The analysis of mitochondrial dynamics within immune cells allows us to understand how fundamental metabolism influences immune cell functions, and how dysregulated immunometabolic processes impact biology and disease pathogenesis. For example, during infections, mitochondrial fission and fusion coincide with effector and memory T-cell differentiation, respectively, resulting in metabolic reprogramming. As frozen cells are generally not optimal for immunometabolic analyses, and given the logistic difficulties of analysis on cells within a few hours of blood collection, we have optimized and validated a simple cryopreservation protocol for peripheral blood mononuclear cells, yielding >95% cellular viability, as well as preserved metabolic and immunologic properties. Combining fluorescent dyes with cell surface antibodies, we demonstrate how to analyze mitochondrial density, membrane potential, and reactive oxygen species production in CD4 and CD8 T cells from cryopreserved clinical samples.The proton electrochemical gradient generated by respiratory chain activity accounts for over 90% of all available ATP and, as such, its evaluation and accurate measurements regarding its total values and fluctuations is an invaluable component in the understanding of mitochondrial functions. Consequently, alterations in electric potential across the inner mitochondrial membrane generated by differential protonic accumulations and transport are known as the mitochondrial membrane potential, or Δψ, and are reflective of the functional metabolic status of mitochondria. There are several experimental approaches to measure Δψ, ranging from fluorometric evaluations to electrochemical probes. Here we discuss the advantages and disadvantages of several of these methods, ranging from one that is dependent on the movement of a particular ion (tetraphenylphosphonium (TPP+) with a selective electrode) to the selection of a fluorescent dye from various types to achieve the same goal. The evaluation of the accumulation and movements of TPP+ across the inner mitochondrial membrane, or the fluorescence of accumulated dye particles, is a sensitive and accurate method of evaluating the Δψ in respiring mitochondria (either isolated or still inside the cell).Dendritic cells (DCs) are the bridge between innate and T cell-dependent adaptive immunity, and are promising therapeutic targets for cancer and immune-mediated disorders. In the recent past, DCs have gained significant interest to manipulate them for the treatment of cancer and immune-mediated disorders. This can be achieved by differentiating them into either immunogenic or tolerogenic DCs (TolDCs), by modulating their metabolic pathways, including glycolysis, oxidative phosphorylation, and fatty acid metabolism, to orchestrate their desired function. For immunogenic DCs, this maturation shifts the metabolic profile to a glycolytic metabolic state and leads to the use of glucose as a carbon source, whereas TolDCs prefer oxidative phosphorylation (OXPHOS) and fatty acid oxidation for their energy resource.Understanding the metabolic regulation of DC subsets and functions at large not only will improve our understanding of DC biology and immune regulation, but can also open up opportunities for treating immune-mediated ailments and cancers by tweaking endogenous T-cell responses through DC-based immunotherapies. Here we describe a method to analyze this dichotomous metabolic reprogramming of the DCs for generating reliable and effective DC cell therapy products. We, hereby, report how to measure the OXPHOS and glycolysis level of DCs. We focus on the metabolic reprogramming of TolDCs using a pharmacological nuclear factor (erythroid-derived 2)-like-2 factor (Nrf2) activator as an example to illustrate the metabolic profile of TolDCs.Metabolism plays an important role in the activation and effector functions of macrophages. Intracellular pathogens, such as Mycobacterium tuberculosis, subvert the immune functions of macrophages to establish an infection by modulating the metabolism of the macrophage. Here, we describe how the Seahorse Extracellular Flux Analyzer (XF) from Agilent Technologies can be used to study the changes in the bioenergetic metabolism of the macrophages induced by infection with mycobacteria. The XF simultaneously measures the oxygen consumption and extracellular acidification of the macrophages noninvasively in real time, and together with the addition of metabolic modulators, substrates, and inhibitors enables measurements of the rates of oxidative phosphorylation, glycolysis, and ATP production.The posttranslational modifications (PTMs) ADP-ribosylation and phosphorylation are important regulators of cellular pathways, and while mass spectrometry (MS)-based methods for the study of protein phosphorylation are well developed, protein ADP-ribosylation methodologies are still in a rapidly developing stage. The method described in this chapter uses immobilized metal affinity chromatography (IMAC), a phosphoenrichment matrix, to enrich ADP-ribosylated peptides which have been cleaved down to their phosphoribose attachment sites by a phosphodiesterase, thus isolating the ADP-ribosylated and phosphorylated proteomes simultaneously. To achieve the robust, relative quantification of PTM-level changes we have incorporated dimethyl labeling, a straightforward and economical choice which can be used on lysate from any cell type, including primary tissue. The entire pipeline has been optimized to work in ADP-ribosylation-compatible buffers and with protease-laden lysate from macrophage cells.Macrophages are professional innate immune cells that are broadly disseminated throughout the body, shape various innate and adaptive immune responses, and play crucial roles in inflammation, homeostasis, wound healing, and tissue remodelling. According to their surrounding microenvironments, macrophages can differentiate themselves in different phenotypes. Over the last two decades, gene expression profiling has been used to decipher new transcripts associated with macrophage phenotypes. This chapter outlines protocols used to isolate and culture murine macrophages and how they can be „polarized” to obtain a specific phenotype. Furthermore, we describe a protocol for gene expression profiling using a quantitative real-time polymerase chain reaction (qPCR), a high-standard technology in the field of gene expression.The co-culture of adipocytes and immune cells, such as macrophages or T cells (CD4+ or CD8+ subsets), is a novel experimental approach used to study paracrine interactions (or the cross talk) between cultured cell types in isolation, in order to understand their role in obese adipose tissue (AT) inflammation and dysfunction. Here we describe the general methodologies required for the co-culture of mature adipocytes (differentiated 3T3-L1 pre-adipocyte cell line) with primary immune cell subsets purified from mouse splenic mononuclear cells using a magnetic MicroBead positive selection, wherein multiple immune cell populations can be purified sequentially from a single mouse spleen, thereby providing diversity in the types of immune cells that can be co-cultured with adipocytes. Additionally, we describe experimental procedures for co-culturing adipocytes and immune cells in two different co-culture systems, including a cell contact-dependent co-culture system, wherein the cells are in direct physical contact, and a cell contact-independent, soluble mediator-driven co-culture system wherein the cells are physically separated by a trans-well semipermeable membrane. Finally, we discuss how these co-culture models can be utilized to recapitulate the AT microenvironment in obesity by utilizing physiologically relevant ratios of adipocytesimmune cells (specifically CDllb+ macrophages, CD4+ T cells, or CD8+ T cells) and lipopolysaccharide stimulation that mimics endotoxin concentrations observed in obesity.Three-dimensional (3D) cultures are better able to reflect the tumor microenvironment than two-dimensional (2D) monolayer cultures by facilitating cell-cell interactions in the appropriate spatial dimensions. Here I describe the isolation and co-culture of immune cells with tumor cell lines in a three-dimensional system facilitated by a basement membrane extract. This allows for further downstream applications to analyze interactions between these cell types.Exosomes are cell-derived vesicles that have been implicated in the pathogenesis of many inflammatory diseases. In the immune system, it has been shown that T lymphocyte-derived exosomes are able to induce diverse cellular responses. There are several methods to isolate and to characterize exosomes, each with their own advantages and disadvantages. Here, we describe a centrifugation approach, combined with mass spectrometry characterization, as a means to study exosomes derived from primary human T lymphocytes. This method is sensitive and therefore can be applied when a limited amount of sample is available.Macrophages play an essential role in diverse biological processes, from the immune response to inflammatory and neurodegenerative disorders, to various cancers. A macrophage subpopulation, known as tumor-associated macrophages (TAMs), has been shown to promote tumorigenesis, metastasis, and immune escape of cancer cells. Some of the pro-tumorigenic effects of TAMs are mediated via the secretion of nano-vesicles (exosomes) from macrophages to neighboring cells. In this chapter, we describe peritoneal macrophage isolation methods, polarization of TAMs, and purification and characterization of macrophage-derived exosomes.Macrophages play a critical role in innate immunity through Toll-like receptor (TLR) signaling. Lipopolysaccharides (LPS) are a ligand of microbial origin that can trigger cell signaling in macrophages through TLRs and production of pro-inflammatory cytokines. Statin, a hypercholesterolemia drug, on the contrary, can reduce inflammatory cytokine production, and inflammation at large. Discovery-based quantitative proteomics is a useful method for unraveling complex protein networks and inter-protein interactions. Here, we describe protocols for studying the inflammatory proteomics network in RAW 264.7 cells (a model murine macrophage cell line) with the singular or sequential treatment of LPS and statin. We provide detailed protocols, including a quantitative proteomic analysis by mass spectrometry data, a protein network analysis by bioinformatics, and a validation of target through biochemical methods (e.g., immunocytochemistry, immunoblotting, gene silencing, and real-time PCR).Fatty acids (FAs) are essential for building complex lipids, posttranslational modifications, and energetics. FAs can be imported from extracellular sources or synthesized by cells. The analysis of fatty acid methyl esters (FAMEs) by gas chromatography-mass spectrometry (GC-MS) allows for the quantitative analysis of long-chain and very-long-chain fatty acid content of cells. When coupled with isotopic labeling, this approach can elucidate the synthetic pathways being engaged by the cells, and the relative contribution of synthesis and import to maintain lipid content. Here, we describe a method for total cellular fatty acid analysis in macrophages.Transcriptome analysis at a single-cell level with single-cell RNA sequencing (scRNA-seq) is a powerful method for detailed characterization of heterogeneous cell populations. Recent developments have enabled parallel analysis of both transcript and protein levels by using antibodies conjugated to barcoded oligonucleotides. These antibodies enable protein levels to be converted into nucleotide format, allowing the sequencing-based detection of both modalities at single-cell level. Here we present a simple and reliable method for conjugation of oligonucleotides with antibodies and a protocol for their use in single-cell transcriptome sequencing.Lactate, the product of aerobic glycolysis, plays a dual role as fuel and intercellular signal in inflammation, immune evasion, and tumor progression. The production of lactate by macrophages has been associated with their polarization and function. Here we describe imaging protocols to characterize the metabolism of cultured human macrophages using a genetically encoded fluorescent sensor-specific for lactate. By superfusing cultures with increasing lactate concentrations and pharmacological inhibitors, it is possible to estimate the kinetic parameters of monocarboxylate transporter 4 (MCT4) and lactate production. Practical advice is given regarding sensor expression, imaging, and data analysis. The spatiotemporal resolution of this technique is amenable to the study of fast events at the single-cell level in different immune and other cell types.Single-cell RNA-sequencing (scRNA-seq) enables a comprehensive analysis of the transcriptome of individual cells by next-generation sequencing. ScRNA-seq offers an unbiased approach to investigate the cellular heterogeneity and dynamics of diverse biological systems, including the immune system. Optimization of the technical procedures performed prior to RNA-seq analysis is imperative to the success of a scRNA-seq experiment. Here, three major experimental procedures are described (1) the isolation of immune CD8a+ T cells from primary murine tissue, (2) the generation of single-cell cDNA libraries using the 10× Genomics Chromium Controller and the Chromium Single Cell 3′ Solution, and (3) cDNA library quality control. In this protocol, CD8a+ T cells are isolated from murine spleen tissue, but any cell type of interest can be enriched and used for single-cell cDNA library generation and subsequent RNA-seq experiments.

Both patients and surrogate decision-makers experience decisional conflict when making a major medical treatment decision with life or death implications. The relationship between health literacy and decisional conflict while making a major medical treatment decision is not understood.

To identify the prevalence of individuals making major medical treatment decisions for themselves or someone else and to explore the relationships between decisional conflict and circumstances of the decision as well as the decision-maker.

Two-phase survey study in phase 1, we screened for who made a major treatment decision; in phase 2, we asked eligible respondents about their experience making the decision.

Address-based random sample of 4000 Wisconsin residents; 1072 completed phase 1 and 464 completed phase 2.

We asked respondents about types of decisions made, the most difficult decision made, and characteristics of the decision-maker and the decision. We included the Decisional Conflict Scale and four domains oelpful.

The need to make major treatment decisions is likely to increase and making decisions on someone else’s behalf appeared to be especially difficult. Improving communication to encourage patient and family engagement in the decision-making conversation, particularly for individuals with limited health literacy, may be helpful.

Little is known about how physicians spend their work time.

To determine how physicians in outpatient care spend their time at work, using an innovative method ecological momentary assessment (EMA).

Physician activity was measured via EMA, using a smartphone app.

Twenty-eight practices across 16 US states. Sixty-one physicians general internal medicine, family medicine, non-interventional cardiology, orthopedics.

Proportions of time spent on 14 activities within 6 broad categories of work direct patient care (including both face-to-face care and other patient care-related activities), electronic health record (EHR) input, administration, teaching/supervising, personal time, and other.

After excluding personal time, physicians spent 66.5% of their time on direct patient care (23.6% multitasking with use of the EHR and 42.9% without the EHR), 20.7% on EHR input alone, 7.7% on administrative activities, and 5.0% on other activities (0.6% using the EHR). In total, physicians spent 44.9% of their time on the EHR.

Unable to measure time spent at home on the EHR or other work tasks; participating physicians were not a random sample of US physicians.

The efficiency of highly trained professionals spending only two-thirds of their time on direct patient care may be questioned. EHR use continues to account for a large proportion of physician time. Further attempts should be made to redesign both EHRs and physician work processes.

The efficiency of highly trained professionals spending only two-thirds of their time on direct patient care may be questioned. EHR use continues to account for a large proportion of physician time. Further attempts should be made to redesign both EHRs and physician work processes.

Though long-term weight loss maintenance is the treatment goal for obesity, weight regain is typical and few studies have evaluated lifestyle habits associated with weight regain.

To identify dietary and physical activity habits associated with 6- and 24-month weight regain among participants in a weight loss maintenance clinical trial.

Secondary analysis of randomized clinical trial data.

Adult primary care patients with recent, intentional weight loss of at least 5%.

Lifestyle habits included consumption of low-fat foods, fish, desserts, sugary beverages, fruits, and vegetables and eating at restaurants from the Connor Diet Habit Survey; moderate-vigorous physical activity by self-report; steps recorded by a pedometer; and sedentary behavior by self-report. The outcome variable was weight change at 6 and 24months. Linear regression models estimated adjusted associations between changes in weight and changes in dietary and physical activity habits.

Overall, participants (mean (SD) 53.4 (12.2) years old; 26% male; 88% white) maintained weight loss at 6months (n = 178, mean (SD) - 0.02 (5.70)% change) but began to regain weight by 24months (n = 157, mean (SD) 4.22 (9.15)% increase). When considered all together, more eating at restaurants, reduced fish consumption, and less physical activity were most consistently associated with weight regain in fully adjusted models at both 6 and 24months of follow-up. In addition, more sedentary behavior was associated with weight regain at 6months while reduced consumption of low-fat foods, and more desserts and sugary beverages were associated with weight regain at 24months.

Consuming less fish, fewer steps per day, and more frequent restaurant eating were most consistently associated with weight regain in primary care patients. Primary care providers may consider addressing specific lifestyle behaviors when counseling patients after successful weight loss.

ClinicalTrials.gov Identifier NCT01946191.

ClinicalTrials.gov Identifier NCT01946191.

Identifying characteristics of primary care practices that perform well on cardiovascular clinical quality measures (CQMs) may point to important practice improvement strategies.

To identify practice characteristics associated with high performance on four cardiovascular disease CQMs.

Longitudinal cohort study among 211 primary care practices in Colorado and New Mexico. Quarterly CQM reports were obtained from 178 (84.4%) practices. There was 100% response rate for baseline practice characteristics and implementation tracking surveys. Follow-up implementation tracking surveys were completed for 80.6% of practices.

Adult patients, staff, and clinicians in family medicine, general internal medicine, and mixed-specialty practices.

Practices received 9 months of practice facilitation and health information technology support, plus biannual collaborative learning sessions.

This study identified practice characteristics associated with overall highest performance using area under the curve (AUC) analysi57).

Multiple strategies-registries, prompts and protocols, patient self-management support, and patient-team partnership activities-were associated with delivering high-quality cardiovascular care over time, measured by CQMs.

ClinicalTrials.gov registration NCT02515578.

ClinicalTrials.gov registration NCT02515578.

The Agency for Healthcare Research and Quality (AHRQ) could devote resources to collate and assess quality improvement studies to support learning health systems (LHS) but there is no reliable data on the consistency of data extraction for important criteria.

We identified quality improvement studies and evaluated the consistency of data extraction from two experienced independent reviewers at three time points baseline, first revision (where explicit instructions for each criterion were created), and final revision (where the instructions were revised). Six investigators looked at the data extracted by the two systematic reviewers and determined the extent of similarity on a scale of 0 to 10 (where 0 represented no similarity and 10 perfect similarity). There were 42 assessments for baseline, 42 assessments for the first revision, and 42 assessments for the final revision. We asked two LHS participants to assess the relative value of our criteria.

The consistency of extraction improved from 1.17 ± 1.85e consistency of data extraction. This is important because it is difficult for LHS to vet these quality improvement studies on their own and they would value AHRQ’s support in that regard.The study describes results of a survey of tomato fields for the presence of begomoviruses from different regions of Peninsular Malaysia. An ORF-based (C2 and C3) study was performed to determine the distribution of begomoviruses associated with a severe leaf curl disease in tomato-growing areas of Peninsular Malaysia. Viral DNA was isolated from symptomatic tomato plants, and begomovirus association was confirmed by PCR using DNA-A degenerate primers. The C2 and C3 sequences of the putative begomoviruses were similar to two corresponded ORFs of different geographically separated strains of begomoviruses Pepper yellow leaf curl Indonesia virus and Tomato yellow leaf curl Kanchanaburi virus. The present study also identified a unique isolate, Ageratum yellow vein Malaysia virus (AYVMV) among above mentioned survey. It has a single-stranded DNA component and its associated betasatellite. The single-stranded DNA component is consisting of 2750 nt with six open reading frames and an organization resembling that of monopartite geminiviruses. The full length of viral single-stranded DNA component genome obtained using next generation sequencing (NGS) showed the highest sequence identity (99%) with Ageratum yellow vein virus (AYVV-BA). The betasatellite component genome obtained by NGS has 1342 nt and showed the highest sequence identity (91%) with the Pepper yellow leaf curl betasatellite. Following ICTV guidelines, Ageratum yellow vein Malaysia virus was assigned the abbreviation AYVMV with sequence and phylogenetic analysis indicating that it might have evolved by recombination of two or more viral ancestors.Neutralizing anti-drug antibody (NAb) assays often have lower drug tolerance (DT) than trough drug concentrations, potentially under-estimating NAb incidence. To improve DT, drug-specific proteins were coupled to magnetic beads to deplete drug in the sample. To avoid interference from carryover, drug-specific proteins that did not interfere in the NAb assay, such as target or non-blocking anti-drug antibodies, were selected. With the drug depletion step, DT improved by > 10-fold in two competitive ligand binding NAb assays. Analysis of anti-drug antibody positive clinical samples with elevated drug levels demonstrated that NAb incidence was under-estimated without the drug depletion step. However, these NAb-positive samples had low titer and no impact on drug concentrations.Rheumatoid arthritis is an autoimmune disorder causing joint deformity and work disability. Several drugs are available to deal with the disease including conventional drugs; biological drugs such as TNFα inhibitors, B cell-targeted drugs, T cell co-stimulation inhibitors, interleukin-6 inhibitors, and interleukin-1 inhibitors; and kinase inhibitory drugs. In spite of the broad spectrum of drugs available, the disease remains uncontrolled in a number of patients and there is a need for new drugs with better efficacy and universal response rate. The failure of the available drugs to control the disease can be owed to the complex pathogenesis with complementary pathways of disease progression. The blockade of one pathway cannot supersede pathogenesis through other complementary pathways. Janus kinase (JAK) and Bruton’s tyrosine kinase (BTK) are the two important mediators of disease which control a number of signaling pathways involved in rheumatoid arthritis pathogenesis. In this study, using the computer-aided drug designing techniques (virtual screening, molecular docking, and molecular dynamics studies), we have designed piperidinyl dipyrrolopyridine-based dual inhibitors of Janus kinase and Bruton’s tyrosine kinase. Dual JAK and BTK inhibitors seem promising to fight the complex pathogenesis of the disease at multiple fronts and can be the future drug for patients unresponsive to current remedies.

To evaluate the current evidence for the effectiveness of transarterial embolization (TAE) in treatment of symptomatic hepatic hemangiomas.

A systematic literature review was conducted in PubMed, CINAHL and Scopus databases to identify studies of hepatic hemangiomas treated with transarterial embolization. Main outcome was defined as the mean difference between pre- and post-TAE hemangioma diameters. Treatment agents were categorized as Lipiodol based [bleomycin (L + BE), pingyangmycin (L + PYG) or ethanol (L + ethanol)] and non-Lipiodol based (polyvinyl-alcohol-only). Conventional random-effect meta-analysis technique was applied to analyze data.

Of 3080 initially inspected publications, 21 studies were included in the meta-analysis comprising of 1450 patients with total of 1871 hemangiomas (36.2% male, mean age 46.3 ± 3.6years). One hundred and twenty-six, 1666, 41 and 38 lesions were treated with L + BE, L + PYG, L + ethanol and PVA, respectively. Median follow-up time after embolization was 12months with Lipiodol is safe and associated with reduced size of hemangiomas resulting in symptoms alleviation.A woman with an upper extremity brachioaxillary arteriovenous dialysis graft presented with a 9-month history of profound ipsilateral arm swelling and numbness secondary to chronic axillosubclavian vein occlusion. Previous endovascular and open venous recanalization attempts were unsuccessful. A totally percutaneous extra-anatomic venous bi-bypass was created to salvage the dialysis access circuit and reconstruct the deep venous system. Using overlapping Viabahn stent-grafts, two parallel bypasses were created from the arteriovenous graft and brachial vein, respectively, to the brachiocephalic vein. The hemodialysis graft regained function. Upper extremity symptoms resolved within 48 h. This is the first reported percutaneous double-barrel technique of extra-anatomic venous bypass creation for simultaneous management of a failed dialysis access and chronic venous occlusive disease.

To search for abscopal effects (AE) distant to the site of radiation after sequential Yittrium-90 (Y-90) radioembolization (RE) of liver malignancies.

In this retrospective analysis, all patients treated by RE between 2007 and 2018 (n = 907) were screened for the following setting/conditions sequential RE of left and right liver lobe in two sessions, liver-specific MRI (MRI1) acquired max. 10days before or after first RE (RE1), liver-specific MRI (MRI2) acquired with a minimum time interval of 20days after MRI1, but before second RE (RE2). No systemic tumor therapies between MRI1 and MRI2. No patients with liver cirrhosis. Metastases > 5mm in untreated liver lobes were compared in MRI1 and MRI2 and rated as follows same size or larger in MRI2 = no abscopal effect (NAE); > 30% shrinkage without Y-90 contamination in SPECT/CT = abscopal effect (AE).

Ninety six of 907 patients met aforementioned criteria. Median time-frame between RE1 and MRI2 was 34 (20-64) days. These 96 cases had 765 metastases which were evaluable (median 5(1-40) metastases per patient). Four patients could be identified with at least one shrinking metastasis of the untreated site one patient with breast cancer (3 metastases 0 NAE; 3 AE), one patient with prostate cancer (6 metastases 3 NAE; 3 metastases > 30% shrinkage but possible Y-90 contamination) and two patients with shrinkage of one metastasis each but less than 30%.

Our retrospective study documents AE after RE of liver tumors in 1 out of 96 cases, 3 other cases remain unclear.

Our retrospective study documents AE after RE of liver tumors in 1 out of 96 cases, 3 other cases remain unclear.

Gingival tissue enlargement is a common side effect of antiepileptic medications (e.g. phenytoin and sodium valproate), immunosuppressing drugs (e.g. cyclosporine) and calcium channel blockers (e.g. nifedipine, verapamil, amlodipine) (Murakami et al. 2018, Clin Periodontol 45S17-S27, 2018). The clinical and histological appearances of lesions caused by these drugs are indistinguishable from one another (Murakami et al. 2018, Clin Periodontol 45S17-S27, 2018). Drug-induced gingival enlargement is rarely seen in edentulous patients.

This case presents a 72-year-old female with a history of squamous cell carcinoma of the floor of the mouth treated with surgical excision and fibula-free flap reconstruction. Following the uncovering of osseointegrated implants placed in the fibular-free flap, the patient developed gingival enlargement of the floor of the mouth. Cessation of amlodipine and switching to an alternative medication lead to a resolution of the enlarged tissue.

This case illustrates that gingival enlargement can occur around dental implants, most notably in rehabilitation cases in patients who have had head and neck cancer. Clinicians should be aware of the risk of gingival enlargement in hypertensive patients taking calcium channel blockers prior to implant placement.

This case illustrates that gingival enlargement can occur around dental implants, most notably in rehabilitation cases in patients who have had head and neck cancer. Clinicians should be aware of the risk of gingival enlargement in hypertensive patients taking calcium channel blockers prior to implant placement.

To build a stronger Pichia pastoris P

promoter and to identify putative transcriptional factor binding sites (TFBSs) on P

that affect the activity of the promoter.

A synthetic library of P

was generated by deleting or duplicating putative TFBS motifs in the promoter sequence. CSRE, MIG1, RAP1 and HAP2/3/4 were found to have important effects on P

activity. The P

variant P4 with a putative binding site of RAP1 on the promoter sequence showed a stronger activity compared with that of the wild-type P

and P

, which is the strongest natural P. pastoris promoter that has been reported. This inference was confirmed with EGFP (enhanced green fluorescent protein) and Candida Antarctica lipase B as the reporters.

The role of the transcriptional regulator RAP1 may be important in P

methanol induction. A stronger P

variant can be constructed by the duplication of the putative binding site of RAP1 on the P

promoter sequence. This P

variant has potential value for heterologous protein production, metabolic engineering, and synthetic biology.

The role of the transcriptional regulator RAP1 may be important in PCAT1 methanol induction. A stronger PCAT1 variant can be constructed by the duplication of the putative binding site of RAP1 on the PCAT1 promoter sequence. This PCAT1 variant has potential value for heterologous protein production, metabolic engineering, and synthetic biology.

In general, a sufficient supply of ATP can promote the synthesis of ATP-driven metabolites, but excessive ATP will lead to the inhibition of cell growth. For enhancing the co-production of glutathione(GSH) and S-adenosylmethionine(SAM), a dynamic ATP regeneration strategy was developed.

The novel ATP regeneration strategy consisting of ATP-sensing riboswitch ydaO motif, polyphosphate kinase (PPK), and Vitreoscilla hemoglobin (VHb) was successfully applied in Escherichia coli. The intracellular ATP level was always around 0.60mg/g dry cell weight during the fermentation process, resulting in significantly enhanced co-production of GSH and SAM. The GSH titer and SAM titer in the strain CGS-2 increased by 137.40% and 82.18% after fermentation for 24h, compared with the control strain.

The ATP regulation strategy is expected to be a favorable tool to improve the efficiency of microbial cell factories. The proposed ATP dynamic regeneration approach may be applicable for cost-effective, high-yield production of ATP-driven metabolites.

The ATP regulation strategy is expected to be a favorable tool to improve the efficiency of microbial cell factories. The proposed ATP dynamic regeneration approach may be applicable for cost-effective, high-yield production of ATP-driven metabolites.

The Spinal Appearance Questionnaire (SAQ), scoliosis specific quality of life questionnaire, was developed to assess the spinal appearance in adolescent idiopathic scoliosis (AIS) patients. The aim of this study is to evaluate the adaptation, validity, and reliability of the Turkish version of the Spinal Appearance Questionnaire (Tr-SAQ).

Tr-SAQ and already validated Turkish SRS-22 were applied to 75AIS patients (56 females) twice within a 2-week interval for test-retest reliability. Validity of the Tr-SAQ was assessed with factor analysis, convergent validity, and discriminant validity. Convergent validity was evaluated by calculating Spearman correlation coefficients between Tr-SAQ and SRS-22 self-image domain. Internal consistency and intraclass correlation coefficient (ICC) were evaluated for the determination of reliability.

Factor analysis indicated that Tr-SAQ had two factors as appearance (items 1-10) and expectations (items 12-15). Convergent validity test showed a significant negative correlation between the Tr-SAQ appearance score and SRS-22 self-image score (Spearman’s r = - 0.6).Test-retest was conducted within a mean of 16.7 (range 14-28) days. Both ICC and Cronbach’s α were found to be high (0.98, 0. 91, respectively). The correlations with the major curve magnitude were stronger for the Tr-SAQ Appearance (r = 0.7) and Tr-SAQ Total (r = 0.6) scores than the correlations between the SRS-22 self-image (r = - 0.5) and SRS-22 Total (r =  - 0.4) scores.

The Turkish version of the SAQ was reliable and valid for clinical use for AIS patients who are native speakers of Turkish.

Level I- diagnostic studies.

Level I- diagnostic studies.

Although a number of factors contribute to racial disparities in breast cancer outcomes, perceived discrimination in healthcare may be a key factor that hinders positive interactions and negatively impacts patient outcomes. The goals of our study were to (1) assess the prevalence of perceived discrimination as reported by breast cancer patients and (2) identify factors related to discrimination in women overall as well as by race.

This study is a secondary analysis of a larger study, „Narrowing the Gaps in Adjuvant Therapy,” where a convenience sample of 359 women completed one telephone survey assessing sociodemographics, and attitudes and beliefs concerning breast cancer treatments and care. Chi-square analysis was used to assess the relationship of categorical variables with perceived discrimination, while the F-test was employed for continuous variables. Logistic regression determined predictors of perceived discrimination, a dichotomous variable (none vs. any).

A majority of women were Black (58%), privately insured (85%), and had at least a Bachelor’s degree (48%). Discrimination was reported by 32.4% of women, with significantly more Black women reporting discrimination than White women. Insurance status, attitudes toward treatment, and distress factors were significantly related to perceived discrimination. In the logistic model, women who were less trusting of their providers (OR = 0.863 [0.751, 0.993], p = .021) and Black women (OR = 7.241 [0.751, 0.993], p = .039) were more likely to report incidences of discrimination.

Our findings suggest a need to understand Black survivor’s experiences with healthcare. Similarly, future work must focus on identifying ways to improve provider trust amongst breast cancer survivors.

Our findings suggest a need to understand Black survivor’s experiences with healthcare. Similarly, future work must focus on identifying ways to improve provider trust amongst breast cancer survivors.The similar socioeconomic position of black and Hispanic women coupled with better birth outcomes among Hispanic women is termed the „Hispanic Paradox.” However, birth outcome disparities among Hispanic women exist by maternal nativity. Persistent unequal exposure over time to stressors contributes to these disparities. We hypothesized that variation in maternal resilience to stressors also exists by race, ethnicity, and nativity. We utilized data from the Spontaneous Prematurity and Epigenetics of the Cervix study in Boston, MA (n = 771) where resilience was measured mid-pregnancy using the Connor Davidson Resilience Scale 25. We assessed resilience differences by race/ethnicity, by nativity then by race, ethnicity, and nativity together. We also assessed the risk of low resilience among foreign-born women by region of origin. We used Poisson regression to calculate risk ratios for low resilience, adjusting for maternal age, education, and insurance. Resilience did not differ significantly across race/ethnicity or by foreign-born status in the overall cohort. US-born Hispanic women were more likely to be in the low resilience tertile compared with their foreign-born Hispanic counterparts (adjusted RR 3.52, 95% CI 1.18-10.49). Foreign-born Hispanic women also had the lowest risk of being in the low resilience tertile compared with US-born non-Hispanic white women (aRR 0.33, 95% CI 0.11-0.98). Resilience did not differ significantly among immigrant women by continent of birth. Overall, foreign-born Hispanic women appear to possess a resilience advantage. Given that this group often exhibits the lowest rates of adverse birth outcomes, our findings suggest a deeper exploration of resilience among immigrant Hispanic women.The Robson Ten-Group Classification System is widely considered to be the gold standard for comparing cesarean section (CS) delivery rates, despite limited adoption in the United States (US). When reporting overall CS rates, Blacks and other minorities are typically reported to have high CS rates but comparing overall CS rates may be misleading as CS may be more common in some higher risk populations. Improved understanding of how CS rates differ by race among standardized groups could highlight differences in care and areas for improvement. The current study examines racial differences in cesarean section delivery rates using the Robson Ten-Group Classification System in a nationwide sample. Data from US vital statistics live birth certificates were used to identify 3,906,088 births which were each classified into one of the ten groups based on five obstetric characteristics identifiable on presentation for delivery including parity, onset of labor, gestational age, fetal presentation, and number of fetuses. Results indicated that Black and Asian mothers had the highest CS rates in groups 1-4 which all contain single, cephalic pregnancies at term with no prior CS and are only differentiated by parity and onset of labor. Black mothers also had the lowest CS rates for groups 6 and 7, containing women with nulliparous and multiparous breech births. Black and Asian mothers show differences in CS rates among groups that could indicate lack of appropriate care. Efforts should be made to prevent unnecessary primary CS among low-risk mothers.

There were 28,055 people living with HIV (PLWH) in Miami-Dade County (MDC) in 2017; 40.1% was either out of care or was not virally suppressed (uncontrolled HIV). The purpose of this study was to determine the association between the social determinants of health (SDOH) and the number of persons with uncontrolled HIV in MDC.

This cross-sectional study included PLWH 15 and older with uncontrolled HIV in MDC, 2017. Data on PLWH’s viral load, age, gender, mode of HIV transmission, and race/ethnicity were aggregated to the ZIP code level. All five SDOH per HealthyPeople 2020 were represented economic stability, education, social and community context, health and healthcare, and neighborhood and built environment.

Descriptive analyses on all study variables and a principal component analysis on the SDOH variables were performed. To account for overdispersion, multivariate negative binomial regressions were run while controlling for confounders and testing for significant interactions.

The results of the reherence, and continuity of care to improve suppression rates in MDC.

We aimed to investigate the characteristics of kidney disease in severely obese Japanese patients with type 2 diabetes mellitus (T2DM).

This was a cross-sectional study of severely obese patients (body mass index ≥35 kg/m

) with T2DM treated at Jinnouchi Hospital, Kumamoto, Japan.

A total of 3128 T2DM patients visited the hospital during the survey period, of whom 55 patients (1.7%) were severely obese and 50 patients were enrolled. In terms of diabetic nephropathy (DN), twenty-five patients were stage 1 (non-DN, 50.0%), sixteen were stage 2 (32.0%), five were stage 3 (10.0%), and four were stage 4 (8.0%). There were significant differences in the presence of urinary occult blood (P = 0.01) and history of cardiovascular disease (CVD) (P = 0.04) between patients with DN (stages 2-4) and those without DN (stage 1). The presence of urinary occult blood (odds ratio [OR], 4.96; 95% confidence interval, 1.32-18.6; P = 0.02) was significantly associated with the presence of DN according to multivariate logistic regression analysis with forced inclusion of age, sex, and CVD history.

Urinary occult blood may be a significant independent factor associated with the presence of nephropathy in severely obese Japanese patients with T2DM. The presence of urinary occult blood could thus be an important pathogenic factor in obesity-related nephropathy in patients with T2DM.

Urinary occult blood may be a significant independent factor associated with the presence of nephropathy in severely obese Japanese patients with T2DM. The presence of urinary occult blood could thus be an important pathogenic factor in obesity-related nephropathy in patients with T2DM.

OsWRKY36 represses plant height and grain size by inhibiting gibberellin signaling. Plant height and grain size are important agronomic traits affecting yield in cereals, including rice. Gibberellins (GAs) are plant hormones that promote plant growth and developmental processions such as stem elongation and grain size. WRKYs are transcription factors that regulate stress tolerance and plant development including height and grain size. However, the relationship between GA signaling and WRKY genes is still poorly understood. Here, we characterized a small grain and semi-dwarf 3 (sgsd3) mutant in rice cv. Hwayoung (WT). A T-DNA insertion in the 5′-UTR of OsWRKY36 induced overexpression of OsWRKY36 in the sgsd3 mutant, likely leading to the mutant phenotype. This was confirmed by the finding that overexpression of OsWRKY36 caused a similar small grain and semi-dwarf phenotype to the sgsd3 mutant whereas knock down and knock out caused larger grain phenotypes. The sgsd3 mutant was also hyposensitive to GA and acice cv. Hwayoung (WT). A T-DNA insertion in the 5′-UTR of OsWRKY36 induced overexpression of OsWRKY36 in the sgsd3 mutant, likely leading to the mutant phenotype. This was confirmed by the finding that overexpression of OsWRKY36 caused a similar small grain and semi-dwarf phenotype to the sgsd3 mutant whereas knock down and knock out caused larger grain phenotypes. The sgsd3 mutant was also hyposensitive to GA and accumulated higher mRNA and protein levels of SLR1 (a GA signaling DELLA-like inhibitor) compared with the WT. Further assays showed that OsWRKY36 enhanced SLR1 transcription by directly binding to its promoter. In addition, we found that OsWRKY36 can protect SLR1 from GA-mediated degradation. We thus identified a new GA signaling repressor OsWRKY36 that represses GA signaling through stabilizing the expression of SLR1.Cytokine polymorphisms can influence their plasma levels and thus affect the immune response in renal transplantation. A total of 146 renal transplant recipients (RTR) were classified into groups according to the estimated glomerular filtration rate (R1  A polymorphism in TNF gene and IL-6 and IL-10 levels would provide a broader and effective view on the clinical monitoring of RTR.

Cancer stem cells (CSCs) are implicated in tumor initiation and development of metastasis. However, whether CSCs also affect the immune system is not fully understood. We investigated correlations between the PD-L1

CSCs, changes in T-cell phenotype in metastatic and non-metastatic lymph nodes (LNs) and response to treatment.

LNs’ aspirates were obtained during the EBUS/TBNA procedure of 20 NSCLC patients at different stages of the disease. CSCs and T-cell characteristics were determined by flow cytometry.

PD-L1

CSCs positively correlated with the percentage of Tregs, PD-1

CD4 T cells and Tim3

CD4

T cells, whereas PD-L1

CSCs were negatively correlated with CD4

T cells and CD28

CD4

T cells. The percentage of PD-L1

CSCs was higher in patients with progressive disease (PD) as compared to patients with stable disease (SD) or partial response (PR). Among T cells, only PD-1

CD4

T cells and Tim3

CD4

T-cell frequencies were higher in patients with PD as compared to patients with SD or PR.

The frequency of PD-L1

CSCs associates with an altered T-cell frequency and phenotype indicating that CSCs can affect the immune system. The higher percentage of PD-L1

CSCs in patients with PD may confirm their resistance to conventional therapy, suggesting that CSCs may be an interesting target for immunotherapy.

The frequency of PD-L1+ CSCs associates with an altered T-cell frequency and phenotype indicating that CSCs can affect the immune system. The higher percentage of PD-L1+ CSCs in patients with PD may confirm their resistance to conventional therapy, suggesting that CSCs may be an interesting target for immunotherapy.Background Community pharmacist-led anticoagulation management service (CPAMS) offers international normalised ratio point-of-care testing of warfarin in a community pharmacy setting. It has now expanded with 7,344 patients enrolled in the service across 164 pharmacies in New Zealand. The clinical benefit of CPAMS has been shown to be superior, but patient satisfaction with the service has not been fully explored. Objective To develop a questionnaire to assess patient satisfaction with CPAMS and evaluate its psychometric properties. Additionally, to determine the level of patient satisfaction with CPAMS and identify determinants of satisfaction with CPAMS. Settings 1071 patients enrolled in CPAMS across New Zealand invited to take part in the study. Main outcome measure Satisfaction with CPAMS service. Methods Adult patients taking warfarin and currently enrolled in CPAMS were recruited through the national international normalised ratio online system and invited to complete a 36-item survey assessing satisfathese differences. Conclusions The high level of patient satisfaction further supports the effectiveness of CPAMS as a delivery model. Patient satisfaction is affected by age, frequency of pharmacy visits, ethnicity, travel distance to pharmacy, and perceived health status. Policy makers and practitioners should consider the characteristics of patients with low levels of satisfaction to improve and enhance CPAMS engagement.Besides lung drastic involvement, SARS-CoV-2 severely affected other systems including liver. Emerging epidemiological studies brought the attentions towards liver injury and impairment as a potential outcome of COVID19. Angiotensin-converting enzyme 2 (ACE2) and Transmembrane serine protease (TMPRSS2) are the main cell entry receptors of SARS-CoV-2. We have tested the ability of medications to regulate expression of SARS-CoV-2 receptors. Understanding that may reflect how such medications may affect the level of infectivity and permissibility of the liver following COVID-19. Using transcriptomic datasets, Toxicogenomic Project-Genomics Assisted Toxicity Evaluation System (Open TG-GATEs) and GSE30351, we have tested the ability of ninety common medications to regulate COVID-19 receptors expression in human primary hepatocytes. Most medications displayed a dose-dependent change in expression of receptors which could hint at a potentially more pronounced change with chronic use. The expression level of TMPRSS2 was increased noticeably with a number of medications such as metformin. Within the analgesics, acetaminophen revealed a dose-dependent reduction in expression of ACE2, while non-steroidal anti-inflammatory drugs had mixed effect on receptors expression. To confirm the observed effects on primary human hepatocytes, rat hepatocyte treatments data was obtained from DrugMatrix toxicogenomic database (GSE57805), which showed a similar ACE2 and TMPRSS2 expression pattern. Treatment of common co-morbidities often require chronic use of multiple medications, which may result in an additive increase in the expression of ACE2 and TMPRSS2. More research is needed to determine the effect of different medications on COVID-19 receptors.

For the group of individuals reaching higher age, living arrangements providing care increase in importance. Increasing dependence can lead to arelocation to anursing home. The attitude towards nursing homes is important in preparing for this event and the psychological response to it but has hardly been studied so far.

The aim of this study was to determine what older people think about and how they evaluate nursing homes depending on their subjective feeling of control, their experiences and on how informed they are.

A total of 150 geriatric rehabilitation patients were interviewed on their attitudes toward nursing homes with afirst version of aquestionnaire (n = 64) or arevised version (n = 86).

Apolarity profile showed amore positive attitude toward nursing homes; however, the participants gave mainly negative statements in afree response format. The majority of the participants had an anxious attitude towards a relocation to a residential home. A stronger feeling of control, ahigher level of information, and positive experiences with nursing homes are related to apositive attitude towards the relocation to anursing home.