By combining isoelectric focusing (IEF) with subsequent gel electrophoresis, two-dimensional electrophoresis (2DE) affords more specific characterization of proteins than each constituent unit ...separation. In a new approach to integrating the two assay dimensions in a microscope slide-sized glass device, we introduce microfluidic 2DE using photopatterned polyacrylamide (PA) gel elements housed in a millimeter-scale, 20-μm-deep chamber. The microchamber minimizes information loss inherent to channel network architectures commonly used for microfluidic 2DE. To define the IEF axis along a “lane” at the top of the chamber, we used free solution carrier ampholytes and immobilized acrylamido buffers in the PA gels. This approach yielded high-resolution (0.1 pH unit) and rapid (<20 min) IEF. Next, protein transfer to the second dimension was accomplished by chemical mobilization perpendicular to the IEF axis. Mobilization drove focused proteins off the IEF lane and into a region for protein gel electrophoresis. Using fluorescently labeled proteins, we observed transfer-induced band broadening factors ∼7.5-fold lower than those observed in microchannel networks. Both native polyacrylamide gel electrophoresis (PAGE) and pore-limit electrophoresis (PLE) were studied as the second assay dimension and completed in <15 min. PLE yields protein molecular mass information without the need for ionic surfactant or reducing agents, simplifying device design and operation. Microchamber-based 2DE unifies two independent separation dimensions in a single device with minimal transfer-associated information losses. Peak capacities for the total assay ranged from 256 to 35 with <1 h assay duration. The rapid microchamber 2DE assay has the potential to bridge an existing gap in targeted proteomics for protein biomarker validation and systems biology that may complement recent innovation in mass spectrometry.
Commercially available, highly passaged pancreatic cancer (PC) cell lines are of limited translational value. Attempts to overcome this limitation have primarily consisted of cancer cell isolation ...and culture directly from human PC specimens. However, these techniques are associated with exceedingly low success rates. Here, we demonstrate a highly reproducible culture of primary PC cell lines (PPCLs) from patient-derived xenografts, which preserve, in part, the intratumoral heterogeneity known to exist in PC. PPCL expansion from patient-derived xenografts was successful in 100% of attempts (5 of 5). Phenotypic analysis was evaluated with flow cytometry, immunofluorescence microscopy, and short tandem repeat profiling. Importantly, tumorigenicity of PPCLs expanded from patient-derived xenografts was assessed by subcutaneous injection into nonobese diabeteic.Cg-Prkdcscid Il2rgtm1Wjl /SzJ mice. Morphologically, subcutaneous injection of all PPCLs into mice yielded tumors with similar characteristics to the parent xenograft. PPCLs uniformly expressed class I human leukocyte antigen, epithelial cell adhesion molecule, and cytokeratin-19. Heterogeneity within each PPCL persisted in culture for the frequency of cells expressing the cancer stem cell markers CD44, CD133, and c-Met and the immunologic markers human leukocyte antigen class II and programmed death ligand 1. This work therefore presents a reliable method for the rapid expansion of primary human PC cells and, thereby, provides a platform for translational investigation and, importantly, potential personalized therapeutic approaches.
South Africa is a semi-arid country which frequently faces water shortages, and experienced a severe drought in the 2016 and 2017 rainfall seasons. Government is under pressure to continue to deliver ...clean water to the growing population at a high assurance of supply. Studies now show that the delivery of water may be sustained not only through built infrastructure such as dams and pipelines, but also through investment in ecological infrastructure (EI). Part 1 of this paper in 2 parts concentrated on the role of EI in delivering water-related ecosystem services, as well as the motivation for this study, and the methods used in modelling and mapping the catchment. Part 2 explores and illustrates the current level of delivery of water-related ecosystem services in different parts of the catchment, with potential hydrological benefits of rehabilitation and protection of EI in the uMngeni catchment. The Mpendle, Lions River, Karkloof, Inanda and Durban sub-catchments are important areas for the generation of streamflows which accumulate downstream (i.e. water yield in the catchment) when annual totals are considered. Modelled annual sediment yield (in tonnes) from naturally vegetated areas is most severe in the lower catchment areas with steeper slopes such as Inanda, and in the high-altitude areas which have both steeper slopes and higher rainfall. The central and eastern parts of the uMngeni catchment were found to contribute the greatest yield of sediment from degraded areas with low protective vegetation cover. This combined modelling and mapping exercise highlighted areas of priority ecosystem service delivery, such as higher altitude grassland areas, which could be recommended for formal conservation, or protection under private partnerships. Generally, these areas confirm the intuitive sense of catchment stakeholders, but provide a robust and more defendable analysis through which water volumes are quantifiable, and potential investment into catchment interventions are justified.
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Despite the enormous advances in the field of clinical pancreatic islet transplantation over the past two decades, the human islet isolation procedure remains suboptimal. Islets are ...extracted (isolated) from the exocrine tissue of donor pancreases using neutral protease (NP) and collagenase-based enzymes, which digest the extracellular matrix (ECM) scaffold surrounding human islets. This process remains highly variable and current isolation enzyme blends are ineffective at digesting pancreases from younger donors with low body mass indexes (BMI). However, age-related differences in pancreatic matrix digestion have not been studied in detail at the molecular level. To address this, we investigated ECM digestion in purified ECM proteins and in pancreatic tissue sections from younger (≤30 years; n = 5) and older (>55 years; n = 5) BMI matched donors, using Raman microspectroscopy (RMS). The Raman spectral profiles for purified collagens I, IV, VI and laminins were significantly altered following controlled enzyme treatment. Pancreatic cryosections were treated with Serva collagenase, NP, or the two enzymes combined, at clinically relevant concentrations. RMS demonstrated that the ECM at the islet-exocrine interface was differentially digested with respect to donor age. The action of collagenase was affected to a greater extent than NP.
RMS is a powerful, marker-independent technology for characterising the human pancreatic ECM and demonstrating differences between donor types. Ongoing detailed studies using RMS will assist the development of donor-specific enzyme blends, increasing the overall success of human islet isolation and benefiting many people with type 1 diabetes worldwide.
Pancreatic islet transplantation is a minimally invasive treatment, which can reverse Type 1 Diabetes Mellitus (T1DM) in selected patients. Islets of Langerhans are extracted (isolated) from the exocrine tissue of human donor pancreases using neutral protease (NP) and collagenase-based enzymes, which digest the extracellular matrix (ECM) scaffold surrounding human islets. This process remains highly variable and current enzymes are ineffective at digesting pancreases from younger donors. Using Raman microspectroscopy we demonstrate that donor age affects the enzymatic digestion of the pancreatic ECM at the molecular level. Collagenase activity is affected to a greater extent than NP. These findings will assist the development of donor-specific enzymes, thereby increasing the overall success of islet isolation and benefiting many people with T1DM worldwide.
Summary
Lower fracture rates in Black men and women compared to their White counterparts are incompletely understood. High-resolution imaging specific to trabecular bone may provide insight. Black ...participants have enhanced trabecular morphology. These differences may contribute to the lower fracture risk in Black versus White individuals.
Introduction
Lower fracture rates in Black men and women compared to their White counterparts may be explained by favorable bone microstructure in Black individuals. Individual trabecular segmentation (ITS) analysis, which characterizes the alignment and plate- and rod-like nature of trabecular bone using high-resolution peripheral quantitative computed tomography (HR-pQCT), may provide insight into trabecular differences by race/ethnic origin.
Purpose
We determined differences in trabecular bone microarchitecture, connectivity, and alignment according to race/ethnic origin and sex in young adults.
Methods
We analyzed HR-pQCT scans of 184 adult (24.2 ± 3.4 years) women (
n
= 51 Black,
n
= 50 White) and men (
n
= 34 Black,
n
= 49 White). We used ANCOVA to compare bone outcomes, and adjusted for age, height, and weight.
Results
Overall, the effect of race on bone outcomes did not differ by sex, and the effect of sex on bone outcomes did not differ by race. After adjusting for covariates, Black participants and men of both races had greater trabecular plate volume fraction, plate thickness, plate number density, plate surface area, and greater axial alignment of trabeculae, leading to higher trabecular bone stiffness compared to White participants and women, respectively (
p
< 0.05 for all).
Conclusion
These findings demonstrate that more favorable bone microarchitecture in Black individuals compared to White individuals and in men compared to women is not unique to the cortical bone compartment. Enhanced plate-like morphology and greater trabecular axial alignment, established in young adulthood, may contribute to the improved bone strength and lower fracture risk in Black versus White individuals and in men compared to women.
The objective of this study was to determine whether identification of the iliolumbar ligaments is of practical use for numbering lumbosacral transitional vertebrae (LSTV).
Five hundred consecutive ...lumbar spine MRI studies were reviewed. A standard protocol of sagittal and axial T1-weighted and T2-weighted spin-echo sequences was used. The sagittal images were assessed for the presence of an LSTV, and axial images were assessed for the level of origin of the iliolumbar ligaments.
Of the 500 patients, 433 (86.6%) had normal lumbosacral segmentation and 67 (13.4%) had a transitional lumbosacral junction. The iliolumbar ligament was identified at L5 in all patients with normal lumbosacral segmentation (n = 433), bilaterally in 432 and unilaterally in one. Using the identification of the iliolumbar ligaments as a marker of the L5 vertebral level, we numbered 46 of the 67 LSTV as L5 transitions and 21 as S1 transitions.
The iliolumbar ligament is readily identifiable on axial lumbar spine MRI and always arises from L5. We suggest that its position can be used to confidently assign lumbar levels in patients with LSTV.
Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which since 2019 has caused over 5 million deaths to date. The pathogenicity of the ...virus is highly variable ranging from asymptomatic to fatal. Evidence from experimental and observational studies suggests that circulating micronutrients may affect COVID-19 outcomes.
To complement and inform observational studies, we investigated the associations of genetically predicted concentrations of 12 micronutrients (β-carotene, calcium, copper, folate, iron, magnesium, phosphorus, selenium, vitamin B-6, vitamin B-12, vitamin D, and zinc) with SARS-CoV-2 infection risk and COVID-19 severity using Mendelian randomization (MR).
Two-sample MR was conducted using 87,870 individuals of European descent with a COVID-19 diagnosis and 2,210,804 controls from the COVID-19 host genetics initiative. Inverse variance-weighted MR analyses were performed with sensitivity analyses to assess the impact of potential violations of MR assumptions.
Compared to the general population, nominally significant associations were noted for higher genetically predicted vitamin B-6 (Odds ratio per standard deviation
: 1.06; 95% confidence interval CI: 1.00, 1.13;
-value = 0.036) and lower magnesium concentrations (
: 0.33; 95%CI: 0.11, 0.96;
= 0.042) with COVID-19 infection risk. However, the association for magnesium was not consistent in some sensitivity analyses, and sensitivity analyses could not be performed for vitamin B-6 as only two genetic instruments were available. Genetically predicted levels of calcium, folate, β-carotene, copper, iron, vitamin B-12, vitamin D, selenium, phosphorus, or zinc were not associated with the outcomes from COVID-19 disease.
These results, though based only on genetically predicated circulating micronutrient concentrations, provide scant evidence for possible associations of micronutrients with COVID-19 outcomes.
Aim To assess the impact of a community pharmacy diabetes service model on patient outcomes in Type 2 diabetes.
Methods The study utilized a multisite, control vs. intervention, repeated‐measures ...design within four states in Australia. Fifty‐six community pharmacies, 28 intervention and 28 control, were randomly selected from a representative sample of urban and rural areas. Intervention pharmacies delivered a diabetes service to patients with Type 2 diabetes, which comprised an ongoing cycle of assessment, management and review, provided at regular intervals over 6 months in the pharmacy. These services included support for self monitoring of blood glucose, education, adherence support, and reminders of checks for diabetes complications. Control pharmacists assessed patients at 0 and 6 months and delivered no intervention.
Results A total of 289 subjects (149 intervention and 140 control) completed the study. For the intervention subjects, the mean blood glucose level decreased over the 6‐month study from 9.4 to 8.5 mmol/l (P < 0.01). Furthermore, significantly greater improvements in glycaemic control were seen in the intervention group compared with the control: the mean reduction in HbA1c in the intervention group was −0.97% (95% CI: −0.8, −1.14) compared with −0.27% (95% CI: −0.15, −0.39) in the control group. Improvements were also seen in blood pressure control and quality of life in the intervention group.
Conclusion A pharmacy diabetes service model resulted in significant improvements in clinical and humanistic outcomes. Thus, community pharmacists can contribute significantly to improving care and health outcomes for patients with Type 2 diabetes. Future research should focus on clarifying the most effective elements of the service model.
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