Antibodies are important reagents for research, diagnostics, and therapeutics. Many examples of chimeric proteins combining the specific target recognition of antibodies with complementing ...functionalities such as fluorescence, toxicity or enzymatic activity have been described. However, antibodies selected solely on the basis of their binding specificities are not necessarily ideal candidates for the construction of chimeras. Here, we describe a high throughput method based on yeast display to directly select antibodies most suitable for conversion to fluorescent chimera. A library of scFv binders was converted to a fluorescent chimeric form, by cloning thermal green protein into the linker between VH and VL, and directly selecting for both binding and fluorescent functionality. This allowed us to directly identify antibodies functional in the single chain TGP format, that manifest higher protein expression, easier protein purification, and one-step binding assays.
Abstract
In vitro display technologies based on phage and yeast have a successful history of selecting single-chain variable fragment (scFv) antibodies against various targets. However, single-chain ...antibodies are often unstable and poorly expressed in Escherichia coli. Here, we explore the feasibility of converting scFv antibodies to an intrinsically fluorescent format by inserting the monomeric, stable fluorescent protein named thermal green, between the light- and heavy-chain variable regions. Our results show that the scTGP format maintains the affinity and specificity of the antibodies, improves expression levels, allows one-step fluorescent assay for detection of binding and is a suitable reagent for epitope binning. We also report the crystal structure of an scTGP construct that recognizes phosphorylated tyrosine on FcεR1 receptor of the allergy pathway.
Quorum sensing (QS) is a form of bacterial communication involved in the production of virulence factors in many species. As a result, inhibition of quorum sensing may be of use in mitigating ...pathogenesis. The signaling molecule indole is currently being investigated as a target for quorum sensing inhibition (QSI) and the indole derivative indole-3-carboxaldehyde (ICA) has been shown to inhibit quorum sensing-mediated behaviors in Escherichia coli. In this study, we investigate bromination as a method of increasing the QSI capabilities of indole carboxaldehydes. The IC50 values of three monobrominated indole carboxaldehydes (5-bromoindole-3-carboxaldehyde, 6-bromoindole-3-carboxaldehyde, and 7-bromoindole-3-carboxaldehyde) were determined and compared to the IC50 value of ICA. The bromination of these indole carboxaldehydes reduced the IC50 values between 2- and 13-fold, indicating that bromination significantly increases the potency of these indole carboxaldehydes.
School-based student health screenings identify issues that may affect physical and intellectual development and are an important way to maintain student health. Nonprofit hospitals can provide a ...unique resource to school districts by assisting in the timely completion of school-based screenings and meet requirements of the Affordable Care Act. This case study describes the collaboration between an academic medical center and a local school district to conduct school-based health screenings.
Penn State Milton S. Hershey Medical Center and Penn State Hershey PRO Wellness Center collaborated with Lebanon School District to facilitate student health screenings, a need identified in part by a community health needs assessment.
From June 2012 through February 2013, district-wide student health screenings were planned and implemented by teams of hospital nursing leadership, school district leadership, and school nurses. In fall 2013, students were screened through standardized procedures for height, weight, scoliosis, vision, and hearing.
In 2 days, 3,105 students (67% of all students in the district) were screened. Letters explaining screening results were mailed to parents of all students screened. Debriefing meetings and follow-up surveys for the participating nurses provided feedback for future screenings.
The 2-day collaborative screening event decreased the amount of time spent by school nurses in screening students throughout the year and allowed them more time in their role as school wellness champion. Additionally, parents found out early in the school year whether their child needed physician follow-up. Partnerships between school districts and hospitals to conduct student health screenings are a practical option for increasing outreach while satisfying community needs.
Health-related quality of life as a predictor of mortality and hospitalization: The Dialysis Outcomes and Practice Patterns Study (DOPPS).
We investigated whether indicators of health-related quality ...of life (HRQOL) may predict the risk of death and hospitalization among hemodialysis patients treated in seven countries, taking into account serum albumin concentration and several other risk factors for death and hospitalization. We also compared HRQOL measures with serum albumin regarding their power to predict outcomes.
We analyzed data from the Dialysis Outcomes and Practice Patterns Study (DOPPS), an international, prospective, observational study of randomly selected hemodialysis patients in the United States (148 facilities), five European countries (101 facilities), and Japan (65 facilities). The total sample size was composed of 17,236 patients. Using the Kidney Disease Quality of Life Short Form (KDQOL-SFTM), we determined scores for three components of HRQOL: (1) physical component summary (PCS), (2) mental component summary (MCS), and (3) kidney disease component summary (KDCS). Complete responses on HRQOL measures were obtained from 10,030 patients. Cox models were used to assess associations between HRQOL and the risk of death and hospitalization, adjusted for multiple sociodemographic variables, comorbidities, and laboratory factors.
For patients in the lowest quintile of PCS, the adjusted risk (RR) of death was 93% higher (RR = 1.93, P < 0.001) and the risk of hospitalization was 56% higher (RR = 1.56, P < 0.001) than it was for patients in the highest quintile level. The adjusted relative risk values of mortality per 10-point lower HRQOL score were 1.13 for MCS, 1.25 for PCS, and 1.11 for KDCS. The corresponding adjusted values for RR for first hospitalization were 1.06 for MCS, 1.15 for PCS, and 1.07 for KDCS. Each RR differed significantly from 1 (P < 0.001). For 1g/dL lower serum albumin concentration, the RR of death adjusted for PCS, MCS, and KDCS and the other covariates was 1.17 (P < 0.01). Albumin was not significantly associated with hospitalization (RR = 1.03, P> 0.5).
Lower scores for the three major components of HRQOL were strongly associated with higher risk of death and hospitalization in hemodialysis patients, independent of a series of demographic and comorbid factors. A 10-point lower PCS score was associated with higher elevation in the adjusted mortality risk, as was a 1g/dL lower serum albumin level. More research is needed to assess whether interventions to improve quality of life lower these risks among hemodialysis patients.
Ischemic stroke causes brain endothelial cell (BEC) death and damages tight junction integrity of the blood-brain barrier (BBB). We harnessed the innate mitochondrial load of BEC-derived ...extracellular vesicles (EVs) and utilized mixtures of EV/exogenous 27 kDa heat shock protein (HSP27) as a one-two punch strategy to increase BEC survival (via EV mitochondria) and preserve their tight junction integrity (via HSP27 effects). We demonstrated that the medium-to-large (m/lEV) but not small EVs (sEV) transferred their mitochondrial load, that subsequently colocalized with the mitochondrial network of the recipient primary human BECs. Recipient BECs treated with m/lEVs showed increased relative ATP levels and mitochondrial function. To determine if the m/lEV-meditated increase in recipient BEC ATP levels was associated with m/lEV mitochondria, we isolated m/lEVs from donor BECs pre-treated with oligomycin A (OGM, mitochondria electron transport complex V inhibitor), referred to as OGM-m/lEVs. BECs treated with naïve m/lEVs showed a significant increase in ATP levels compared to untreated OGD cells, OGM-m/lEVs treated BECs showed a loss of ATP levels suggesting that the m/lEV-mediated increase in ATP levels is likely a function of their innate mitochondrial load. In contrast, sEV-mediated ATP increases were not affected by inhibition of mitochondrial function in the donor BECs. Intravenously administered m/lEVs showed a reduction in brain infarct sizes compared to vehicle-injected mice in a mouse middle cerebral artery occlusion model of ischemic stroke. We formulated binary mixtures of human recombinant HSP27 protein with EVs: EV/HSP27 and ternary mixtures of HSP27 and EVs with a cationic polymer, poly (ethylene glycol)-b-poly (diethyltriamine): (PEG-DET/HSP27)/EV. (PEG-DET/HSP27)/EV and EV/HSP27 mixtures decreased the paracellular permeability of small and large molecular mass fluorescent tracers in oxygen glucose-deprived primary human BECs. This one-two punch approach to increase BEC metabolic function and tight junction integrity may be a promising strategy for BBB protection and prevention of long-term neurological dysfunction post-ischemic stroke.
Display omitted
•Medium-to-large extracellular vesicles (m/lEVs), not small EVs contain mitochondria.•m/lEVs increased ATP and mitochondrial function in brain endothelial cells (BECs).•m/lEVs from BECs with deficient mitochondria did not increase recipient BEC ATP levels.•Intravenously injected m/lEVs reduced brain infarct sizes in a mouse stroke model.•EV/HSP27 mixtures reduced small and large dextran molecule permeability across BECs.
Reports relating meat intake to prostate cancer risk are inconsistent. Associations between these dietary factors and prostate cancer were examined in a consortium of 15 cohort studies. During ...follow‐up, 52,683 incident prostate cancer cases, including 4,924 advanced cases, were identified among 842,149 men. Cox proportional hazard models were used to calculate study‐specific relative risks (RR) and then pooled using random effects models. Results do not support a substantial effect of total red, unprocessed red and processed meat for all prostate cancer outcomes, except for a modest positive association for tumors identified as advanced stage at diagnosis (advanced(r)). For seafood, no substantial effect was observed for prostate cancer regardless of stage or grade. Poultry intake was inversely associated with risk of advanced and fatal cancers (pooled multivariable RR MVRR, 95% confidence interval, comparing ≥45 vs. <5 g/day: advanced 0.83, 0.70–0.99; trend test p value 0.29), fatal, 0.69, 0.59–0.82, trend test p value 0.16). Participants who ate ≥25 versus <5 g/day of eggs (1 egg ∼ 50 g) had a significant 14% increased risk of advanced and fatal cancers (advanced 1.14, 1.01–1.28, trend test p value 0.01; fatal 1.14, 1.00–1.30, trend test p value 0.01). When associations were analyzed separately by geographical region (North America vs. other continents), positive associations between unprocessed red meat and egg intake, and inverse associations between poultry intake and advanced, advanced(r) and fatal cancers were limited to North American studies. However, differences were only statistically significant for eggs. Observed differences in associations by geographical region warrant further investigation.
What's New?
The debate over red meat consumption and cancer risk is longstanding. In this consortium of 15 cohorts from North America, Europe, Australia and Asia, the authors examined over 50,000 cases of prostate cancer and the associated intake of unprocessed red and processed meat, seafood, eggs and poultry. Overall no substantial risk for unprocessed red and processed meat intake and prostate cancer was found. Interestingly, positive associations between intake of unprocessed red meat as well as eggs and advanced or fatal prostate cancers were detected only in participants living in North America, a finding which warrants further investigation into meat and egg composition, consumption and potential differences in lifestyle and screening practices between continents.
Abstract Objective To describe self-reported decision-making styles and associated pathways through end-of-life (EOL) decision-making for African-American, Caucasian, and Hispanic seriously ill male ...Veterans, and to examine potential relationships of race/ethnicity on these styles. Methods Forty-four African American, White, and Hispanic male Veterans with advanced serious illnesses participated in 8 racially/ethnically homogenous focus groups. Transcripts were qualitatively analyzed to identify major themes, with particular attention to themes that might be unique to each of the racial/ethnic groups. Results Patients described two main decision-making styles, deciding for oneself and letting others decide, leading to five variants that we labeled Autonomists, Altruists, Authorizers, Absolute Trusters, and Avoiders. These variants, with exception of avoiders (not found among White patients), were found across all racial/ethnic groups. The variants suggested different ‘implementation strategies’, i.e., how clear patients made decisions and whether or not they then effectively communicated them. Conclusion These identified decision-making styles and variants generate strategies for clinicians to better address individualized advance care planning. Practice implications Physicians should elicit seriously ill patients’ decision-making styles and consider potential implementation strategies these styles may generate, thus tailoring individualized recommendations to assist patients in their advance care planning. Patient-centered EOL decision-making can ensure that patient preferences are upheld.
The efficient delivery of reactive and toxic gaseous reagents to organic reactions was studied using metal‐organic frameworks (MOFs). The simultaneous cargo vehicle and catalytic capabilities of ...several MOFs were probed for the first time using the examples of aromatization, aminocarbonylation, and carbonylative Suzuki–Miyaura coupling reactions. These reactions highlight that MOFs can serve a dual role as a gas cargo vehicle and a catalyst, leading to product formation with yields similar to reactions employing pure gases. Furthermore, the MOFs can be recycled without sacrificing product yield, while simultaneously maintaining crystallinity. The reported findings were supported crystallographically and spectroscopically (e.g., diffuse reflectance infrared Fourier transform spectroscopy), foreshadowing a pathway for the development of multifunctional MOF‐based reagent‐catalyst cargo vessels for reactive gas reagents as an attractive alternative to the use of toxic pure gases or gas generators.
Multifaceted metal‐organic frameworks (MOFs) can act as a food truck to adsorb, store, and deliver toxic gases to organic reactions. We showcase the versatility of MOFs by employing them as a safe‐delivery tool, while simultaneously catalyzing organic reactions, producing yields and conversions that are comparable to the reactions utilizing the toxic gases directly.
Background: Few risk factors have been implicated in pancreatic cancer etiology. Alcohol has been theorized to promote carcinogenesis.
However, epidemiologic studies have reported inconsistent ...results relating alcohol intake to pancreatic cancer risk.
Methods: We conducted a pooled analysis of the primary data from 14 prospective cohort studies. The study sample consisted
of 862,664 individuals among whom 2,187 incident pancreatic cancer cases were identified. Study-specific relative risks and
95% confidence intervals were calculated using Cox proportional hazards models and then pooled using a random effects model.
Results: A slight positive association with pancreatic cancer risk was observed for alcohol intake (pooled multivariate relative
risk, 1.22; 95% confidence interval, 1.03-1.45 comparing ≥30 to 0 grams/day of alcohol; P value, test for between-studies heterogeneity = 0.80). For this comparison, the positive association was only statistically
significant among women although the difference in the results by gender was not statistically significant ( P value, test for interaction = 0.19). Slightly stronger results for alcohol intake were observed when we limited the analysis
to cases with adenocarcinomas of the pancreas. No statistically significant associations were observed for alcohol from wine,
beer, and spirits comparing intakes of ≥5 to 0 grams/day. A stronger positive association between alcohol consumption and
pancreatic cancer risk was observed among normal weight individuals compared with overweight and obese individuals ( P value, test for interaction = 0.01).
Discussion: Our findings are consistent with a modest increase in risk of pancreatic cancer with consumption of 30 or more
grams of alcohol per day. (Cancer Epidemiol Biomarkers Prev 2009;18(3):765–76)
PDF
