Activation of Mitofusin2 by Smad2-RIN1 Complex during Mitochondrial Fusion.

Smads are nuclear-shuttling transcriptional mediators of transforming growth factor-ß (TGF-ß) signaling. Although their essential nuclear roles in gene regulation during development and carcinogenesis are well established, whether they have important cytoplasmic functions remains unclear. Here we report that Smad2 is a critical determinant of mitochondrial dynamics. We identified mitofusin2 (MFN2) and Rab and Ras Interactor 1 (RIN1) as new Smad2 binding partners required for mitochondrial fusion. Unlike TGF-ß-induced Smad2/3 transcriptional responses underlying mitochondrial fragmentation and apoptosis, inactive cytoplasmic Smad2 rapidly promotes mitochondrial fusion by recruiting RIN1 into a complex with MFN2. We demonstrate that Smad2 is a key scaffold, allowing RIN1 to act as a GTP exchange factor for MFN2-GTPase activation to promote mitochondrial ATP synthesis and suppress superoxide production. These results reveal functional implications between Smads and mitochondrial dysfunction in cancer and metabolic and neurodegenerative disorders.

External quality assessment practices in medical laboratories: an IFCC global survey of member societies.

OBJECTIVES: Clinical laboratory results are required for critical medical decisions, underscoring the importance of quality results. As part of total quality management, external quality assessment (EQA) is a vital component to ensure laboratory accuracy. The goal of this survey was to evaluate the current status of global laboratory quality systems and assess the need for implementation, expansion, or harmonization of EQA programs (EQAP) for Clinical Chemistry and Laboratory Medicine. METHODS: The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Task Force on Global Laboratory Quality (TF-GLQ) conducted a survey of IFCC full and affiliate members (n=110) on laboratory quality practice. A total of 41 (37.3%) countries representing all IFCC regions except North America provided responses about EQA availability and practices. RESULTS: All 41 countries perform EQA, 38 reported that their laboratories had EQA policies and procedures, and 39 further act/evaluate unacceptable EQA results. 39 countries indicated they have international and/or national EQAP and 30 use alternative performance assessments. EQA frequency varied among countries. Generally, an EQAP provided the EQA materials (40/41) with four countries indicating that they did not have an EQAP in their country. CONCLUSIONS: Globally, most laboratories participate in an EQAP and have defined quality procedures for EQA. There remain gaps in EQA material availability and implementation of EQA as a part of a total laboratory quality system. This survey highlights the need for education, training, and harmonization and will guide efforts of the IFCC TF-GLQ in identifying areas for enhancing global laboratory quality practices.

Quality standards and internal quality control practices in medical laboratories: an IFCC global survey of member societies.

OBJECTIVES: The trueness and precision of clinical laboratory results are ensured through total quality management systems (TQM), which primarily include internal quality control (IQC) practices. However, quality practices vary globally. To understand the current global state of IQC practice and IQC management in relation to TQM the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Task Force on Global Laboratory Quality (TF-GLQ) conducted a survey of IFCC member countries on IQC practices and management. METHODS: The survey included 16 questions regarding IQC and laboratory TQM practices and was distributed to IFCC full and affiliate member countries (n=110). A total of 46 (41.8 %) responses were received from all regions except North America. RESULTS: Of the responding countries, 78.3 % (n=36) had legislative regulations or accreditation requirements governing medical laboratory quality standards. However, implementation was not mandatory in 46.7 % (n=21) of responding countries. IQC practices varied considerably with 57.1 % (n=28) of respondents indicating that they run 2 levels of IQC, 66.7 % (n=24) indicating they run IQC every 24 h and 66.7 % (n=28) using assay manufacturer IQC material sources. Only 29.3 % (n=12) of respondents indicated that every medical laboratory in their country has written IQC policies and procedures. By contrast, 97.6 % (n=40) of responding countries indicated they take corrective action and result remediation in the event of IQC failure. CONCLUSIONS: The variability in TQM and IQC practices highlights the need for more formal programs and education to standardize and improve TQM in medical laboratories.

Implementation and challenges of portable blood gas measurements in air medical transport.

OBJECTIVES: Ventilator management in prehospital settings using end-tidal CO2 can lead to inappropriate ventilation in the absence of point of care blood gas (POCBG) measurements. Implementation of POCBG testing in helicopter Emergency Medical Services (HEMS) is limited in part because of concern for preanalytical and analytical errors due to altitude, vibration, and other associated environmental factors and due to insufficient documentation of implementation challenges. METHODS: We performed accuracy and precision verification studies using standard materials tested pre-, in-, and post-flight (n=10) in a large HEMS agency. Quality assurance error log data were extracted and summarized for common POCBG errors during the first 31 months of use and air medical transport personnel were surveyed regarding POCBG use (n=63). RESULTS: No clinically significant differences were found between pre-, in-, and post-flight blood gas measurements. Error log data demonstrated a reduction in device errors over time. Survey participants found troubleshooting device errors and learning new clinical processes to be the largest barriers to implementation. Continued challenges for participants coincided with error log data including temperature and sampling difficulties. Survey participants indicated that POCBG testing improved patient management. CONCLUSIONS: POCBG testing does not appear to be compromised by the HEMS environment. Temperature excursions can be reduced by use of insulated transport bags with heating and cooling packs. Availability of POCBG results in air medical transport appeared to improve ventilator management, increase recognition of ventilation-perfusion mismatch, and improve patient tolerance of ventilation.

Evaluating the incidence of engraftment syndrome with different melphalan formulations in adult multiple myeloma and immunoglobulin light chain amyloidosis patients undergoing autologous hematopoietic cell transplantation.

BACKGROUND: Engraftment syndrome (ES) is a common complication of autologous hematopoietic cell transplantation (HCT). The difference in incidence of ES between melphalan formulations has not been widely reported throughout the literature and would allow for a more comprehensive understanding of the advantages and disadvantages of both melphalan formulations. PATIENTS AND METHODS: This retrospective, single-center, observational study evaluated 83 adult multiple myeloma and immunoglobulin light chain amyloidosis patients who received either propylene glycol-containing (PG) or propylene glycol-free (PG-free) melphalan 140 mg/m2 as single-agent conditioning chemotherapy for autologous HCT from May 31, 2015 to May 31, 2019. The primary outcome was to assess the incidence of ES, as defined using the Maiolino criteria, with both melphalan formulations. Secondary outcomes included an analysis of potential risk factors for the development of ES, as well as an evaluation of overall length of stay (LOS). RESULTS: The incidence of ES for PG and PG-free melphalan did not differ significantly, 14/39 (35.9%) and 12/44 (27.3%) (P = 0.4), respectively. No potential risk factors for ES were identified on multivariate logistic regression analysis. A statistically significant difference in number of days to engraftment was identified for PG and PG-free melphalan, 15.56 vs. 13.82 days (P = 0.01), respectively; although, this did not translate to a decrease in LOS, 19.9 vs. 18.59 days (P = 0.14). CONCLUSIONS: The incidence of ES did not differ significantly between melphalan formulations. Future research is needed to determine whether the faster time to engraftment seen with PG-free melphalan may translate to a decrease in LOS.

Glycemic relapse in a collaborative primary care-based type 2 diabetes management program.

OBJECTIVES: The purpose of this study is to evaluate the incidence of glycemic relapse in patients who attained their glycosylated hemoglobin (A1C) goal through a health system-wide collaborative primary care-based pharmacist- and Certified Diabetes Care and Education Specialist (CDCES)-led type 2 diabetes (T2D) management program and to identify relapse risk factors. METHODS: This retrospective cohort study examined patients with T2D in the diabetes management program with a baseline A1C of at least 9% who attained their A1C goal. The primary outcome was incidence of glycemic relapse. Time to relapse was estimated using Kaplan-Meier curve, and a cox proportional hazards model was fitted to identify the risk factors for glycemic relapse. RESULTS: Three hundred sixty-two patients were followed-up for a median of 10.5 (interquartile range 12.1) months after program completion; 38 patients (10.5%) experienced a glycemic relapse. Kaplan-Meier analysis estimated a 12-month relapse rate of 8.3%. The presence of a medication adherence barrier, presence of a higher number of chronic medications at baseline, presence of a baseline body mass index (BMI) of 30-39.9, and use of insulin at program completion increased risk for glycemic relapse in a univariate model. In multivariate regression, baseline BMI of 30-39.9 remained statistically significant. Older age at baseline was associated with a statistically significantly decreased relapse risk in both models. CONCLUSION: This study highlights a low incidence of glycemic relapse for patients with T2D who reach their A1C goal through a collaborative primary care-based pharmacist- and CDCES-led T2D management program. The presence of risk factors for glycemic relapse may indicate a need for ongoing intensive care despite achieving A1C goal.

SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected hospitalized COVID-19 patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), emerged at the end of 2019 and by mid-June 2020 the virus had spread to at least 215 countries, caused more than 8 000 000 confirmed infections and over 450 000 deaths, and overwhelmed healthcare systems worldwide. Like severe acute respiratory syndrome coronavirus (SARS-CoV), which emerged in 2002 and caused a similar disease, SARS-CoV-2 is a betacoronavirus. Both viruses use human angiotensin-converting enzyme 2 (hACE2) as a receptor to enter cells. However, the SARS-CoV-2 spike (S) glycoprotein has a novel insertion that generates a putative furin cleavage signal and this has been postulated to expand the host range. Two low-passage (P) strains of SARS-CoV-2 (Wash1 : P4 and Munich : P1) were cultured twice in Vero E6 cells and characterized virologically. Sanger and MinION sequencing demonstrated significant deletions in the furin cleavage signal of Wash1 : P6 and minor variants in the Munich : P3 strain. Cleavage of the S glycoprotein in SARS-CoV-2-infected Vero E6 cell lysates was inefficient even when an intact furin cleavage signal was present. Indirect immunofluorescence demonstrated that the S glycoprotein reached the cell surface. Since the S protein is a major antigenic target for the development of neutralizing antibodies, we investigated the development of neutralizing antibody titres in serial serum samples obtained from COVID-19 human patients. These were comparable regardless of the presence of an intact or deleted furin cleavage signal. These studies illustrate the need to characterize virus stocks meticulously prior to performing either in vitro or in vivo pathogenesis studies.

A Model of Dormant-Emergent Metastatic Breast Cancer Progression Enabling Exploration of Biomarker Signatures.

Breast cancer mortality predominantly results from dormant micrometastases that emerge as fatal outgrowths years after initial diagnosis. In order to gain insights concerning factors associated with emergence of liver metastases, we recreated spontaneous dormancy in an all-human ex vivo hepatic microphysiological system (MPS). Seeding this MPS with small numbers (<0.05% by cell count) of the aggressive MDA-MB-231 breast cancer cell line, two populations formed: actively proliferating ("growing"; EdU+), and spontaneously quiescent ("dormant"; EdU-). Following treatment with a clinically standard chemotherapeutic, the proliferating cells were eliminated and only quiescent cells remained; this residual dormant population could then be induced to a proliferative state ("emergent"; EdU+) by physiologically-relevant inflammatory stimuli, lipopolysaccharide (LPS) and epidermal growth factor (EGF). Multiplexed proteomic analysis of the MPS effluent enabled elucidation of key factors and processes that correlated with the various tumor cell states, and candidate biomarkers for actively proliferating (either primary or secondary emergence) versus dormant metastatic cells in liver tissue. Dormancy was found to be associated with signaling reflective of cellular quiescence even more strongly than the original tumor-free liver tissue, whereas proliferative nodules presented inflammatory signatures. Given the minimal tumor burden, these markers likely represent changes in the tumor microenvironment rather than in the tumor cells. A computational decision tree algorithm applied to these signatures indicated the potential of this MPS for clinical discernment of each metastatic stage from blood protein analysis.

Emerging Roles of Transforming Growth Factor ß Signaling in Diabetic Retinopathy.

Diabetic retinopathy (DR) is a serious complication of diabetes mellitus affecting about one third of diabetic adults. Despite its prevalence, treatment options are limited and often implemented only in the later stages of the disease. To date, the pathogenesis of DR has been extensively characterized in the context of elevated glucose, insulin, and VEGF signaling, although a growing number of other growth factors and molecules, including transforming growth factor ß (TGF-ß) are being recognized as important contributors and/or therapeutic targets. Here, we review the complex roles of TGF-ß signaling in DR pathogenesis and progression. J. Cell. Physiol. 232: 486-489, 2017. © 2016 Wiley Periodicals, Inc.

Liver protects metastatic prostate cancer from induced death by activating E-cadherin signaling.

Liver is one of the most common sites of cancer metastasis. Once disseminated, the prognosis is poor as these tumors often display generalized chemoresistance, particularly for carcinomas that derive not from the aerodigestive tract. When these cancers seed the liver, the aggressive cells usually undergo a mesenchymal to epithelial reverting transition that both aids colonization and renders the tumor cells chemoresistant. In vitro studies demonstrate that hepatocytes drive this phenotypic shift. However, the in vivo evidence and the molecular signals that protect these cells from induced death are yet to be defined. Herein, we report that membrane surface E-cadherin-expressing prostate cancer cells were resistant to cell death by chemotherapeutic drugs but E-cadherin null cells or those expressing E-cadherin only in the cytoplasm were sensitive to death signals and chemotherapies both in vitro and in vivo. While cell-cell E-cadherin ligandation reduced mitogenesis, this chemoprotection was proliferation-independent as killing of both 5-ethynyl-2'-deoxyuridine-positive (or Ki67+ ) and 5-ethynyl-2'-deoxyuridine-negative (Ki67- ) cells was inversely related to membrane-bound E-cadherin. Inhibiting the canonical survival kinases extracellular signal-regulated protein kinases, protein kinase B, and Janus kinase, which are activated by chemotherapeutics in epithelial cell-transitioned prostate cancer, abrogated the chemoresistance both in cell culture and in animal models of metastatic cancer. For disseminated tumors, protein kinase B disruption in itself had no effect on tumor survival but was synergistic with chemotherapy, leading to increased killing. CONCLUSION: Liver microenvironment-driven phenotypic switching of carcinoma cells and subsequent survival signaling results in activation of canonical survival pathways that protect the disseminated prostate cancer liver micrometastases in a proliferation-independent manner, and these pathways can be targeted as an adjuvant treatment to improve the efficacy of traditional chemotherapeutics (Hepatology 2016;64:1725-1742).

Cadmium attenuates the macrophage response to LPS through inhibition of the NF-κB pathway.

Chronic obstructive pulmonary disease (COPD) in the U.S. is primarily caused by cigarette smoking. COPD patients are highly susceptible to respiratory infections in part due to alveolar macrophage dysfunction despite a substantial increase in macrophages in the lung. Cadmium (Cd) is a toxic metal that is concentrated within tobacco and accumulates in the lung of smokers. We hypothesized that Cd uptake into macrophages alters immune function thereby impairing the macrophage response to invading pathogens. Our hypothesis was tested by comparing primary human monocytes and macrophages, primary mouse bronchoalveolar lavage myeloid cells, and related cell lines. Strikingly, Cd exposure followed by LPS stimulation resulted in a dose-dependent, significant decrease in nuclear p65 activity in macrophages that was not observed in monocytes. This corresponded with Cd-mediated inhibition of IKKß and an impaired ability to transcribe and release cytokines in response to LPS challenge in vivo. These findings provide novel evidence that Cd has the capacity to disrupt macrophage immune function compared with monocytes. Importantly, Cd results in immune dysfunction in macrophages through inhibition of the NF-κB signaling pathway. Based on these findings, we provide new evidence that Cd contributes to immune dysfunction in the lung of COPD subjects and may increase susceptibility to infection.

Activated alveolar epithelial cells initiate fibrosis through secretion of mesenchymal proteins.

Fibrosis is characterized by accumulation of activated fibroblasts and pathological deposition of fibrillar collagens. Activated fibroblasts overexpress matrix proteins and release factors that promote further recruitment of activated fibroblasts, leading to progressive fibrosis. The contribution of epithelial cells to this process remains unknown. Epithelium-directed injury may lead to activation of epithelial cells with phenotypes and functions similar to activated fibroblasts. Prior reports that used a reporter gene fate-mapping strategy are limited in their ability to investigate the functional significance of epithelial cell-derived mesenchymal proteins during fibrogenesis. We found that lung epithelial cell-derived collagen I activates fibroblast collagen receptor discoidin domain receptor-2, contributes significantly to fibrogenesis, and promotes resolution of lung inflammation. Alveolar epithelial cells undergoing transforming growth factor-ß-mediated mesenchymal transition express several other secreted profibrotic factors and are capable of activating lung fibroblasts. These studies provide direct evidence that activated epithelial cells produce mesenchymal proteins that initiate a cycle of fibrogenic effector cell activation, leading to progressive fibrosis. Therapy targeted at epithelial cell production of type I collagen offers a novel pathway for abrogating this progressive cycle and for limiting tissue fibrosis but may lead to sustained lung injury/inflammation.

Clinical Significance of Uncommon, Non-Clinical, and Novel Autoantibodies.

Autoantibodies are a common mark of autoimmune reaction and their identification in the patients' serum, cerebrospinal fluid, or tissues is generally believed to represent diagnostic or prognostic biomarkers of autoimmune diseases or autoinflammatory conditions. Traditionally, autoantibody testing is an important part of the clinical examination of suspected patients, and in the absence of reliable T cell tests, characterization of autoantibody responses might be suitable in finding causes of specific autoimmune responses, their strength, and sometimes commencement of autoimmune disease. Autoantibodies are also useful for prognostic stratification in clinically diverse groups of patients if checked repeatedly. Antibody discoveries are continuing, with important consequences for verifying autoimmune mechanisms, diagnostic feasibility, and clinical management. Adding newly identified autoantibody-autoantigen pairs to common clinical laboratory panels should help upgrade and harmonize the identification of systemic autoimmune rheumatic disorders and other autoimmune conditions. Herein, we aim to summarize our current knowledge of uncommon and novel autoantibodies in the context of discussing their validation, diagnostic practicability, and clinical relevance. The regular updates within the field are important and well justified.

Comparison of quantitative and qualitative anti-dsDNA assays.

OBJECTIVE: In evaluation of systemic lupus erythematosus (SLE), anti-double-stranded DNA antibodies (anti-dsDNA) play a significant role in diagnosis, monitoring SLE activity, and assessing prognosis. However, evaluations of the performance and limitations for recently developed methods for anti-dsDNA assessment are sparse. METHODS: Specimens used for antinuclear antibody testing (n = 129) were evaluated for anti-dsDNA assay comparability across 4 medical centers in the United States. The methods compared were Werfen Quanta Lite dsDNA, Zeus Scientific dsDNA Enzyme Immunoassay, Bio-Rad multiplex immunoassay (MIA) dsDNA, ImmunoConcepts Crithidia, and Bio-Rad Laboratories Crithidia. RESULTS: For quantitative anti-dsDNA measurements, Spearman's correlation coefficient was highest between Zeus and Werfen (ρ = 0.86; CI, 0.81-0.90; P < .0001). Comparison of MIA to Werfen or Zeus yielded similar results to each other (ρ = 0.58; CI, 0.44-0.68; P < .0001; and ρ = 0.59; CI, 0.46-0.69; P < .0001, respectively), but lower than the correlation between Zeus and Werfen. Positive concordance between assays ranged from 31.4% to 97.1%, and negative concordance between assays ranged from 58.5% to 100%. The detection of anti-dsDNA in those with SLE diagnosis ranged from 50.9% to 77.4% for quantitative assays and 15.1% to 24.5% for Crithidia assays. CONCLUSION: Current quantitative anti-dsDNA assays are not interchangeable for patient follow-up. Crithidia-based assays demonstrate high negative concordance and lack positive concordance among the methods.

Copeptin Stimulation by Combined Intravenous Arginine and Oral LevoDopa/Carbidopa in Healthy Short Children and Children with the Polyuria-Polydipsia Syndrome.

INTRODUCTION: Stimulated copeptin may provide an alternative to water deprivation testing (WDT) in the evaluation of polyuria-polydipsia syndrome (PPS). Though best studied, arginine stimulation alone produces a modest copeptin response in children. We investigated the effectiveness of the arginine + LevoDopa/Carbidopa stimulation test (ALD-ST) for copeptin. METHODS: 47 healthy short children (controls), 10 children with primary polydipsia, and 10 children with AVP deficiency received arginine hydrochloride (500 mg/kg intravenously over 30 min) and Levodopa/carbidopa (10:1 ratio; 175 mg of l-Dopa/m2 BSA) orally. Serum copeptin was measured at 0, 60, 90, and 120 min. RESULTS: In controls, ALD-ST increased copeptin from a median of 7.0 pmol/L (IQR 5.0-10.0) to a peak of 44.0 pmol/L (IQR 21.4-181.0) between 60 and 120 min (p < 0.001). Copeptin peak was higher in subjects who experienced nausea or vomiting (57%) than in those who did not (131.0 pmol/L [IQR 42.5-193.8] vs. 22.7 pmol/L [IQR 16.0-33.7], p < 0.001). While subjects with primary polydipsia had similar baseline (8.5 pmol/L [IQR 8.0-11.0]) and stimulated (125.2 pmol/L [IQR 87.6-174.0]) copeptin levels as controls, subjects with AVP deficiency had lower baseline (2.5 pmol/L [IQR 2.0-3.1]) and peak levels (4.6 pmol/L [IQR 2.4-6.0]). A peak copeptin of ≥9.3 pmol/L best predicted absence of complete or partial AVP deficiency with a sensitivity of 100% and specificity of 80%. CONCLUSIONS: ALD-ST induced a robust peak copeptin in healthy short children and children with primary polydipsia. Nausea/vomiting, a side effect of ALD-ST, amplified the copeptin response. The ALD-ST may be a suitable initial screening test in children with PPS.