Trafficking in eukaryotic cells is a tightly regulated process to ensure correct cargo delivery to the proper destination organelle or plasma membrane. In this review, we focus on how the vesicle fusion machinery, the SNARE complex, is regulated by the interplay of the multisubunit tethering complexes (MTC) with the SNAREs and Sec1/Munc18 (SM) proteins. Although these factors are used in different stages of membrane trafficking, e.g., Golgi to plasma membrane transport vs. vacuolar fusion, and in a variety of diverse eukaryotic cell types, many commonalities between their functions are being revealed. We explore the various protein-protein interactions and findings from functional reconstitution studies in order to highlight both their common features and the differences in their modes of regulation. These studies serve as a starting point for mechanistic explorations in other systems.
Receptor interacting protein kinase 3 (RIPK3) is a crucial inducer of necroptosis. Its activity is controlled by interaction with other signal adaptors through the "RIP homotypic interaction motif" (RHIM). Recent studies revealed a critical function for RIPK3 in the maintenance of epithelial tissue integrity. In mice with genetic deficiency of the apoptosis adaptors FADD or caspase 8, RIPK3 promotes necroptotic cell death of epithelial cells, leading to excessive and lethal inflammation. In contrast, when FADD and caspase 8 functions are intact, RIPK3 serves as a protector of intestinal epithelial integrity by promoting injury-induced wound repair. In the latter case, RIPK3 promotes optimal cytokine expression by cells of hematopoietic origin. Specifically, bone marrow derived dendritic cells (BMDCs) have an obligate requirement for RIPK3 for optimal secretion of mature IL-1beta and other inflammatory cytokines in response to toll-like receptor 4 (TLR4) stimulation. RIPK3 promotes cytokine expression through two complementary mechanisms: NF-kappaB dependent gene transcription and processing of pro-IL-1beta. We propose that RIPK3 functions in different cell compartments to mediate inflammation through distinct mechanisms.
Regulation of chaperone binding and nucleosome dynamics by key residues within the globular domain of histone H3
BACKGROUND: Nucleosomes have an important role in modulating access of DNA by regulatory factors. The role specific histone residues have in this process has been shown to be an important mechanism of transcription regulation. Previously, we identified eight amino acids in histones H3 and H4 that are required for nucleosome occupancy over highly transcribed regions of the genome.
RESULTS: We investigate the mechanism through which three of these previously identified histone H3 amino acids regulate nucleosome architecture. We find that histone H3 K122, Q120, and R49 are required for Spt2, Spt6, and Spt16 occupancies at genomic locations where transcription rates are high, but not over regions of low transcription rates. Furthermore, substitution at one residue, K122, located on the dyad axis of the nucleosome, results in improper reassembly and disassembly of nucleosomes, likely accounting for the transcription rate-dependent regulation by these mutant histones.
CONCLUSIONS: These data show that when specific amino acids of histone proteins are substituted, Spt2, Spt6, and Spt16 occupancies are reduced and nucleosome dynamics are altered. Therefore, these data support a mechanism for histone chaperone binding where these factors interact with histone proteins to promote their activities during transcription.
OBJECTIVES: The purpose of this study was to examine decade-long trends (2001-2011) in, and factors associated with, door-to-balloon time within 90 minutes of hospital presentation among patients hospitalized with ST-segment elevation myocardial infarction (STEMI) who received a primary percutaneous coronary intervention (PCI).
METHODS: Residents of central Massachusetts hospitalized with STEMI who received a primary PCI at two major PCI-capable medical centers in central Massachusetts on a biennial basis between 2001 and 2011 comprised the study population (n=629). Multivariable regression analyses were used to examine factors associated with failing to receive a primary PCI within 90 minutes after emergency department (ED) arrival.
RESULTS: The average age of this patient population was 61.9 years; 30.5% were women, and 91.7% were White. During the years under study, 50.9% of patients received a primary PCI within 90 minutes of ED arrival; this proportion increased from 2001/2003 (17.2%) to 2009/2011 (70.5%) (P < 0.001). Having previously undergone coronary artery bypass graft surgery, arriving at the ED by car/walk-in and during off-hours were significantly associated with a higher risk of failing to receive a primary PCI within 90 minutes of ED arrival.
CONCLUSION: The likelihood of receiving a timely primary PCI in residents of central Massachusetts hospitalized with STEMI at the major teaching/community medical centers increased dramatically during the years under study. Several groups were identified for purposes of heightened surveillance and intervention efforts to reduce the likelihood of failing to receive a timely primary PCI among patients acutely diagnosed with STEMI.
In human cells, the basal body (BB) core comprises a ninefold microtubule-triplet cylindrical structure. Distal and subdistal appendages are located at the distal end of BB, where they play indispensable roles in cilium formation and function. Most cells that arrest in the G0 stage of the cell cycle initiate BB docking at the plasma membrane followed by BB-mediated growth of a solitary primary cilium, a structure required for sensing the extracellular environment and cell signaling. In addition to the primary cilium, motile cilia are present in specialized cells, such as sperm and airway epithelium. Mutations that affect BB function result in cilia dysfunction. This can generate syndromic disorders, collectively called ciliopathies, for which there are no effective treatments. In this review, we focus on the features and functions of BBs and centrosomes in Homo sapiens.
BACKGROUND: Endocrine-cerebro-osteodysplasia (ECO) syndrome [MIM:612651] caused by a recessive mutation (p.R272Q) in Intestinal cell kinase (ICK) shows significant clinical overlap with ciliary disorders. Similarities are strongest between ECO syndrome, the Majewski and Mohr-Majewski short-rib thoracic dysplasia (SRTD) with polydactyly syndromes, and hydrolethalus syndrome. In this study, we present a novel homozygous ICK mutation in a fetus with ECO syndrome and compare the effect of this mutation with the previously reported ICK variant on ciliogenesis and cilium morphology.
RESULTS: Through homozygosity mapping and whole-exome sequencing, we identified a second variant (c.358G > T; p.G120C) in ICK in a Turkish fetus presenting with ECO syndrome. In vitro studies of wild-type and mutant mRFP-ICK (p.G120C and p.R272Q) revealed that, in contrast to the wild-type protein that localizes along the ciliary axoneme and/or is present in the ciliary base, mutant proteins rather enrich in the ciliary tip. In addition, immunocytochemistry revealed a decreased number of cilia in ICK p.R272Q-affected cells.
CONCLUSIONS: Through identification of a novel ICK mutation, we confirm that disruption of ICK causes ECO syndrome, which clinically overlaps with the spectrum of ciliopathies. Expression of ICK-mutated proteins result in an abnormal ciliary localization compared to wild-type protein. Primary fibroblasts derived from an individual with ECO syndrome display ciliogenesis defects. In aggregate, our findings are consistent with recent reports that show that ICK regulates ciliary biology in vitro and in mice, confirming that ECO syndrome is a severe ciliopathy.
Bare metal stents (BMS) continue to be widely used in patients with coronary artery disease undergoing percutaneous revascularization. Progressive luminal renarrowing has been reported late after BMS implantation resulting in a significant rate of stent failure events. We present a case of very late BMS failure due to in-stent restenosis where optical coherence tomography (OCT) was used to demonstrate neoatherosclerosis as the underlying mechanism. We provide a brief review of neoatherosclerosis and showcase salient features on OCT evaluation.
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While T cell immunity initially limits Mycobacterium tuberculosis infection, why T cell immunity fails to sterilize the infection and allows recrudescence is not clear. One hypothesis is that T cell exhaustion impairs immunity and is detrimental to the outcome of M. tuberculosis infection. Here we provide functional evidence for the development T cell exhaustion during chronic TB. Second, we evaluate the role of the inhibitory receptor T cell immunoglobulin and mucin domain-containing-3 (TIM3) during chronic M. tuberculosis infection. We find that TIM3 expressing T cells accumulate during chronic infection, co-express other inhibitory receptors including PD1, produce less IL-2 and TNF but more IL-10, and are functionally exhausted. Finally, we show that TIM3 blockade restores T cell function and improves bacterial control, particularly in chronically infected susceptible mice. These data show that T cell immunity is suboptimal during chronic M. tuberculosis infection due to T cell exhaustion. Moreover, in chronically infected mice, treatment with anti-TIM3 mAb is an effective therapeutic strategy against tuberculosis.
SUMO-Targeted Ubiquitin Ligase (STUbL) Slx5 regulates proteolysis of centromeric histone H3 variant Cse4 and prevents its mislocalization to euchromatin
Centromeric histone H3, CENP-ACse4, is essential for faithful chromosome segregation. Stringent regulation of cellular levels of CENP-ACse4 restricts its localization to centromeres. Mislocalization of CENP-ACse4 is associated with aneuploidy in yeast, flies and tumorigenesis in human cells; thus, defining pathways that regulate CENP-A levels is critical for understanding how mislocalization of CENP-A contributes to aneuploidy in human cancers. Previous work in budding yeast has shown that ubiquitination of overexpressed Cse4 by Psh1, an E3 ligase, partially contributes to proteolysis of Cse4. Here, we provide the first evidence that Cse4 is sumoylated by E3 ligases Siz1 and Siz2 in vivo and in vitro. Ubiquitination of Cse4 by Small Ubiquitin-related Modifier (SUMO)-Targeted Ubiquitin Ligase (STUbL) Slx5 plays a critical role in proteolysis of Cse4 and prevents mislocalization of Cse4 to euchromatin under normal physiological conditions. Accumulation of sumoylated Cse4 species and increased stability of Cse4 in slx5 strains suggest that sumoylation precedes ubiquitin-mediated proteolysis of Cse4. Slx5-mediated Cse4 proteolysis is independent of Psh1 since slx5 psh1 strains exhibit higher levels of Cse4 stability and mislocalization compared to either slx5 or psh1 strains. Our results demonstrate a role for Slx5 in ubiquitin-mediated proteolysis of Cse4 to prevent its mislocalization and maintain genome stability.
Implementation of a Computerized Screening Inventory: Improved Usability Through Iterative Testing and Modification
BACKGROUND: The administration of health screeners in a hospital setting has traditionally required (1) clinicians to ask questions and log answers, which can be time consuming and susceptible to error, or (2) patients to complete paper-and-pencil surveys, which require third-party entry of information into the electronic health record and can be vulnerable to error and misinterpretation. A highly promising method that avoids these limitations and bypasses third-party interpretation is direct entry via a computerized inventory.
OBJECTIVE: To (1) computerize medical and behavioral health screening for use in general medical settings, (2) optimize patient acceptability and feasibility through iterative usability testing and modification cycles, and (3) examine how age relates to usability.
METHODS: A computerized version of 15 screeners, including behavioral health screeners recommended by a National Institutes of Health Office of Behavioral and Social Sciences Research collaborative workgroup, was subjected to systematic usability testing and iterative modification. Consecutive adult, English-speaking patients seeking treatment in an urban emergency department were enrolled. Acceptability was defined as (1) the percentage of eligible patients who agreed to take the assessment (initiation rate) and (2) average satisfaction with the assessment (satisfaction rate). Feasibility was defined as the percentage of the screening items completed by those who initiated the assessment (completion rate). Chi-square tests, analyses of variance, and Pearson correlations were used to detect whether improvements in initiation, satisfaction, and completion rates were seen over time and to examine the relation between age and outcomes.
RESULTS: Of 2157 eligible patients approached, 1280 agreed to complete the screening (initiation rate=59.34%). Statistically significant increases were observed over time in satisfaction (F3,1061=3.35, P=.019) and completion rates (F3,1276=25.44, P < .001). Younger age was associated with greater initiation (initiated, mean [SD], 46.6 [18.7] years; declined: 53.0 [19.5] years, t2,155=-7.6, P < .001), higher completion (r=-.20, P < .001), and stronger satisfaction (r=-.23, P < .001).
CONCLUSIONS: In a rapid-paced emergency department with a heterogeneous patient population, 59.34% (1280/2157) of all eligible patients initiated the computerized screener with a completion rate reaching over 90%. Usability testing revealed several critical principles for maximizing usability of the computerized medical and behavioral health screeners used in this study. Further work is needed to identify usability issues pertaining to other screeners, racially and ethnically diverse patient groups, and different health care settings.
The unconventional myosin CRINKLED and its mammalian orthologue MYO7A regulate caspases in their signalling roles
Caspases provide vital links in non-apoptotic regulatory networks controlling inflammation, compensatory proliferation, morphology and cell migration. How caspases are activated under non-apoptotic conditions and process a selective set of substrates without killing the cell remain enigmatic. Here we find that the Drosophila unconventional myosin CRINKLED (CK) selectively interacts with the initiator caspase DRONC and regulates some of its non-apoptotic functions. Loss of CK in the arista, border cells or proneural clusters of the wing imaginal discs affects DRONC-dependent patterning. Our data indicate that CK acts as substrate adaptor, recruiting SHAGGY46/GSK3-beta to DRONC, thereby facilitating caspase-mediated cleavage and localized modulation of kinase activity. Similarly, the mammalian CK counterpart, MYO7A, binds to and impinges on CASPASE-8, revealing a new regulatory axis affecting receptor interacting protein kinase-1 (RIPK1) > CASPASE-8 signalling. Together, our results expose a conserved role for unconventional myosins in transducing caspase-dependent regulation of kinases, allowing them to take part in specific signalling events.
Spontaneous coronary artery dissection in a patient with autosomal dominant polycystic kidney disease: a case report
BACKGROUND: Spontaneous coronary artery dissection is an uncommon syndrome. Its prevalence among patients with polycystic kidney disease is very rare, with no previously reported involvement of the right posterior descending coronary artery.
CASE PRESENTATION: We describe the case of a middle-aged Caucasian woman with polycystic kidney disease who presented with a non-ST elevation myocardial infarction. Cardiac catheterization revealed a dissection of her right posterior descending coronary artery. She was treated with dual antiplatelet therapy and had a favorable outcome.
CONCLUSION: We report a rare and interesting case of spontaneous coronary artery dissection of the right posterior descending coronary artery in a patient with polycystic kidney disease. It is important to consider spontaneous coronary artery dissection in the differential diagnosis of patients with polycystic kidney disease who present with an acute coronary syndrome.
Integrative analyses reveal a long noncoding RNA-mediated sponge regulatory network in prostate cancer
Mounting evidence suggests that long noncoding RNAs (lncRNAs) can function as microRNA sponges and compete for microRNA binding to protein-coding transcripts. However, the prevalence, functional significance and targets of lncRNA-mediated sponge regulation of cancer are mostly unknown. Here we identify a lncRNA-mediated sponge regulatory network that affects the expression of many protein-coding prostate cancer driver genes, by integrating analysis of sequence features and gene expression profiles of both lncRNAs and protein-coding genes in tumours. We confirm the tumour-suppressive function of two lncRNAs (TUG1 and CTB-89H12.4) and their regulation of PTEN expression in prostate cancer. Surprisingly, one of the two lncRNAs, TUG1, was previously known for its function in polycomb repressive complex 2 (PRC2)-mediated transcriptional regulation, suggesting its sub-cellular localization-dependent function. Our findings not only suggest an important role of lncRNA-mediated sponge regulation in cancer, but also underscore the critical influence of cytoplasmic localization on the efficacy of a sponge lncRNA.
BACKGROUND: Persistent thromboxane (TX) generation while receiving aspirin therapy is associated with an increased risk of cardiovascular events. The Reduction in Graft Occlusion Rates (RIGOR) study found that aspirin-insensitive TXA2 generation, indicated by elevated urine 11-dehydro-TXB2 (UTXB2) 6 months after coronary artery bypass graft surgery, was a potent risk factor for vein graft thrombosis and originated predominantly from nonplatelet sources. Our goal was to identify risks factors for nonplatelet TXA2 generation.
METHODS AND RESULTS: Multivariable modeling was performed by using clinical and laboratory variables obtained from 260 RIGOR subjects with verified aspirin-mediated inhibition of platelet TXA2 generation. The strongest variable associated with UTXB2 6 months after surgery, accounting for 47.2% of the modeled effect, was urine 8-iso-prostaglandin (PG)F2alpha, an arachidonic acid metabolite generated nonenzymatically by oxidative stress (standardized coefficient 0.442, P < 0.001). Age, sex, race, lipid therapy, creatinine, left ventricular ejection fraction, and aspirin dose were also significantly associated with UTXB2 (P < 0.03), although they accounted for only 4.8% to 10.2% of the modeled effect. Urine 8-iso-PGF2alpha correlated with risk of vein graft occlusion (odds ratio 1.67, P=0.001) but was not independent of UTXB2. In vitro studies revealed that endothelial cells generate TXA2 in response to oxidative stress and direct exposure to 8-iso-PGF2alpha.
CONCLUSIONS: Oxidative stress-induced formation of 8-iso-PGF2alpha is strongly associated with nonplatelet thromboxane formation and early vein graft thrombosis after coronary artery bypass graft surgery. The endothelium is potentially an important source of oxidative stress-induced thromboxane generation. These findings suggest therapies that reduce oxidative stress could be useful in reducing cardiovascular risks associated with aspirin-insensitive thromboxane generation.
NEMO Prevents RIP Kinase 1-Mediated Epithelial Cell Death and Chronic Intestinal Inflammation by NF-kappaB-Dependent and -Independent Functions
Intestinal epithelial cells (IECs) regulate gut immune homeostasis, and impaired epithelial responses are implicated in the pathogenesis of inflammatory bowel diseases (IBD). IEC-specific ablation of nuclear factor kappaB (NF-kappaB) essential modulator (NEMO) caused Paneth cell apoptosis and impaired antimicrobial factor expression in the ileum, as well as colonocyte apoptosis and microbiota-driven chronic inflammation in the colon. Combined RelA, c-Rel, and RelB deficiency in IECs caused Paneth cell apoptosis but not colitis, suggesting that NEMO prevents colon inflammation by NF-kappaB-independent functions. Inhibition of receptor-interacting protein kinase 1 (RIPK1) kinase activity or combined deficiency of Fas-associated via death domain protein (FADD) and RIPK3 prevented epithelial cell death, Paneth cell loss, and colitis development in mice with epithelial NEMO deficiency. Therefore, NEMO prevents intestinal inflammation by inhibiting RIPK1 kinase activity-mediated IEC death, suggesting that RIPK1 inhibitors could be effective in the treatment of colitis in patients with NEMO mutations and possibly in IBD.
A Delphi process to address medication appropriateness for older persons with multiple chronic conditions
BACKGROUND: Frameworks exist to evaluate the appropriateness of medication regimens for older patients with multiple medical conditions (MCCs). Less is known about how to translate the concepts of the frameworks into specific strategies to identify and remediate inappropriate regimens.
METHODS: Modified Delphi method involving iterative rounds of input from panel members. Panelists (n = 9) represented the disciplines of nursing, medicine and pharmacy. Included among the physicians were two geriatricians, one general internist, one family practitioner, one cardiologist and two nephrologists. They participated in 3 rounds of web-based anonymous surveys.
RESULTS: The panel reached consensus on a set of markers to identify problems with medication regimens, including patient/caregiver report of non-adherence, medication complexity, cognitive impairment, medications identified by expert opinion as inappropriate for older persons, excessively tight blood sugar and blood pressure control among persons with diabetes mellitus, patient/caregiver report of adverse medication effects or medications not achieving desired outcomes, and total number of medications. The panel also reached consensus on approaches to address these problems, including endorsement of strategies to discontinue medications with known benefit if necessary because of problems with feasibility or lack of alignment with patient goals.
CONCLUSIONS: The results of the Delphi process provide the basis for an algorithm to improve medication regimens among older persons with MCCs. The algorithm will require assessment not only of medications and diagnoses but also cognition and social support, and it will support discontinuation of medications both when risks outweigh benefits and when regimens are not feasible or do not align with goals.
BACKGROUND: Microenvironments called niches maintain resident stem cell populations by balancing self-renewal with differentiation, but the genetic regulation of this process is unclear. The niche of the Drosophila testis is well-characterized and genetically tractable, making it ideal for investigating the molecular regulation of stem cell biology. The JAK/STAT pathway, activated by signals from a niche component called the hub, maintains both germline and somatic stem cells.
RESULTS: This study investigated the molecular regulation of the JAK/STAT pathway in the stem cells of the Drosophila testis. We determined that the transcriptional regulator Apontic (Apt) acts in the somatic (cyst) stem cells (CySCs) to balance differentiation and maintenance. We found Apt functions as a negative feedback inhibitor of STAT activity, which enables cyst cell maturation. Simultaneous loss of the STAT regulators apt and Socs36E, or the Stat92E-targeting microRNA miR-279, expanded the somatic stem cell-like population.
CONCLUSIONS: Genetic analysis revealed that a conserved genetic regulatory network limits JAK/STAT activity in the somatic stem cells of Drosophila testis. In these cells, we determined JAK/STAT signaling promotes apt expression. Then, Apt functions through Socs36E and miR-279 to attenuate pathway activation, which is required for timely CySC differentiation. We propose that Apt acts as a core component of a STAT-regulatory circuit to prevent stem cell overpopulation and allow stem cell maturation.
Does the Mutant CAG Expansion in Huntingtin mRNA Interfere with Exonucleolytic Cleavage of its First Exon
BACKGROUND: Silencing mutant huntingtin mRNA by RNA interference (RNAi) is a therapeutic strategy for Huntington's disease. RNAi induces specific endonucleolytic cleavage of the target HTT mRNA, followed by exonucleolytic processing of the cleaved mRNA fragments.
OBJECTIVES: We investigated the clearance of huntingtin mRNA cleavage products following RNAi, to find if particular huntingtin mRNA sequences persist. We especially wanted to find out if the expanded CAG increased production of a toxic mRNA species by impeding degradation of human mutant huntingtin exon 1 mRNA.
METHODS: Mice expressing the human mutant HTT transgene with 128 CAG repeats (YAC128 mice) were injected in the striatum with self-complementary AAV9 vectors carrying a miRNA targeting exon 48 of huntingtin mRNA (scAAV-U6-miRNA-HTT-GFP). Transgenic huntingtin mRNA levels were measured in striatal lysates after two weeks. For qPCR, we used species specific primer-probe combinations that together spanned 6 positions along the open reading frame and untranslated regions of the human huntingtin mRNA. Knockdown was also measured in the liver following tail vein injection.
RESULTS: Two weeks after intrastriatal administration of scAAV9-U6-miRNA-HTT-GFP, we measured transgenic mutant huntingtin in striatum using probes targeting six different sites along the huntingtin mRNA. Real time PCR showed a reduction of 29% to 36% in human HTT. There was no significant difference in knockdown measured at any of the six sites, including exon 1. In liver, we observed a more pronounced HTT mRNA knockdown of 70% to 76% relative to the untreated mice, and there were also no significant differences among sites.
CONCLUSIONS: Our results demonstrate that degradation is equally distributed across the human mutant huntingtin mRNA following RNAi-induced cleavage.
Total Elbow Arthroplasty in the United States: Evaluation of Cost, Patient Demographics, and Complication Rates
Total elbow arthroplasty (TEA) is utilized in the treatment of rheumatoid and post-traumatic elbow arthritis. TEA is a relatively low volume surgery in comparison to other types of arthroplasty and therefore little is known about current surgical utilization, patient demographics and complication rates in the United States. The purpose of our study is to evaluate the current practice trends and associated in-patient complications of TEA at academic centers in the United States. We queried the University Health Systems Consortium administrative database from 2007 to 2011 for patients who underwent an elective TEA. A descriptive analysis of demographics was performed which included patient age, sex, race, and insurance status. We also evaluated the following patient clinical benchmarks: hospital length of stay (LOS), hospital direct cost, in-hospital mortality, complications, and 30-day readmission rates. Our cohort consisted of 3146 adult patients (36.5% male and 63.5% female) with an average age of 58 years who underwent a total elbow arthroplasty (159 academic medical centers) in the United States. The racial demographics included 2334 (74%) Caucasian, 285 (9%) black, 236 (7.5%) Hispanic, 16 (0.5%) Asian, and 283 (9%) other patients. The mean LOS was 4.2+/-5 days and the mean total direct cost for the hospital was 16,300+/-4000 US Dollars per case. The overall inpatient complication rate was 3.1% and included mortality < 1%, DVT (0.8%), re-operation (0.5%), and infection (0.4%). The 30-day readmission rate was 4.4%. TEA is a relatively uncommon surgery in comparison to other forms of arthroplasty but is associated with low in-patient and 30-day perioperative complication rate. Additionally, the 30-day readmission rate and overall hospital costs are comparable to the traditional total hip and knee arthroplasty surgeries.