Production of High-Yield Adeno Associated Vector Batches Using HEK293 Suspension Cells.

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, demonstrate extensive transduction of the CNS by intravenous injection in mice. To achieve comparable transduction, a 100-fold higher titer (minimally 1 x 1011 genome copies/mouse) is needed compared to direct injection in the CNS parenchyma. In our group, AAV production, including AAV.PHP.eB relies on adherent HEK293T cells and the triple transfection method. Achieving high yields of AAV with adherent cells entails a labor- and material-intensive process. This constraint prompted the development of a protocol for suspension-based cell culture in conical tubes. AAVs generated in adherent cells were compared to the suspension production method. Culture in suspension using transfection reagents Polyethylenimine or TransIt were compared. AAV vectors were purified by iodixanol gradient ultracentrifugation followed by buffer exchange and concentration using a centrifugal filter. With the adherent method, we achieved an average of 2.6 x 1012 genome copies (GC) total, whereas the suspension method and Polyethylenimine yielded 7.7 x 1012 GC in total, and TransIt yielded 2.4 x 1013 GC in total. There is no difference in in vivo transduction efficiency between vectors produced with adherent compared to the suspension cell system. In summary, a suspension HEK293 cell based AAV production protocol is introduced, resulting in a reduced amount of time and labor needed for vector production while achieving 3 to 9 times higher yields using components available from commercial vendors for research purposes.

SKAP2 acts downstream of CD11b/CD18 and regulates neutrophil effector function.

Background: The importance of CD11b/CD18 expression in neutrophil effector functions is well known. Beyond KINDLIN3 and TALIN1, which are involved in the induction of the high-affinity binding CD11b/CD18 conformation, the signaling pathways that orchestrate this response remain incompletely understood. Method: We performed an unbiased screening method for protein selection by biotin identification (BioID) and investigated the KINDLIN3 interactome. We used liquid chromatography with tandem mass spectrometry as a powerful analytical tool. Generation of NB4 CD18, KINDLIN3, or SKAP2 knockout neutrophils was achieved using CRISPR-Cas9 technology, and the cells were examined for their effector function using flow cytometry, live cell imaging, microscopy, adhesion, or antibody-dependent cellular cytotoxicity (ADCC). Results: Among the 325 proteins significantly enriched, we identified Src kinase-associated phosphoprotein 2 (SKAP2), a protein involved in actin polymerization and integrin-mediated outside-in signaling. CD18 immunoprecipitation in primary or NB4 neutrophils demonstrated the presence of SKAP2 in the CD11b/CD18 complex at a steady state. Under this condition, adhesion to plastic, ICAM-1, or fibronectin was observed in the absence of SKAP2, which could be abrogated by blocking the actin rearrangements with latrunculin B. Upon stimulation of NB4 SKAP2-deficient neutrophils, adhesion to fibronectin was enhanced whereas CD18 clustering was strongly reduced. This response corresponded with significantly impaired CD11b/CD18-dependent NADPH oxidase activity, phagocytosis, and cytotoxicity against tumor cells. Conclusion: Our results suggest that SKAP2 has a dual role. It may restrict CD11b/CD18-mediated adhesion only under resting conditions, but its major contribution lies in the regulation of dynamic CD11b/CD18-mediated actin rearrangements and clustering as required for cellular effector functions of human neutrophils.

No Association Between Hospital Volume and Early Second Revision Rate in Revision Total Knee Arthroplasty in the Dutch Orthopaedic Register.

BACKGROUND: Revision knee arthroplasty (R-KA) is rising globally. Technical difficulty of R-KA varies from liner exchange to full revision. Centralization has been shown to reduce mortality and morbidity rates. The present study aimed to evaluate the association between hospital R-KA volume and overall second revision rate, as well as revision rate for different types of revision. METHODS: The R -KAs between 2010 and 2020 with available data on the primary KA in the Dutch Orthopaedic Arthroplasty Register were included. Minor revisions were excluded. Implant data and anonymous patient characteristics were obtained from the Dutch Orthopaedic Arthroplasty Register. Survival analyses and competing risk analysis were performed per volume category (≤12, 13 to 24, or ≥25 cases/year) at 1, 3, and 5 years following R-KA. There were 8,072 R-KA cases available. Median follow-up was 3.7 years (range 0 to 13.7 years). There were a total of 1,460 second revisions (18.1%) at the end of follow-up. RESULTS: There were no statistically significant differences between second revision rates of the three volume groups. Adjusted hazard ratio for second revision were 0.97 (Confidence Interval (CI) 0.86 to 1.11) for hospitals with 13 to 24 cases/year and 0.94 (CI 0.83 to 1.07) with ≥25 cases/year compared to low volume (≤12 cases/year). Type of revision did not influence second revision rate. CONCLUSION: Second revision rate of R-KA does not seem to be dependent on hospital volume or type of revision in the Netherlands. LEVEL OF EVIDENCE: Level IV, Observational registry study.

Cancer cells resist antibody-mediated destruction by neutrophils through activation of the exocyst complex.

BACKGROUND: Neutrophils kill antibody-opsonized tumor cells using trogocytosis, a unique mechanism of destruction of the target plasma. This previously unknown cytotoxic process of neutrophils is dependent on antibody opsonization, Fcγ receptors and CD11b/CD18 integrins. Here, we demonstrate that tumor cells can escape neutrophil-mediated cytotoxicity by calcium (Ca2+)-dependent and exocyst complex-dependent plasma membrane repair. METHODS: We knocked down EXOC7 or EXOC4, two exocyst components, to evaluate their involvement in tumor cell membrane repair after neutrophil-induced trogocytosis. We used live cell microscopy and flow cytometry for visualization of the host and tumor cell interaction and tumor cell membrane repair. Last, we reported the mRNA levels of exocyst in breast cancer tumors in correlation to the response in trastuzumab-treated patients. RESULTS: We found that tumor cells can evade neutrophil antibody-dependent cellular cytotoxicity (ADCC) by Ca2+-dependent cell membrane repair, a process induced upon neutrophil trogocytosis. Absence of exocyst components EXOC7 or EXOC4 rendered tumor cells vulnerable to neutrophil-mediated ADCC (but not natural killer cell-mediated killing), while neutrophil trogocytosis remained unaltered. Finally, mRNA levels of exocyst components in trastuzumab-treated patients were inversely correlated to complete response to therapy. CONCLUSIONS: Our results support that neutrophil attack towards antibody-opsonized cancer cells by trogocytosis induces an active repair process by the exocyst complex in vitro. Our findings provide insight to the possible contribution of neutrophils in current antibody therapies and the tolerance mechanism of tumor cells and support further studies for potential use of the exocyst components as clinical biomarkers.

Identifying main uncertainties in estimating past and present radiative forcing of peatlands.

Reconstructions of past climate impact, that is, radiative forcing (RF), of peatland carbon (C) dynamics show that immediately after peatland initiation the climate warming effect of CH4 emissions exceeds the cooling effect of CO2 uptake, but thereafter the net effect of most peatlands will move toward cooling, when RF switches from positive to negative. Reconstructing peatland C dynamics necessarily involves uncertainties related to basic assumptions on past CO2  flux, CH4 emission and peatland expansion. We investigated the effect of these uncertainties on the RF of three peatlands, using either apparent C accumulation rates, net C balance (NCB) or NCB plus C loss during fires as basis for CO2 uptake estimate; applying a plausible range for CH4 emission; and assuming linearly interpolated expansion between basal dates or comparatively early or late expansion. When we factored that some C would only be stored temporarily (NCB and NCB+fire), the estimated past cooling effect of CO2 uptake increased, but the present-day RF was affected little. Altering the assumptions behind the reconstructed CO2  flux or expansion patterns caused the RF to peak earlier and advanced the switch from positive to negative RF by several thousand years. Compared with NCB, including fires had only small additional effect on RF lasting less than 1000 year. The largest uncertainty in reconstructing peatland RF was associated with CH4 emissions. As shown by the consistently positive RF modelled for one site, and in some cases for the other two, peatlands with high CH4 emissions and low C accumulation rates may have remained climate warming agents since their initiation. Although uncertainties in present-day RF were mainly due to the assumed CH4 emission rates, the uncertainty in lateral expansion still had a significant effect on the present-day RF, highlighting the importance to consider uncertainties in the past peatland C balance in RF reconstructions.

Long-Term Outcome Following Revision Total Knee Arthroplasty is Associated With Indication for Revision.

BACKGROUND: There is limited information about long-term clinical outcomes following revision total knee arthroplasty (TKA) in relation to the indication for revision. Previously, a clear relation between indication for revision and clinical outcome was shown after 2 years. Present study evaluated (1) whether the reported association at 2 year remains present at 7.5 years, and (2) how clinical outcome at 7.5 years developed compared to baseline and 2-year follow-up, and (3) whether patients had additional adverse events. METHODS: A cohort of 129 patients with a total system revision TKA was selected. Range of motion, Visual Analog Scale for pain and satisfaction, and clinical and functional Knee Society Score were obtained preoperatively, at 3 months, 1, 2, and 7.5 years. Reasons for revision were septic loosening, aseptic loosening, malposition, instability, and severe stiffness. RESULTS: Patients revised for severe stiffness had significantly worse outcomes. No difference was found between the other indications. The clinical outcome after revision TKA at 7.5 years remained stable for septic and aseptic loosening, malposition, and instability but deteriorated slightly for the severe stiffness group. Visual Analog Scale satisfaction remained constant for all indications. There were 11 additional complications between 2- and 7.5-year follow-up, 9 of which necessitated reoperation. CONCLUSION: All indications except severe stiffness had a similar clinical outcome which was maintained up to 7.5-year follow-up. The severe stiffness group had worse outcomes and deteriorated slightly at longer follow-up. Outcome at 3 months seems predictive for long-term outcome. Additional complications did not differ significantly for the different reasons for revision. LEVEL OF EVIDENCE: Level III, prognostic study.

Evaluating the patient experience of an emergency burns assessment service in a UK burn unit using a service user evaluation questionnaire and process mapping.

An evaluation of the patient experience, from referral to first assessment, at an outpatient emergency burns assessment service in a UK burn unit. All patients attending their first appointment were invited to complete a questionnaire, covering patient expectations following referral, their journey to the hospital and an evaluation of the appointment. Process mapping was used to map the patient journey within the department and identify functional bottlenecks and waits. 35 new patients completed the questionnaire over a four-week period in February 2019. 70% of respondents had received no printed information about their condition or the hospital prior to the appointment and 28% of patients did not know what to expect from attending the clinic. Patients incurred high direct and indirect costs in order to attend their appointments. 86% patients felt more confident about looking after their injury following their appointment. The patient journey through the clinic was observed for 19 patients; four functional bottlenecks were identified. The longest waits were for clinical photography and completion of nursing paperwork. A multimodal approach to this quality improvement project has enabled the service to identify process bottlenecks and through consultation with stakeholders, develop staff training and patient information to improve the service.

Factors affecting the harmonization of disease-related metabolic brain pattern expression quantification in [18F]FDG-PET (PETMETPAT).

INTRODUCTION: The implementation of spatial-covariance [18F]fluorodeoxyglucose positron emission tomography-based disease-related metabolic brain patterns as biomarkers has been hampered by intercenter imaging differences. Within the scope of the JPND-PETMETPAT working group, we illustrate the impact of these differences on Parkinson's disease-related pattern (PDRP) expression scores. METHODS: Five healthy controls, 5 patients with idiopathic rapid eye movement sleep behavior disorder, and 5 patients with Parkinson's disease were scanned on one positron emission tomography/computed tomography system with multiple image reconstructions. In addition, one Hoffman 3D Brain Phantom was scanned on several positron emission tomography/computed tomography systems using various reconstructions. Effects of image contrast on PDRP scores were also examined. RESULTS: Human and phantom raw PDRP scores were systematically influenced by scanner and reconstruction effects. PDRP scores correlated inversely to image contrast. A Gaussian spatial filter reduced contrast while decreasing intercenter score differences. DISCUSSION: Image contrast should be considered in harmonization efforts. A Gaussian filter may reduce noise and intercenter effects without sacrificing sensitivity. Phantom measurements will be important for correcting PDRP score offsets.

Plant communities control long term carbon accumulation and biogeochemical gradients in a Patagonian bog.

Peat carbon accumulation is controlled by both large scale factors, such as climate and hydrological setting, and small scale factors, such as microtopography and plant community. These small scale factors commonly vary within peatlands and can cause variation in biogeochemical traits and carbon accumulation within the same site. To understand these within-site variations, we investigated long term carbon accumulation, peat decomposition, biogeochemistry of pore water and plant macrofossils along a transect in an ombrotrophic bog in southern Patagonia. An additional question we addressed is how historical deposition of volcanic ash on the peatland has affected its carbon balance. Variability in plant community and water table led to differences in long term peat and carbon accumulation (peat moss > cushion plant), organic matter decomposition (cushion plant > peat moss), and methane production (peat moss > cushion plant). Macrofossil analysis and radiocarbon dating indicated a relationship between plant community and carbon accumulation or decomposition during the historical succession of vegetation in the peatland. C/N ratio and isotopic signatures reflected variability in plant community as litter source, and DOC concentrations were controlled by humification level. Volcanic ash deposition had only limited effect on plant composition, but it was associated with increased decomposition in overlying peat layers. This study highlights the importance of understanding how plant communities develop, as changes in communities could significantly affect the potential of ombrotrophic peatlands as C sink.

Improved clinical outcomes after revision arthroplasty with a hinged implant for severely stiff total knee arthroplasty.

PURPOSE: Management of the severely stiff total knee arthroplasty (TKA) is challenging, with the outcome of revision arthroplasty being inferior compared to the outcome for other indications. The aim of this study was to analyse the outcome after revision TKA with hinged-type implants for severely stiff TKA [range of motion (ROM) ≤ 70°] at 2 years. METHODS: A cohort of 38 patients with a hinged-type revision TKA (Waldemar Link or RT-Plus) and preoperative ROM ≤ 70° were selected from a prospectively collected database. ROM, visual analogue scale (VAS) for pain and satisfaction and Knee Society Score (KSS) were obtained preoperatively and at 3 months, 1 year and 2 years. Pre- and postoperative outcome were compared at 2 years. RESULTS: There was a significant increase in ROM and KSS. VAS pain scores did not differ significantly. The median ROM at 2 years was 90° (range 50°-125°) with a median gain of 45° (range 5°-105°). Median VAS pain was 28.5 (range 0-96) points and median VAS satisfaction was 72 (range 0-100) points at 2 years. Twelve patients suffered a complication. Recurrent stiff knee was the most frequently reported complication (n = 5). CONCLUSIONS: Hinged-type revision TKA following a severely stiff TKA renders a significant, although moderate, clinical improvement at 2 years. LEVEL OF EVIDENCE: Retrospective case series. Level IV.

Development and validation of the Burnt Hand Outcome Tool (BHOT): A patient-led questionnaire for adults with hand burns.

Hand burns are unique in their functional, aesthetic and emotional impact on patients. Measuring the progress of a patient's ability to carry out essential and desired tasks, their emotional state, interaction with society as well as scar, pain and itch as their burn heals, has until now relied on the use of a combination of several different tools. We have developed a questionnaire specifically to address the multiple different aspects of the impact of a hand burn on a patient. This has been validated in a study of adult patients with hand burns in a UK unit, by a variety of psychometric tests. Ninety-four patients entered the study and questionnaires were completed over the course of a year at five time points. The total BHOT and DASH questionnaires completed at each time point was as follows: 86 before; 52 healed; 29 at 3 months; 31 at 6 months; 28 at 1 year, i.e. 226 DASH and 226 BHOT questionnaires in total. The questionnaire has been shown to have excellent reliability, criterion validity, construct validity, and responsiveness. The result is the Burnt Hand Outcome Tool (BHOT), a patient reported, quick and easy to use yet comprehensive questionnaire specifically for adult patients with burns to the hand.

Gastrointestinal cancer incidence in type 2 diabetes mellitus; results from a large population-based cohort study in the UK.

BACKGROUND: Patients with type 2 diabetes mellitus (T2DM) have been shown to have higher incidences of liver, pancreatic, and colorectal cancer compared to non-diabetic individuals. Current evidence is conflicting for other gastrointestinal (GI) cancers. Therefore, we aimed to determine incidence rates (IRs) of all GI cancers in patients with and without T2DM. METHODS: A cohort study was performed using the UK Clinical Practice Research Datalink (1988-2012). A cohort of antidiabetic drug users was matched at baseline to a non-diabetic cohort, by age, sex, and practice. Crude IRs and 95% confidence intervals (95% CI) of GI cancers per 100,000 person-years were calculated stratified by age, sex, and calendar year. RESULTS: 333,438 T2DM and 333,438 non-diabetic individuals were analyzed. IRs of liver (IR 26, 95% CI 24-28 vs. 8.9, 95% CI 7.7-10), pancreatic (IR 65, 95% CI 62-69 vs. 31, 95% CI 28-34), and colon cancer (IR 119, 95% CI 114-124 vs. 109, 95% CI 104-114) were significantly higher in the diabetic compared to the non-diabetic cohort, whereas the IR of oesophageal cancer was significantly lower (IR 41, 95% CI 39-44 vs. 47, 95% CI 44-51). Sex-specific IRs of colon cancer remained significantly higher in men with T2DM, and IRs of esophageal cancer remained significantly lower in women with T2DM. CONCLUSION: In this study, T2DM patients were shown to have higher crude IRs of liver, pancreatic and colon cancer, but not of gastric, biliary, and rectal cancer. Moreover, the lower observed IRs of oesophageal cancer in diabetic patients warrants further investigation.

Ocean biogeochemistry modeled with emergent trait-based genomics.

Marine ecosystem models have advanced to incorporate metabolic pathways discovered with genomic sequencing, but direct comparisons between models and "omics" data are lacking. We developed a model that directly simulates metagenomes and metatranscriptomes for comparison with observations. Model microbes were randomly assigned genes for specialized functions, and communities of 68 species were simulated in the Atlantic Ocean. Unfit organisms were replaced, and the model self-organized to develop community genomes and transcriptomes. Emergent communities from simulations that were initialized with different cohorts of randomly generated microbes all produced realistic vertical and horizontal ocean nutrient, genome, and transcriptome gradients. Thus, the library of gene functions available to the community, rather than the distribution of functions among specific organisms, drove community assembly and biogeochemical gradients in the model ocean.

Uterine Artery Embolization for Symptomatic Adenomyosis: 7-Year Clinical Follow-up Using UFS-Qol Questionnaire.

PURPOSE: The purpose of this study was to assess clinical outcomes 7 years after uterine artery embolization (UAE) in the treatment of symptomatic adenomyosis. MATERIALS AND METHODS: In this prospective cohort study, one specialized hospital in the Netherlands recruited patients with symptomatic adenomyosis or adenomyosis in combination with fibroids for UAE. The 7-year post-intervention outcomes were health-related quality of life (HRQOL), symptom severity scores (SSS), satisfaction, menopause and re-interventions. RESULTS: Twenty-nine patients with adenomyosis (15 with fibroids) were treated with UAE between September 2006 and January 2010. The 7-year questionnaire was mailed in November 2016. The mean follow-up was 95 months (SD 9.0) at a mean age of 50 (SD 5.4). Questionnaires were returned by 24/29 patients (83%). The remaining five patients were contacted through telephone. One of these patients was untraceable. Seven years after treatment 5 of 28 patients (18%) underwent a secondary hysterectomy. The HRQOL and SSS scores as measured by UFS-QOL at 3 months after UAE showed significant improvement of -57 points (score: 15) and +40 points (score: 91), respectively. These scores remained comparable stable up unto 7 years. The SSS showed a significant difference of 17 points (0-100) in favor of the adenomyosis in combination with fibroids group (p = 0.020). Menopause was reported by 10/28 patients (36%). Twenty-one of 29 (72%) patients declared to be at least fairly satisfied about UAE. CONCLUSIONS: After 7 years of follow-up, in 82% of UAE-treated patients with symptomatic adenomyosis a hysterectomy was avoided.

Trends in breast cancer incidence among women with type-2 diabetes in British general practice.

AIMS: To quantify breast cancer incidence in women with type-2 diabetes and assess age-standardized trends in invasive breast cancer incidence over time and by age groups. METHODS: A population-based cohort study was conducted using the British general practice database (Clinical Practice Research Datalink) using data from 1989 to 2012. All adult women prescribed anti-hyperglycemic medication were selected and matched (1:1) on age and clinical practice to a reference cohort without diabetes. RESULTS: During approximately 1.6 million person years (py), 2371 breast cancer cases were diagnosed in the diabetes cohort (n=147,998) and 2252 in the reference cohort (n=147,998). Incidence of breast cancer, overall or by age groups, among women with diabetes remained stable over time. The (overall) age-standardized breast cancer IR per 100,000 py of the diabetes cohort (150, 95%CI:143-157) resembled that observed in the reference cohort (148, 95%CI:141-156); with an incidence rate ratio (IRR) of 1.01 (95%CI:0.94-1.08, p>0.05). CONCLUSIONS: Currently, around 2880 women with type-2 diabetes are diagnosed with breast cancer per year in the United Kingdom. However, breast cancer incidence remained stable in the last 10 years and seems to be comparable in women with and without diabetes.

Cardiac Overexpression of S100A6 Attenuates Cardiomyocyte Apoptosis and Reduces Infarct Size After Myocardial Ischemia-Reperfusion.

BACKGROUND: Cardiomyocyte-specific transgenic mice overexpressing S100A6, a member of the family of EF-hand calcium-binding proteins, develop less cardiac hypertrophy, interstitial fibrosis, and myocyte apoptosis after permanent coronary ligation, findings that support S100A6 as a potential therapeutic target after acute myocardial infarction. Our purpose was to investigate S100A6 gene therapy for acute myocardial ischemia-reperfusion. METHODS AND RESULTS: We first performed in vitro studies to examine the effects of S100A6 overexpression and knockdown in rat neonatal cardiomyocytes. S100A6 overexpression improved calcium transients and protected against apoptosis induced by hypoxia-reoxygenation via enhanced calcineurin activity, whereas knockdown of S100A6 had detrimental effects. For in vivo studies, human S100A6 plasmid or empty plasmid was delivered to the left ventricular myocardium by ultrasound-targeted microbubble destruction in Fischer-344 rats 2 days prior to a 30-minute ligation of the left anterior descending coronary artery followed by reperfusion. Control animals received no therapy. Pretreatment with S100A6 gene therapy yielded a survival advantage compared to empty-plasmid and nontreated controls. S100A6-pretreated animals had reduced infarct size and improved left ventricular systolic function, with less myocyte apoptosis, attenuated cardiac hypertrophy, and less cardiac fibrosis. CONCLUSIONS: S100A6 overexpression by ultrasound-targeted microbubble destruction helps ameliorate myocardial ischemia-reperfusion, resulting in lower mortality and improved left ventricular systolic function post-ischemia-reperfusion via attenuation of apoptosis, reduction in cardiac hypertrophy, and reduced infarct size. Our results indicate that S100A6 is a potential therapeutic target for acute myocardial infarction.

Insulin glargine use and breast cancer risk: Associations with cumulative exposure.

BACKGROUND: This study was aimed to assess the risk of breast cancer associated with exposure to insulin glargine in women with type 2 diabetes and evaluate whether the pattern of risk concurs with the hypothesized trend of an increase in risk with longer duration of use, taking into account previous cumulative exposure to other types of insulin. METHODS: We performed a restrospective cohort study (2002-2013) in the Clinical Practice Research Datalink among adult female patients with a first ever insulin prescription (n = 12 468). Time-dependent exposure measures were used to assess associations with duration of use of: (1) other insulin types before glargine was first prescribed (i.e. among switchers); and (2) of glargine during follow-up. Analyses were performed separately for insulin-naïve glargine users and patients switched to glargine. Cox proportional hazards models were used to derive p-trends, hazard ratios (HR) and 95% confidence intervals (CI) for breast cancer associated with glargine use. RESULTS: During 66 151 person years, 186 breast cancer cases occurred; 76 in glargine users (3.0/1000 years) and 110 in users of other insulins (2.7/1000 years). Among insulin-naïve women, no association with cumulative glargine use was observed (p-trend = 0.91), even after ≥5 years (HR = 1.06, 95% CI 0.48-2.33). Among switchers, a linear trend with years of prior exposure to other insulins was found (p-trend = 0.02). An increased risk was observed in glargine users with extensive (>3 years) past exposure to other insulins (HR = 3.17, 95% CI 1.28-7.84). A non-significant trend with cumulative glargine exposure was found among switchers (p-trend = 0.24). CONCLUSIONS: Exposure to glargine was not associated with an increased breast cancer risk in insulin-naïve patients. Exposure to other insulins prior to the start of glargine appears to be relevant when studying breast cancer risk associated with glargine use.

Therapeutic Angiogenesis by Ultrasound-Mediated MicroRNA-126-3p Delivery.

OBJECTIVE: MicroRNAs are involved in many critical functions, including angiogenesis. Ultrasound-targeted microbubble destruction (UTMD) is a noninvasive technique for targeted vascular transfection of plasmid DNA and may be well suited for proangiogenic microRNA delivery. We aimed to investigate UTMD of miR-126-3p for therapeutic angiogenesis in chronic ischemia. APPROACH AND RESULTS: The angiogenic potential of miR-126-3p was tested in human umbilical vein endothelial cells in vitro. UTMD of miR-126-3p was tested in vivo in Fischer-344 rats before and after chronic left femoral artery ligation, evaluating target knockdown, miR-126-3p and miR-126-5p expression, phosphorylated Tie2 levels, microvascular perfusion, and vessel density. In vitro, miR-126-3p-transfected human umbilical vein endothelial cells showed repression of sprouty-related protein-1 and phosphatidylinositol-3-kinase regulatory subunit 2, negative regulators of vascular endothelial growth factor and angiopoietin-1 signaling, increased phosphorylated Tie2 mediated by knockdown of phosphatidylinositol-3-kinase regulatory subunit 2 and greater angiogenic potential mediated by both vascular endothelial growth factor/vascular endothelial growth factor R2 and angiopoietin-1 /Tie2 effects. UTMD of miR-126-3p resulted in targeted vascular transfection, peaking early after delivery and lasting for >3 days, and resulting in inhibition of sprouty-related protein-1 and phosphatidylinositol-3-kinase regulatory subunit 2, with minimal uptake in remote organs. Finally, UTMD of miR-126-3p to chronic ischemic hindlimb muscle resulted in improved perfusion, vessel density, enhanced arteriolar formation, pericyte coverage, and phosphorylated Tie2 levels, without affecting miR-126-5p or delta-like 1 homolog levels. CONCLUSIONS: UTMD of miR-126 results in improved tissue perfusion and vascular density in the setting of chronic ischemia by repressing sprouty-related protein-1 and phosphatidylinositol-3-kinase regulatory subunit 2 and enhancing vascular endothelial growth factor and angiopoietin-1 signaling, with no effect on miR-126-5p. UTMD is a promising platform for microRNA delivery, with applications for therapeutic angiogenesis.

Sand patties provide evidence for the presence of Deepwater Horizon oil on the beaches of the West Florida Shelf.

The ecological consequences of the Deepwater Horizon (DWH) oil spill are both long-term and pervasive. The distribution of toxicity and mutagenicity in the Gulf of Mexico suggests oil from the DWH spill could have contaminated the West Florida Shelf (WFS). We utilized polycyclic aromatic hydrocarbon (PAH) analysis to determine presence and potential origin of oil contaminants in beach sand patty samples. PAH profiles from WFS beaches were statistically significantly similar to DWH contaminated samples from the Northeast Gulf of Mexico (Gulf Shores, AL; Ft. Pickens, FL). Dioctyl sodium sulfosuccinate (DOSS), a major component of Corexit 9500 dispersant was also detected in the sediments. DOSS concentrations ranged from 1.6 to 5.5ngg(-1) dry weight. Additionally, two samples from DWH oil contaminated beaches were acutely toxic and one WFS beach sediment sample was mutagenic. These observations provide support for the theory that DWH oil made its way onto beaches of the WFS.

The risk of colorectal cancer in patients with type 2 diabetes: associations with treatment stage and obesity.

OBJECTIVE: To assess the risk of colorectal cancer associated with type 2 diabetes, as compared with a nondiabetic reference population, and to study additional associations between treatment stage and duration of obesity and colorectal cancer risk. RESEARCH DESIGN AND METHODS: We conducted an observational population-based cohort study within the Clinical Practice Research Datalink (1987-2012). All patients (≥18 years) with at least one prescription for an antidiabetic drug (n = 300,039) were matched (1:1) by birth year, sex, and practice to a comparison cohort without diabetes. Cox proportional hazards models were used to derive adjusted hazard ratios (HRs) for colorectal cancer associated with type 2 diabetes. Within the diabetic cohort, associations of colorectal cancer with treatment stages and duration of obesity (BMI ≥30 kg/m(2)) were studied. RESULTS: After a median follow-up of 4.5 years, 2,759 cases of colorectal cancer were observed among the diabetic study population. Type 2 diabetes was associated with a 1.3-fold increased risk of colorectal cancer (HR 1.26 [95% CI 1.18-1.33]). Among diabetic patients, no association was found with treatment stages. A trend of increased colorectal cancer risk was observed with longer duration of obesity. Risk of colorectal cancer was significantly increased for patients with recorded duration of obesity of 4-8 years (HR 1.19 [1.06-1.34]) and >8 years (1.28 [1.11-1.49]). CONCLUSIONS: Type 2 diabetes is associated with a moderately increased risk of colorectal cancer. Among diabetic patients, an increased risk was observed for patients who suffered from obesity for a total duration of 4 years or more.