Scaffolded Antigens in Yeast Cell Particle Vaccines Provide Protection against Systemic Polyoma Virus Infection
Background. U65, a self-aggregating peptide scaffold, traps fused protein antigens in yeast cells. Conversion to Yeast Cell Particle (YCP) vaccines by partial removal of surface mannoproteins exposes beta-glucan, mediating efficient uptake by antigen-presenting cells (APCs). YCP vaccines are inexpensive, capable of rapid large-scale production and have potential for both parenteral and oral use. Results. YCP processing by alkaline hydrolysis exposes up to 20% of the glucan but converts scaffolded antigen and internal yeast proteins into a common aggregate, preventing selective yeast protein removal. For U65-green fluorescent protein (GFP) or U65-Apolipoprotein A1 (ApoA1) subcutaneous vaccines, maximal IgG responses in mice required 10% glucan exposure. IgG responses to yeast proteins were 5-fold lower. Proteolytic mannoprotein removal produced YCPs with only 6% glucan exposure, insufficiently porous for selective removal of even native yeast proteins. Vaccine efficacy was reduced 10-fold. Current YCP formulations, therefore, are not suitable for human use but have considerable potential for use in feed animal vaccines. Significantly, a YCP vaccine expressing a GFP fusion to VP1, the murine polyoma virus major capsid protein, after either oral or subcutaneous administration, protected mice against an intraperitoneal polyoma virus challenge, reducing viral DNA levels in spleen and liver by > 98%.
Increasing evidence shows that long noncoding RNAs (lncRNAs) have important roles in the regulation of multiple cellular processes, including cell division, cell growth, and apoptosis, as well as cancer metastasis and neurological disease progression; however, the mechanism of how lncRNAs regulate these processes is not well established. In this study, we demonstrated that downregulating the expression of the lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) in breast cancer cells inhibited cell growth and induced cell apoptosis. In addition, the RNA-binding protein fused in sarcoma/translocated in liposarcoma (FUS/TLS) physically interacted with NEAT1, and reducing the expression of FUS/TLS also induced cell apoptosis. Multiple miRNAs were identified as regulators of NEAT1, but only overexpression of miR-548ar was able to decrease NEAT1 expression and promote apoptosis. These results indicate a novel interaction between NEAT1, miR-548ar-3p, and FUS and their role in the regulation of breast cancer cell apoptosis.
Influenza viral evolution presents a formidable challenge to vaccination due to the virus' ability to rapidly mutate to evade immune responses. Live influenza infections generate large and diverse CD4 effector T cell responses that yield highly protective, long-lasting CD4 T cell memory that can target conserved viral epitopes. We review advances in our understanding of mechanisms involved in generating CD4 T cell responses against the influenza A virus (IAV), focusing on specialized follicular helper (TFH) and CD4 cytotoxic (ThCTL) effector subsets and on CD4 T cell memory. We also discuss two recent findings in context of enhancing vaccine responses. First, helper T cells require priming with APC secreting high levels of IL-6. Second, the transition of IAV-generated effectors to memory depends on IL-2, costimulation and antigen signals, just before effectors reach peak numbers, defined as the "memory checkpoint." The need for these signals during the checkpoint could explain why many current influenza vaccines are poorly effective and elicit poor cellular immunity. We suggest that CD4 memory generation can be enhanced by re-vaccinating at this time. Our best hope lies in a universal vaccine that will not need to be formulated yearly against seasonal antigenically novel influenza strains and will also be protective against a pandemic strain. We suggest a vaccine approach that elicits a powerful T cell response, by initially inducing high levels of APC activation and later providing antigen at the memory checkpoint, may take us a step closer to such a universal influenza vaccine.
Defining the Binding Region in Factor H to Develop a Therapeutic Factor H-Fc Fusion Protein against Non-Typeable Haemophilus influenzae
Non-typeable Haemophilus influenzae (NTHi) cause a range of illnesses including otitis media, sinusitis, and exacerbation of chronic obstructive pulmonary disease, infections that contribute to the problem of antibiotic resistance and are themselves often intractable to standard antibiotic treatment regimens. We investigated a strategy to exploit binding of the complement inhibitor Factor H (FH) to NTHi as a functional target for an immunotherapeutic containing the NTHi binding domain of FH fused to the Fc domain of IgG1. Chimeric proteins containing the regions that most FH-binding bacteria use to engage human FH, domains 6 and 7 (FH6,7/Fc) and/or 18 through 20 (FH18-20/Fc), were evaluated for binding to NTHi. FH6,7/Fc bound strongly to each of seven NTHi clinical isolates tested and efficiently promoted complement-mediated killing by normal human serum. FH18-20/Fc bound weakly to three of the strains but did not promote complement dependent killing. Outer-membrane protein P5 has been implicated in FH binding by NTHi, and FH6,7/Fc binding was greatly diminished in five of seven P5 deficient isogenic mutant strains tested, implicating an alternative FH binding protein in some strains. Binding of FH18-20/Fc was decreased in the P5 mutant of one strain. A murine model was used to evaluate potential therapeutic application of FH6,7/Fc. FH6,7/Fc efficiently promoted binding of C3 to NTHi exposed to mouse serum, and intranasal delivery of FH6,7/Fc resulted in significantly enhanced clearance of NTHi from the lung. Moreover, a P5 deficient mutant was attenuated for survival in the lung model, suggesting that escape mutants lacking P5 would be less likely to replace strains susceptible to FH6,7/Fc. These results provide evidence for the potential utility of FH6,7/Fc as a therapeutic against NTHi lung infection. FH binding is a common property of many respiratory tract pathogens and FH/Fc chimeras may represent promising alternative or adjunctive therapeutics against such infections, which are often polymicrobial.
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.