Tribbles 3 deficiency promotes atherosclerotic fibrous cap thickening and macrophage-mediated extracellular matrix remodelling.

Tribbles 3 (TRIB3) modulates lipid and glucose metabolism, macrophage lipid uptake, with a gain-of-function variant associated with increased cardiovascular risk. Here we set out to examine the role of this pseudokinase in atherosclerotic plaque development. Human endarterectomy atherosclerotic tissue specimens analysed by immunofluorescence showed upregulated TRIB3 in unstable plaques and an enrichment in unstable regions of stable plaques. Atherosclerosis was induced in full body Trib3KO and Trib3WT littermate mice by injecting mPCSK9 expressing adeno-associated virus and western diet feeding for 12 weeks. Trib3KO mice showed expanded visceral adipose depot while circulatory lipid levels remained unaltered compared to wildtype mice. Trib3KO mice aortae showed a reduced plaque development and improved plaque stability, with increased fibrous cap thickness and collagen content, which was accompanied by increased macrophage content. Analysis of both mouse and human macrophages with reduced TRIB3 expression showed elongated morphology, increased actin expression and altered regulation of genes involved in extracellular matrix remodelling. In summary, TRIB3 controls plaque development and may be atherogenic in vivo. Loss of TRIB3 increases fibrous cap thickness via altered metalloproteinase expression in macrophages, thus inhibiting collagen and elastic fibre degradation, suggesting a role for TRIB3 in the formation of unstable plaques.

TRIB1 regulates tumor growth via controlling tumor-associated macrophage phenotypes and is associated with breast cancer survival and treatment response.

Molecular mechanisms that regulate tumor-associated macrophage (TAM) phenotype and function are incompletely understood. The pseudokinase TRIB1 has been reported as a regulator of macrophage phenotypes, both in mouse and human systems. Methods: Bioinformatic analysis was used to investigate the link between TRIB1 expression in breast cancer and therapeutic response to chemotherapy. In vivo models of breast cancer included immune-competent mice to characterize the consequences of altered (reduced or elevated) myeloid Trib1 expression on tumor growth and composition of stromal immune cell populations. Results: TRIB1 was highly expressed by TAMs in breast cancer and high TRIB1 expression correlated with response to chemotherapy and patient survival. Both overexpression and knockout of myeloid Trib1 promote mouse breast tumor growth, albeit through different molecular mechanisms. Myeloid Trib1 deficiency led to an early acceleration of tumor growth, paired with a selective reduction in perivascular macrophage numbers in vivo and enhanced oncogenic cytokine expression in vitro. In contrast, elevated levels of Trib1 in myeloid cells led to an increased late-stage mammary tumor volume, coupled with a reduction of NOS2 expressing macrophages and an overall reduction of macrophages in hypoxic tumor regions. In addition, we show that myeloid Trib1 is a previously unknown, negative regulator of the anti-tumor cytokine IL-15, and that increased myeloid Trib1 expression leads to reduced IL-15 levels in mammary tumors, with a consequent reduction in the number of T-cells that are key to anti-tumor immune responses. Conclusions: Together, these results define a key role for TRIB1 in chemotherapy responses for human breast cancer and provide a mechanistic understanding for the importance of the control of myeloid TRIB1 expression in the development of this disease.

Predicting Hospital Resource Use During COVID-19 Surges: A Simple but Flexible Discretely Integrated Condition Event Simulation of Individual Patient-Hospital Trajectories.

OBJECTIVES: To assist with planning hospital resources, including critical care (CC) beds, for managing patients with COVID-19. METHODS: An individual simulation was implemented in Microsoft Excel using a discretely integrated condition event simulation. Expected daily cases presented to the emergency department were modeled in terms of transitions to and from ward and CC and to discharge or death. The duration of stay in each location was selected from trajectory-specific distributions. Daily ward and CC bed occupancy and the number of discharges according to care needs were forecast for the period of interest. Face validity was ascertained by local experts and, for the case study, by comparing forecasts with actual data. RESULTS: To illustrate the use of the model, a case study was developed for Guy's and St Thomas' Trust. They provided inputs for January 2020 to early April 2020, and local observed case numbers were fit to provide estimates of emergency department arrivals. A peak demand of 467 ward and 135 CC beds was forecast, with diminishing numbers through July. The model tended to predict higher occupancy in Level 1 than what was eventually observed, but the timing of peaks was quite close, especially for CC, where the model predicted at least 120 beds would be occupied from April 9, 2020, to April 17, 2020, compared with April 7, 2020, to April 19, 2020, in reality. The care needs on discharge varied greatly from day to day. CONCLUSIONS: The DICE simulation of hospital trajectories of patients with COVID-19 provides forecasts of resources needed with only a few local inputs. This should help planners understand their expected resource needs.

Tribbles-1 Expression and Its Function to Control Inflammatory Cytokines, Including Interleukin-8 Levels are Regulated by miRNAs in Macrophages and Prostate Cancer Cells.

The pseudokinase TRIB1 controls cell function in a range of contexts, by regulating MAP kinase activation and mediating protein degradation via the COP1 ubiquitin ligase. TRIB1 regulates polarization of macrophages and dysregulated Trib1 expression in murine models has been shown to alter atherosclerosis burden and adipose homeostasis. Recently, TRIB1 has also been implicated in the pathogenesis of prostate cancer, where it is often overexpressed, even in the absence of genetic amplification. Well described TRIB1 effectors include MAP kinases and C/EBP transcription factors, both in immune cells and in carcinogenesis. However, the mechanisms that regulate TRIB1 itself remain elusive. Here, we show that the long and conserved 3'untranslated region (3'UTR) of TRIB1 is targeted by miRNAs in macrophage and prostate cancer models. By using a systematic in silico analysis, we identified multiple "high confidence" miRNAs potentially binding to the 3'UTR of TRIB1 and report that miR-101-3p and miR-132-3p are direct regulators of TRIB1 expression and function. Binding of miR-101-3p and miR-132-3p to the 3'UTR of TRIB1 mRNA leads to an increased transcription and secretion of interleukin-8. Our data demonstrate that modulation of TRIB1 by miRNAs alters the inflammatory profile of both human macrophages and prostate cancer cells.

Neutrophil microvesicles drive atherosclerosis by delivering miR-155 to atheroprone endothelium.

Neutrophils are implicated in the pathogenesis of atherosclerosis but are seldom detected in atherosclerotic plaques. We investigated whether neutrophil-derived microvesicles may influence arterial pathophysiology. Here we report that levels of circulating neutrophil microvesicles are enhanced by exposure to a high fat diet, a known risk factor for atherosclerosis. Neutrophil microvesicles accumulate at disease-prone regions of arteries exposed to disturbed flow patterns, and promote vascular inflammation and atherosclerosis in a murine model. Using cultured endothelial cells exposed to disturbed flow, we demonstrate that neutrophil microvesicles promote inflammatory gene expression by delivering miR-155, enhancing NF-κB activation. Similarly, neutrophil microvesicles increase miR-155 and enhance NF-κB at disease-prone sites of disturbed flow in vivo. Enhancement of atherosclerotic plaque formation and increase in macrophage content by neutrophil microvesicles is dependent on miR-155. We conclude that neutrophils contribute to vascular inflammation and atherogenesis through delivery of microvesicles carrying miR-155 to disease-prone regions.

Myeloid Tribbles 1 induces early atherosclerosis via enhanced foam cell expansion.

Macrophages drive atherosclerotic plaque progression and rupture; hence, attenuating their atherosclerosis-inducing properties holds promise for reducing coronary heart disease (CHD). Recent studies in mouse models have demonstrated that Tribbles 1 (Trib1) regulates macrophage phenotype and shows that Trib1 deficiency increases plasma cholesterol and triglyceride levels, suggesting that reduced TRIB1 expression mediates the strong genetic association between the TRIB1 locus and increased CHD risk in man. However, we report here that myeloid-specific Trib1 (mTrib1) deficiency reduces early atheroma formation and that mTrib1 transgene expression increases atherogenesis. Mechanistically, mTrib1 increased macrophage lipid accumulation and the expression of a critical receptor (OLR1), promoting oxidized low-density lipoprotein uptake and the formation of lipid-laden foam cells. As TRIB1 and OLR1 RNA levels were also strongly correlated in human macrophages, we suggest that a conserved, TRIB1-mediated mechanism drives foam cell formation in atherosclerotic plaque and that inhibiting mTRIB1 could be used therapeutically to reduce CHD.

MKL1-actin pathway restricts chromatin accessibility and prevents mature pluripotency activation.

Actin cytoskeleton is well-known for providing structural/mechanical support, but whether and how it regulates chromatin and cell fate reprogramming is far less clear. Here, we report that MKL1, the key transcriptional co-activator of many actin cytoskeletal genes, regulates genomic accessibility and cell fate reprogramming. The MKL1-actin pathway weakens during somatic cell reprogramming by pluripotency transcription factors. Cells that reprogram efficiently display low endogenous MKL1 and inhibition of actin polymerization promotes mature pluripotency activation. Sustained MKL1 expression at a level seen in typical fibroblasts yields excessive actin cytoskeleton, decreases nuclear volume and reduces global chromatin accessibility, stalling cells on their trajectory toward mature pluripotency. In addition, the MKL1-actin imposed block of pluripotency can be bypassed, at least partially, when the Sun2-containing linker of the nucleoskeleton and cytoskeleton (LINC) complex is inhibited. Thus, we unveil a previously unappreciated aspect of control on chromatin and cell fate reprogramming exerted by the MKL1-actin pathway.

Multiplexed fluctuation-dissipation-theorem calibration of optical tweezers inside living cells.

In order to apply optical tweezers-based force measurements within an uncharacterized viscoelastic medium such as the cytoplasm of a living cell, a quantitative calibration method that may be applied in this complex environment is needed. We describe an improved version of the fluctuation-dissipation-theorem calibration method, which has been developed to perform in situ calibration in viscoelastic media without prior knowledge of the trapped object. Using this calibration procedure, it is possible to extract values of the medium's viscoelastic moduli as well as the force constant describing the optical trap. To demonstrate our method, we calibrate an optical trap in water, in polyethylene oxide solutions of different concentrations, and inside living fission yeast (S. pombe).

Using a Cageside Device for Testing Glycosylated Hemoglobin in Cynomolgus Macaques (Macaca fascicularis).

Nonhuman primates naturally develop type 2 diabetes mellitus and exhibit clinical features that are similar to those observed in humans, including obesity, insulin resistance, dyslipidemia, and pancreatic pathology. The glycosylated hemoglobin (HbA1C) test is the primary test used for diabetes management in humans because it reflects the average blood glucose levels over the previous 3 mo. The HbA1C results are a better predictor of potential risk of complications than are single or episodic measures of glucose levels. HbA1C levels have proven useful for the diagnosis and monitoring of blood glucose levels in NHP, but for testing by a commercial laboratory, the test requires a vial of whole blood, results are not available for several days, and the test is expensive. The cageside device requires a single drop of blood, it displays the HbA1C percentage in 5 min, and the cost per sample is less than for sending it to a commercial lab. We therefore assessed the correlation between a cageside test using a handheld unit and the commercial lab test for measuring HbA1C in cynomolgus macaques. From both normal and confirmed diabetic animals, 4 mL blood was collected from a peripheral vessel and sent to a commercial lab for HbA1C testing. At the same time, a drop of capillary blood was collected and tested immediately in the HbA1C cageside test. A comparison of the results revealed significant correlation between the cageside and commercial lab tests. Therefore, we feel that the HbA1C test using handheld device may help to rule out nondiabetics and indicate which animals require additional testing.

Evidence for a role of TRIB3 in the regulation of megakaryocytopoiesis.

Megakaryocytopoiesis is a complex differentiation process driven by the hormone thrombopoietin by which haematopoietic progenitor cells give rise to megakaryocytes, the giant bone marrow cells that in turn break down to form blood platelets. The Tribbles Pseudokinase 3 gene (TRIB3) encodes a pleiotropic protein increasingly implicated in the regulation of cellular differentiation programmes. Previous studies have hinted that TRIB3 could be also involved in megakaryocytopoiesis but its role in this process has so far not been investigated. Using cellular model systems of haematopoietic lineage differentiation here we demonstrate that TRIB3 is a negative modulator of megakaryocytopoiesis. We found that in primary cultures derived from human haematopoietic progenitor cells, thrombopoietin-induced megakaryocytic differentiation led to a time and dose-dependent decrease in TRIB3 mRNA levels. In the haematopoietic cell line UT7/mpl, silencing of TRIB3 increased basal and thrombopoietin-stimulated megakaryocyte antigen expression, as well as basal levels of ERK1/2 phosphorylation. In primary haematopoietic cell cultures, silencing of TRIB3 facilitated megakaryocyte differentiation. In contrast, over-expression of TRIB3 in these cells inhibited the differentiation process. The in-vitro identification of TRIB3 as a negative regulator of megakaryocytopoiesis suggests that in-vivo this gene could be important for the regulation of platelet production.

Impact of the NAP-1 strain on disease severity, mortality, and recurrence of healthcare-associated Clostridium difficile infection.

OBJECTIVES: Studies are conflicting regarding the association of the North American pulsed-field gel electrophoresis type 1 (NAP1) strain in Clostridium difficile infection (CDI) and outcomes. We evaluated the association of NAP1 with healthcare-associated CDI disease severity, mortality, and recurrence at our academic medical center. METHODS: Healthcare-associated CDI cases were identified from November 1, 2011 through January 31, 2013. Multivariable regression models were used to evaluate the associations of NAP1 with severe disease (based on the Hines VA severity score index), mortality, and recurrence. RESULTS: Among 5424 stool specimens submitted to the Clinical Microbiology Laboratory, 292 (5.4%) were positive for C. difficile by polymerase chain reaction (PCR) on or after hospital day 4; 70 (24%) of these specimens also tested positive for NAP1. During the study period, 247 (85%) patients had non-severe disease and 45 (15%) patients had severe disease. Among patients with non-severe disease, 65 (26%) had NAP1 and among patients with severe disease, 5 (11%) had NAP1. After controlling for potential confounders, NAP1 was not associated with an increased likelihood of severe disease (adjusted odds ratio [aOR] = 0.35; 95% confidence interval [CI], 0.13-0.93), in-hospital mortality (aOR = 1.02; 95% CI, 0.53-1.96), or recurrence (aOR = 1.16, 95% CI, 0.36-3.77). CONCLUSIONS: The NAP1 strain did not increase disease severity, mortality, or recurrence in this study, although the incidence of NAP1-positive healthcare associated-CDI was low. The role of strain typing in outcomes and treatment selection in patients with healthcare-associated CDI remains uncertain.

Collagen V haploinsufficiency in a murine model of classic Ehlers-Danlos syndrome is associated with deficient structural and mechanical healing in tendons.

Classic Ehlers-Danlos syndrome (EDS) patients suffer from connective tissue hyperelasticity, joint instability, skin hyperextensibility, tissue fragility, and poor wound healing due to heterozygous mutations in COL5a1 or COL5a2 genes. This study investigated the roles of collagen V in establishing structure and function in uninjured patellar tendons as well as in the injury response using a Col5a1+/- mouse, a model for classic EDS. These analyses were done comparing tendons from a classic EDS model (Col5a1+/- ) with wild-type controls. Tendons were subjected to mechanical testing, histological, and fibril analysis before injury as well as 3 and 6 weeks after injury. We found that Col5a1+/- tendons demonstrated diminished recovery of mechanical competency after injury as compared to normal wild-type tendons, which recovered their pre-injury values by 6 weeks post injury. Additionally, the Col5a1+/- tendons demonstrated altered fibril morphology and diameter distributions compared to the wild-type tendons. This study indicates that collagen V plays an important role in regulating collagen fibrillogenesis and the associated recovery of mechanical integrity in tendons after injury. In addition, the dysregulation with decreased collagen V expression in EDS is associated with a diminished injury response. The results presented herein have the potential to direct future targeted therapeutics for classic EDS patients. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2707-2715, 2017.

Prospective evaluation of peripherally inserted central catheter complications in both inpatient and outpatient settings.

We describe a prospective observational cohort (N = 187) to evaluate peripherally inserted central catheter line complications concurrently from the time of placement until removal. A significantly higher percentage of patients who experienced intraluminal thrombosis were receiving total parenteral nutrition (P ≤ .001) and had a dual lumen catheter (P = .01). Among patients with a confirmed or suspected infection, a significantly higher proportion received total parenteral nutrition (P = .01), had dual-lumen catheters (P = .04), and were neutropenic (P = .04).

Deconstructing Clinical Workflow: Identifying Teaching-Learning Principles for Barcode Electronic Medication Administration With Nursing Students.

The purpose of this quality improvement project was to better understand how to teach medication administration underpinned by an electronic medication administration record (eMAR) system used in simulated, prelicensure nursing education. Methods included a workflow and integration analysis and a detailed process mapping of both an oral and a sublingual medication administration. Procedural and curriculum development considerations related to medication administration using eMAR technology are presented for nurse educators.

Non-genetic diversity modulates population performance.

Biological functions are typically performed by groups of cells that express predominantly the same genes, yet display a continuum of phenotypes. While it is known how one genotype can generate such non-genetic diversity, it remains unclear how different phenotypes contribute to the performance of biological function at the population level. We developed a microfluidic device to simultaneously measure the phenotype and chemotactic performance of tens of thousands of individual, freely swimming Escherichia coli as they climbed a gradient of attractant. We discovered that spatial structure spontaneously emerged from initially well-mixed wild-type populations due to non-genetic diversity. By manipulating the expression of key chemotaxis proteins, we established a causal relationship between protein expression, non-genetic diversity, and performance that was theoretically predicted. This approach generated a complete phenotype-to-performance map, in which we found a nonlinear regime. We used this map to demonstrate how changing the shape of a phenotypic distribution can have as large of an effect on collective performance as changing the mean phenotype, suggesting that selection could act on both during the process of adaptation.

Recent refinements to cranial implants for rhesus macaques (Macaca mulatta).

The advent of cranial implants revolutionized primate neurophysiological research because they allow researchers to stably record neural activity from monkeys during active behavior. Cranial implants have improved over the years since their introduction, but chronic implants still increase the risk for medical complications including bacterial contamination and resultant infection, chronic inflammation, bone and tissue loss and complications related to the use of dental acrylic. These complications can lead to implant failure and early termination of study protocols. In an effort to reduce complications, we describe several refinements that have helped us improve cranial implants and the wellbeing of implanted primates.

Collagenoma in an African Clawed Frog (Xenopus laevis).

A 3-y-old female Xenopus laevis was reported for a gray mass on the abdomen. The frog was used for egg collection and was otherwise experimentally naïve. On physical exam, the frog was bright and active and had a firm, gray, lobulated mass (1.5 cm × 0.5 cm × 0.5 cm) in the cutaneous tissue of the left lateral abdomen. An excisional biopsy was performed under anesthesia, and the entire mass was removed and processed for histopathology. Microscopically, the dermis was greatly expanded by connective tissue with a marked decrease in the number of glands, and occasional degenerative glands were present. When stained with Masson trichrome, the excessive connective tissue stained blue, indicating that it was composed of collagen. With Verhoeff-van Gieson staining, the connective tissue stained bright red with an absence of black-staining material, demonstrating the presence of collagen and ruling out elastic fibers. In light of the morphology of the mass and the results of the special stains, the mass was diagnosed as a collagenoma. To our knowledge, this report is the first description of a collagenoma in X. laevis.

Neurocysticercosis in a Rhesus Macaque (Macaca mulatta).

An 8-y-old, intact, male rhesus macaque (Macaca mulatta) was sedated to undergo MRI in preparation for the implantation of cranial hardware. During imaging, 9 focal lesions were noted in the brain and musculature of the head. The lesions were hyperechoic with hypoechoic rims. The animal was deemed inappropriate for neuroscience research, and euthanasia was elected. Gross examination revealed multiple round, thick-walled, fluid-filled cysts (diameter, approximately 0.5 cm) in multiple tissues: one each in the left caudal lung lobe, left masseter muscle, and the dura overlying the brain and 8 throughout the gray and white matter of the brain parenchyma. Formalin-fixed sections of cyst-containing brain were stained with hematoxylin and eosin. Microscopic examination and molecular analysis of the COX1 (COI) gene recognized the causative organism as Taenia solium at 99.04% identity.