. Sensitivity estimates for CT scan ranged from 0.69 to 1.00 and for RT-PCR varied ranging from 0.47 to 1.00. The pooled estimates of sensitivity for CT and RT-PCR were 0.91 [95% CI (0.84–0.97)] and 0.84 [95% CI (0.71–0.94)], respectively. On subgroup analysis, pooled sensitivity of CT and RT-PCR was 0.95 [95% CI (0.88–0.98)] and 0.91 [95% CI (0.80–0.96), p = o.ooo1]. The pooled specificity of CT and RT-PCR was 0.31 [95% CI (0.035–0.84)] and 1.00 [95% CI (0.96–1.00)]. Conclusion: CT is more sensitive than RT-PCR in detecting COVID-19 infection, but has a very low specificity. Advances in knowledge: Since the results of a CT scan are available quickly, it can be used as an adjunctive initial diagnostic test for patients with a history of positive contact or epidemiological history.

Objective: This paper discusses the challenges and successes of engaging young people in a project aimed at developing an online counselling intervention for young people affected by cancer. Context: For younger people with a diagnosis of cancer or who are caring for someone with cancer the psychosocial consequences can create significant challenges for their social and educational development. Whilst young people have been shown to be reluctant to make use of traditional face-to-face counselling, research is beginning to suggest that effective therapeutic relationships can be formed with young people online. Design: The first phase of the study involved working with a “Young Persons Panel” of healthy school pupils and university students to develop and pilot an online counselling intervention and study materials in preparation for a pilot evaluation of the intervention. Intervention: An avatar-based virtual reality counselling world was created where young people can create their own avatar and receive counselling over the internet from a qualified counsellor via an avatar in a virtual reality world. Findings: The process of engaging young people in the C:EVOLVE project enabled a unique intervention to be developed and demonstrated positive developmental opportunities. However, despite the rigorous approach to the development of the intervention, initial attempts within the pilot evaluation phase of the study showed difficulties recruiting to the study and this phase of the study has currently ceased whilst further exploratory work takes place Conclusion: This study has demonstrated the complexities of intervention development and evaluation research targeted at young people and the challenges created when attempting to bring clinical practice and research evaluation together. Keywords: online counselling, cancer, young people, intervention development, avatar counselling.

Nintedanib and pirfenidone are approved treatments for idiopathic pulmonary fibrosis (IPF). This open-label, two-group trial investigated the pharmacokinetic drug–drug interaction between these two drugs in patients with IPF.Subjects not treated with antifibrotics at screening (group 1, n=20) received a single nintedanib dose (150 mg) followed by pirfenidone (titrated to 801 mg thrice daily) for 3 weeks, with a further single nintedanib dose (150 mg) on the last day (day 23). Subjects treated with pirfenidone at screening (group 2, n=17) continued to receive pirfenidone alone (801 mg thrice daily) for 7 days, then co-administered with nintedanib (150 mg twice daily) for a further 7 days, before single doses of both treatments on day 16.In group 1, adjusted geometric mean (gMean) ratios (with/without pirfenidone) were 88.6% and 80.6% for nintedanib area under the plasma concentration–time curve (AUC) and maximum plasma concentration (Cmax), respectively. In group 2, gMean ratios (with/without nintedanib) were 97.2% and 99.5% for pirfenidone AUC and Cmax, respectively. For all parameters, the 90% confidence intervals included 100%, suggesting similar exposure for administration alone and when co-administered. Both treatments were well tolerated.These data indicate there is no relevant pharmacokinetic drug–drug interaction between nintedanib and pirfenidone when co-administered in IPF patients.

Study objective Online educational resources such as blogs are increasingly used for education by emergency medicine clinicians. The Social Media Index was developed to quantify their relative impact. The Medical Education Translational Resources: Indicators of Quality (METRIQ) study was conducted in part to determine the association between the Social Media Index score and quality as measured by gestalt and previously derived quality instruments. Methods Ten blogs were randomly selected from a list of emergency medicine and critical care Web sites. The 2 most recent clinically oriented blog posts published on these blogs were evaluated with gestalt, the Academic Life in Emergency Medicine Approved Instructional Resources (ALiEM AIR) score, and the METRIQ-8 score. Volunteer raters (including medical students, emergency medicine residents, and emergency medicine attending physicians) were identified with a multimodal recruitment methodology. The Social Media Index was calculated in February 2016, November 2016, April 2017, and December 2017. Pearson's correlations were calculated between the Social Media Index and the average rater gestalt, ALiEM AIR score, and METRIQ-8 score. Results A total of 309 of 330 raters completed all ratings (93.6%). The Social Media Index correlated moderately to strongly with the mean rater gestalt ratings (range 0.69 to 0.76) and moderately with the mean rater ALiEM AIR score (range 0.55 to 0.61) and METRIQ-8 score (range 0.53 to 0.57) during the month of the blog post's selection and for 2 years after. Conclusion The Social Media Index's correlation with multiple quality evaluation instruments over time supports the hypothesis that it is associated with overall Web site quality. It can play a role in guiding individuals to high-quality resources that can be reviewed with critical appraisal techniques.

Pediatric-type follicular lymphoma and pediatric marginal zone lymphoma are two of the rarest B-cell lymphomas. These lymphomas occur predominantly in the pediatric population and show features distinct from their more common counterparts in adults: adult-type follicular lymphoma and adult-type nodal marginal zone lymphoma. Here we report a detailed whole-exome deep sequencing analysis of a cohort of pediatric-type follicular lymphomas and pediatric marginal zone lymphomas. This analysis revealed a recurrent somatic variant encoding p.Lys66Arg in the transcription factor interferon regulatory factor 8 (IRF8) in 3 of 6 cases (50%) of pediatric-type follicular lymphoma. This specific point mutation was not detected in pediatric marginal zone lymphoma or in adult-type follicular lymphoma. Additional somatic point mutations in pediatric-type follicular lymphoma were observed in genes involved in transcription, intracellular signaling, and cell proliferation. In pediatric marginal zone lymphoma, no recurrent mutation was identified; however, somatic point mutations were observed in genes involved in cellular adhesion, cytokine regulatory elements, and cellular proliferation. A somatic variant in AMOTL1, a recurrently mutated gene in splenic marginal zone lymphoma, was also identified in a case of pediatric marginal zone lymphoma. The overall non-synonymous mutational burden was low in both pediatric-type follicular lymphoma and pediatric marginal zone lymphoma (4.6 mutations per exome). Altogether, these findings support a distinctive genetic basis for pediatric-type follicular lymphoma and pediatric marginal zone lymphoma when compared with adult subtypes and to one another. Moreover, identification of a recurrent point mutation in IRF8 provides insight into a potential driver mutation in the pathogenesis of pediatric-type follicular lymphoma with implications for novel diagnostic or therapeutic strategies.

Study objective Open educational resources such as blogs are increasingly used for medical education. Gestalt is generally the evaluation method used for these resources; however, little information has been published on it. We aim to evaluate the reliability of gestalt in the assessment of emergency medicine blogs. Methods We identified 60 English-language emergency medicine Web sites that posted clinically oriented blogs between January 1, 2016, and February 24, 2016. Ten Web sites were selected with a random-number generator. Medical students, emergency medicine residents, and emergency medicine attending physicians evaluated the 2 most recent clinical blog posts from each site for quality, using a 7-point Likert scale. The mean gestalt scores of each blog post were compared between groups with Pearson's correlations. Single and average measure intraclass correlation coefficients were calculated within groups. A generalizability study evaluated variance within gestalt and a decision study calculated the number of raters required to reliably (>0.8) estimate quality. Results One hundred twenty-one medical students, 88 residents, and 100 attending physicians (93.6% of enrolled participants) evaluated all 20 blog posts. Single-measure intraclass correlation coefficients within groups were fair to poor (0.36 to 0.40). Average-measure intraclass correlation coefficients were more reliable (0.811 to 0.840). Mean gestalt ratings by attending physicians correlated strongly with those by medical students (r=0.92) and residents (r=0.99). The generalizability coefficient was 0.91 for the complete data set. The decision study found that 42 gestalt ratings were required to reliably evaluate quality (>0.8). Conclusion The mean gestalt quality ratings of blog posts between medical students, residents, and attending physicians correlate strongly, but individual ratings are unreliable. With sufficient raters, mean gestalt ratings provide a community standard for assessment.

Objectives: The rate of plasma product wastage for the United States in 2011 was approximately 1.8%. The plasma wastage rate at our institution was higher, mainly due to products returned out of temperature range from procedural areas. A process review and intervention to reduce plasma wastage was undertaken, which included modifications to our transport cooler. Methods: A new cooler system was designed, and this device was implemented alongside an updated protocol for delivering plasma while also enhancing the previous RBC cooler validation time. We audited plasma and RBC product wastage prior to these interventions, from January 2013 to February 2014, vs after the intervention from April 2014 to March 2015. Results: After the intervention, the monthly plasma wastage rate declined 60% (12.6 units/100 units transfused preintervention vs 5.0 units/100 units transfused postintervention; P

The impact of failed cerebrospinal fluid leak (CSF) leak repair in endoscopic skull base surgery has not been adequately studied.In this investigation we reviewed patients who had undergone endoscopic skull base surgery between 2002 and 2014 at 7 international centers. Demographic variables, comorbidities, tumor characteristics, and repair techniques were evaluated to determine association with successful repair of CSF leak. Postoperative complications and length of stay were compared among groups.Data were collected on 2097 patients who were divided into 3 groups: (1) those with no intraoperative leak (n = 1533); (2) those with successful repair of their intraoperative leak (n = 452); and (3) those with failed repair (n = 112). Compared with successful repair, failed repair was associated with an increased risk of intracranial infection (odds ratio [OR], 5.6; 95% confidence interval [CI], 5.3-13.15), pneumocephalus (OR, 16; 95% CI, 5.8-44.4), 30-day readmission (OR, 8.4; 95% CI, 5.3-13.5), reoperation (OR, 115.4; 95% CI, 56.3-236.8), and prolonged hospital stay (14.9 vs 7.0 days, p0.01). Outcomes in patients who had successful repairs of intraoperative leaks were similar to those who never had leakage. Intraoperative use of pedicled nasoseptal flaps was associated with successful repair (OR, 0.60; 95% CI, 0.34-0.92).Intraoperative CSF leaks are a frequent and expected occurrence during endoscopic skull base surgery. Failed CSF leak repair has a significant impact on patient outcomes, with increased rates of postoperative pneumocephalus, intracranial infections, reoperation, deep vein thrombosis, readmission, and prolonged hospital stay. Recognition and repair of intraoperative CSF leaks reduces postoperative complications. Use of pedicled nasoseptal flaps improves outcomes in reconstructing defects at higher risk for postoperative leak.

Alterations in gene dosage due to copy-number variation (CNV) are associated with autism spectrum disorder (ASD), intellectual disability (ID) and other psychiatric disorders. The nervous system is so acutely sensitive to the dose of methyl-CpG-binding protein 2 (MeCP2) that even a two-fold change in MeCP2 protein level—either increased or decreased—results in distinct disorders with overlapping features including ID, autistic behavior and severe motor dysfunction. Rett syndrome is caused by loss-of-function mutations in MECP2, whereas duplications spanning the MECP2 locus result in MECP2 Duplication Syndrome (MDS) which accounts for ~1% of X-linked ID. Despite evidence from mouse models that restoring MeCP2 levels can reverse the course of disease, there are currently no FDA-approved therapies available to clinically modulate MeCP2 abundance. In this study we used a forward genetic screen against all known human kinases and phosphatases to identify druggable regulators of MeCP2 stability. Two putative modulators of MeCP2 levels, HIPK2 and protein phosphatase PP2A, were validated as stabilizers of MeCP2 in vivo. Further, pharmacological inhibition of PP2A in vivo reduced MeCP2 levels within the nervous system and rescued both overexpression and motor abnormalities in a mouse model of MDS. Our findings reveal potential therapeutic targets for treating disorders of altered MECP2 dosage and establish a potent strategy to identify druggable candidates for the broader category of neurologic disease resulting from CNVs.

Intravenous delivery of adenoviruses is the optimal route for many gene therapy applications. Once in the blood, coagulation factor X (FX) binds to the adenovirus capsid and protects the virion from natural antibody and classical complement-mediated neutralisation in mice. However, to date no studies have examined the relevance of this FX/viral immune protective mechanism in human samples. In this study we assessed the effects of blocking FX on adenovirus type 5 (Ad5) activity in the presence of human serum. FX prevented human IgM binding directly to the virus. In individual human sera samples (n=25), approximately half of those screened inhibited adenovirus transduction only when the Ad5:FX interaction was blocked, demonstrating that FX protected the virus from neutralising components in a large proportion of human sera. In contrast, the remainder of sera tested had no inhibitory effects on Ad5 transduction and FX armament was not required for effective gene transfer. In human sera in which FX had a protective role, Ad5 induced lower levels of complement activation in the presence of FX. We therefore demonstrate for the first time the importance of Ad:FX protection in human samples and highlight subject variability and species-specific differences as key considerations for adenoviral gene therapy.Gene Therapy accepted article preview online, 25 March 2016. doi:10.1038/gt.2016.32.

Despite 20 years of published medical society guidelines for blood transfusion and a pivotal clinical trial in 1999 providing Level 1 evidence that restrictive transfusion practices can be utilized safely, blood transfusions did not begin to decline in the United States until 2010. Widespread adoption of electronic medical records allowed implementation of computerized systems such as clinical decision support (CDS) with best practice alerts to improve blood utilization. We describe our own experience using well-designed and highly targeted CDS to promote restrictive transfusion practices and improve red blood cell utilization, with a 42% reduction in blood transfusions from 2009 through 2015, accompanied by improved clinical outcomes.

A 3.5-year-old girl presented to our clinic experiencing pain in her maxillary central incisors following traumatic injury during a fall. Radiographic examination revealed both primary maxillary central incisors with mid-root and apical third horizontal root fractures, respectively. Splinting with orthodontic brackets and stainless steel wire was performed. At 2 weeks, resorption of the apical fragments in both injured teeth was observed, and after 3 months, almost complete resorption was noted on radiographs. Tooth mobility at this point was back to physiologic levels and the splint was removed. After 2.5 years, the primary maxillary incisors were replaced by permanent incisors demonstrating normal tooth color, position, and root development. Although this case illustrated the favorable prognosis of two primary teeth with root fractures and severely mobile coronal fragments by a conservative approach, more scientific evidences are needed and frequent recalls are necessary when primary root fractures are attempted to be managed with splinting.

Background and Purpose— Cerebral vasospasm continues to be a major cause of poor outcome in patients with ruptured aneurysms. Prophylactic Transluminal Balloon Angioplasty (pTBA) appeared to prevent delayed ischemic neurological deficit in a pilot study. A phase II multicenter randomized clinical trial was subsequently designed. Methods— One hundred and seventy patients with Fisher Grade III subarachnoid hemorrhage were enrolled in the study. Of these, 85 patients were randomized to the treatment group and underwent pTBA within 96 hours after subarachnoid hemorrhage. Main end points of the study included the 3-month dichotomized Glasgow Outcome Score (GOS), development of delayed ischemic neurological deficit (DIND), occurrence of Transcranial Doppler (TCD) vasospasm, and length of stay in the ICU and hospital. Results— The incidence of DIND was lower in the pTBA group ( P =0.30) and fewer patients required therapeutic angioplasty to treat DIND ( P =0.03). Overall pTBA resulted in an absolute risk reduction of 5.9% and a relative risk reduction of 10.4% unfavorable outcome ( P =0.54). Good grade patients had absolute and relative risk reductions of respectively 9.5 and 29.4% ( P =0.73). Length of stay in ICU and hospital was similar in both groups. Four patients had a procedure-related vessel perforation, of which three patients died. Conclusions— While the trial is unsuccessful as defined by the primary end point (GOS), proof of concept is confirmed by these results. Fewer patients tend to develop vasospasm after treatment with pTBA and there is a statistically significantly decreased need for therapeutic angioplasty. pTBA does not improve the poor outcome of patients with Fisher grade III subarachnoid hemorrhage.

Two severe, progressive neurological disorders characterized by intellectual disability, autism, and developmental regression, Rett syndrome and MECP2 duplication syndrome, result from loss and gain of function, respectively, of the same critical gene, methyl-CpG-binding protein 2 (MECP2). Neurons acutely require the appropriate dose of MECP2 to function properly but do not die in its absence or overexpression. Instead, neuronal dysfunction can be reversed in a Rett syndrome mouse model if MeCP2 function is restored. Thus, MECP2 disorders provide a unique window into the delicate balance of neuronal health, the power of mouse models, and the importance of chromatin regulation in mature neurons. In this Review, we will discuss the clinical profiles of MECP2 disorders, the knowledge acquired from mouse models of the syndromes, and how that knowledge is informing current and future clinical studies.

Methyl-CpG binding protein 2 (MeCP2) is a nuclear protein with important roles in regulating chromatin structure and gene expression, and mutations in MECP2 cause Rett syndrome (RTT). Within the MeCP2 protein sequence, the nuclear localization signal (NLS) is reported to reside between amino acids 255-271, and certain RTT-causing mutations overlap with the MeCP2 NLS, suggesting that they may alter nuclear localization. One such mutation, R270X, is predicted to interfere with the localization of MeCP2, but recent in vivo studies have demonstrated that this mutant remains entirely nuclear. To clarify the mechanism of MeCP2 nuclear import, we isolated proteins that interact with the NLS and identified karyopherin α 3 (KPNA3 or Kap-α3) and karyopherin α 4 (KPNA4 or Kap-α4) as key binding partners of MeCP2. MeCP2-R270X did not interact with KPNA4, consistent with a requirement for an intact NLS in this interaction. However, this mutant retains binding to KPNA3, accounting for the normal localization of MeCP2-R270X to the nucleus. These data provide a mechanism for MeCP2 nuclear import and have implications for the design of therapeutics aimed at modulating the function of MeCP2 in RTT patients.

Prostate cancer (PCa) is the most prevalent cancer amongst men and the second most common cause of cancer related-deaths in the USA. Prostate cancer is a heterogeneous disease ranging from indolent asymptomatic cases to very aggressive life threatening forms. The goal of this study was to identify differentially expressed metabolites and lipids in prostate cells with different tumorigenic phenotypes. We have used mass spectrometry metabolomic profiling, lipidomic profiling, bioinformatic and statistical methods to identify, quantify and characterize differentially regulated molecules in five prostate derived cell lines. We have identified potentially interesting species of different lipid subclasses including phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), glycerophosphoinositols (PIs) and other metabolites that are significantly upregulated in prostate cancer cells derived from distant metastatic sites. Transcriptomic and biochemical analysis of key enzymes that are involved in lipid metabolism demonstrate the significant upregulation of choline kinase alpha in the metastatic cells compared to the non-malignant and non-metastatic cells. This suggests that different de novo lipogenesis and other specific signal transduction pathways are activated in aggressive metastatic cells as compared to normal and non-metastatic cells.

This study reports concentrations of BDEs 47, 99, 100, 153, and 154 in outdoor air [median sigmaPBDE (sum of BDEs 47, 99, 100, 153, and 154) = 18 pg m(-3)] in air from a range of office and home indoor microenvironments (median sigmaPBDE = 762 pg m(-3)) and vegan and omnivorous duplicate diet samples (median sigmaPBDE = 154 and 181 pg g(-1) dryweightforvegan and omnivorous diets, respectively). Median daily human exposure to sigmaPBDE via inhalation is 6.9 ng/person and 90.5 ng/person via diet but the relative significance of these pathways may vary considerably between individuals. Median concentrations in indoor air were higher in workplace (sigmaPBDE = 1082 pg m(-3)) than in domestic (sigmaPBDE = 128 pg m(-3)) microenvironments, and substantial differences in concentrations in air from different rooms in the same office building were found. When data from the only mechanically ventilated room was excluded, a significant positive correlation (p < 0.001) was observed between PBDE concentrations and both the number of electrical appliances and polyurethane foam-containing chairs. Concentrations of sigmaPBDE and BDEs 47 and 99 were significantly higher (p < 0.1) in omnivorous diet samples than in vegan diet samples, implying that while plant-based foods contribute appreciably, higher exposure occurs via ingestion of animal-based comestibles.

Tissue inhibitor of metalloproteinases-3 (TIMP3) is one of four members of a family of proteins that were originally classified according to their ability to inhibit matrix metalloproteinases (MMP). TIMP3, which encodes a potent angiogenesis inhibitor, is mutated in Sorsby fundus dystrophy, a macular degenerative disease with submacular choroidal neovascularization. In this study we demonstrate the ability of TIMP3 to inhibit vascular endothelial factor (VEGF)-mediated angiogenesis and identify the potential mechanism by which this occurs: TIMP3 blocks the binding of VEGF to VEGF receptor-2 and inhibits downstream signaling and angiogenesis. This property seems to be independent of its MMP-inhibitory activity, indicating a new function for this molecule.

Stable complexes of cationic liposomes with plasmid DNA were prepared by (1) including a small amount of poly(ethylene glycol)-phospholipid conjugate or (2) condensing the DNA with polyamines prior to the formation of liposome-plasmid complexes. These preparations were stable for months at 4°C and gave reproducible high transfection activity for in vivo gene delivery after intravenous injection in mice. Under these conditions, the expression of marker gene (luciferase) was primarily in the lungs (reaching values up to 3 ng expression per mg tissue protein), but also in other tissues to a lesser extent. Non-stabilized formulations lost all their transfection activity in 4 days. In these formulations cholesterol, not dioleoylphosphatidylethanolamine, was the helper lipid effective for sustaining high transfection activity in vivo. These new developments in formulation technology should enhance the potential for liposome-mediated gene therapy.

Objectives To investigate patients’ and family members’ perceptions and experiences of disclosure of healthcare incidents and to derive principles of effective disclosure. Design Retrospective qualitative study based on 100 semi-structured, in depth interviews with patients and family members. Setting Nationwide multisite survey across Australia. Participants 39 patients and 80 family members who were involved in high severity healthcare incidents (leading to death, permanent disability, or long term harm) and incident disclosure. Recruitment was via national newspapers (43%), health services where the incidents occurred (28%), two internet marketing companies (27%), and consumer organisations (2%). Main outcome measures Participants’ recurrent experiences and concerns expressed in interviews. Results Most patients and family members felt that the health service incident disclosure rarely met their needs and expectations. They expected better preparation for incident disclosure, more shared dialogue about what went wrong, more follow-up support, input into when the time was ripe for closure, and more information about subsequent improvement in process. This analysis provided the basis for the formulation of a set of principles of effective incident disclosure. Conclusions Despite growing prominence of open disclosure, discussion about healthcare incidents still falls short of patient and family member expectations. Healthcare organisations and providers should strengthen their efforts to meet patients’ (and family members’) needs and expectations.

In recent discussions regarding concerns about the academic achievement of US students, educational policy makers have suggested the implementation of certain teacher policies. To address the limited empirical research on the putative educational impact of such policies, this study used multilevel structural equation models to investigate the longitudinal associations between teacher evaluation and reward policies, and student mathematics achievement and dropout with a national sample of students (n = 7,779) attending one of 431 public high schools. The student sample included an equal number of boys and girls averaging 16 years of age, and included a White (53%) majority. This study examined whether associations between teacher policies and student achievement were mediated by the teacher–student relationship climate. Results of this study were threefold. First, teacher evaluation policies that allowed students to evaluate their teachers were associated with more positive student reports of the classroom teaching climate. Second, schools with teacher reward policies that included assigning higher performing teachers with higher performing students had a negative association with student perceptions of the teaching climate. Lastly, schools with better student perceptions of the teaching climate were associated with lower student dropout rates by students’ senior year. These findings are discussed in light of their educational policy implications.

Summary Restoration of lost opposition in the context of significant thenar soft tissue defects represents a tremendous reconstructive challenge. Free functioning muscle transfer has been described in this context and has the advantage of providing both a functioning muscle unit as well as soft tissue coverage in a single reconstructive procedure. It adds to the injured limb, and by sparing donor tendons avoids the need for re-education of motor function. We describe the use of a free innervated gracilis muscle flap for functional thenar reconstruction in two unique cases following extensive traumatic loss of thenar skin and musculature. Crucially, in each case, the recurrent motor branch of the median nerve had been destroyed at its point of insertion into the thenar muscle remnants. Aim To date, the main reported disadvantages of free functioning muscle transfer in thenar reconstruction include difficult flap dissections, donor site morbidity, inadequate strength and excursion of the transplanted muscle and excessively bulky flaps. Our aim was, as far as possible, to address these issues. Surgical procedure Each thenar defect was measured and a corresponding segment of gracilis muscle, measured in situ, was raised on the proximal neurovascular pedicle. End-side microvascular anastomosis was performed between the medial circumflex femoral artery and the radial artery. The venae comitantes of the pedicle were anastomosed end-end with those of the radial artery and also with the cephalic vein. Epineural anastomosis was performed between the motor branch of the obturator nerve and the recurrent motor branch of the median nerve. Each flap was covered with a split thickness skin graft. Results Both flaps survived without any complication. Both patients regained excellent voluntary thumb opposition, sufficient to allow return to full-time employment, and had restoration of sufficient thenar bulk. This was achieved with minimal donor site morbidity. Conclusions Restoration of lost opposition, in the context of significant thenar soft tissue defects, can be achieved using a free functional gracilis flap. This produces clinically excellent functional results and can be carried out as a single stage reconstructive procedure. This is a novel application of a tremendously versatile donor muscle in functioning free muscle transfer.