gga-miR-375 Plays a Key Role in Tumorigenesis Post Subgroup J Avian Leukosis Virus Infection

Avian leukosis is a neoplastic disease caused in part by subgroup J avian leukosis virus J (ALV-J). Micro ribonucleic acids (miRNAs) play pivotal oncogenic and tumour-suppressor roles in tumour development and progression. However, little is known about the potential role of miRNAs in avian leukosis tumours. We have found a novel tumour-suppressor miRNA, gga-miR-375, associated with avian leukosis tumorigenesis by miRNA microarray in a previous report. We have also previously studied the biological function of gga-miR-375; Overexpression of gga-miR-375 significantly inhibited DF-1 cell proliferation, and significantly reduced the expression of yes-associated protein 1 (YAP1) by repressing the activity of a luciferase reporter carrying the 3′-untranslated region of YAP1. This indicates that gga-miR-375 is frequently downregulated in avian leukosis by inhibiting cell proliferation through YAP1 oncogene targeting. Overexpression of gga-miR-375 markedly promoted serum starvation induced apoptosis, and there may be the reason why the tumour cycle is so long in the infected chickens. In vivo assays, gga-miR-375 was significantly downregulated in chicken livers 20 days after infection with ALV-J, and YAP1 was significantly upregulated 20 days after ALV-J infection (P<0.05). We also found that expression of cyclin E, an important regulator of cell cycle progression, was significantly upregulated (P<0.05). Drosophila inhibitor of apoptosis protein 1 (DIAP1), which is related to caspase-dependent apoptosis, was also significantly upregulated after infection. Our data suggests that gga-miR-375 may function as a tumour suppressor thereby regulating cancer cell proliferation and it plays a key role in avian leukosis tumorigenesis.     

A Combination of Serological Assays to Detect Human Antibodies to the Avian Influenza A H7N9 Virus

Human infection with avian influenza A H7N9 virus was first identified in March 2013 and represents an ongoing threat to public health. There is a need to optimize serological methods for this new influenza virus. Here, we compared the sensitivity and specificity of the hemagglutinin inhibition (HI), microneutralization (MN), and Western blot (WB) assays for the detection of human antibodies against avian influenza A (H7N9) virus. HI with horse erythrocytes (hRBCs) and a modified MN assay possessed greater sensitivity than turkey erythrocytes and the standard MN assay, respectively. Using these assays, 80% of tested sera from confirmed H7N9 cases developed detectable antibody to H7N9 after 21 days. To balance sensitivity and specificity, we found serum titers of ≥20 (MN) or 160 (HI) samples were most effective in determining seropositive to H7N9 virus. Single serum with HI titers of 20–80 or MN titer of 10 could be validated by each other or WB assay. Unlike serum collected from adult or elderly populations, the antibody response in children with mild disease was low or undetectable. These combinations of assays will be useful in case diagnosis and serologic investigation of human cases.     

Age at Menarche and Natural Menopause and Number of Reproductive Years in Association with Mortality: Results from a Median Follow-Up of 11.2 Years among 31,955 Naturally Menopausal Chinese Women

Background

Studies conducted in Western countries suggest that early age at menarche and early age at menopause are both associated with increased total mortality, but limited data are available for Asian populations. We examined associations of age at menarche and natural menopause and duration of the reproductive span with mortality in a population-based cohort study of Chinese women.

Methods

We evaluated the effects of age at menarche, age at natural menopause, and number of reproductive years on total and cause-specific mortality among 31,955 naturally menopausal Chinese women who participated in the Shanghai Women''s Health Study, a population-based, prospective cohort study.

Results

A total of 3,158 deaths occurred during a median follow-up of 11.2 years. Results from Cox proportional hazards models showed that younger age at menopause (<46.64 years) was associated with higher risk of total mortality (P_trend  = 0.02). Younger age at menarche (<14 years) was associated with higher risk of mortality from stroke (P_trend  = 0.03) and diabetes (P_trend = 0.02) but lower risk of mortality from respiratory system cancer (P_trend  = 0.01). Women with a shorter reproductive span had lower risk of mortality from gynecological cancers (P_trend = 0.03).

Conclusions

Our study found that menstrual characteristics are important predictors of mortality, suggesting an important role of sex hormones in biological aging.     

Impact of Body Mass Index on Plasma N-Terminal ProB-Type Natriuretic Peptides in Chinese Atrial Fibrillation Patients without Heart Failure

Background

An inverse relationship between body mass index (BMI) and circulating levels of N-terminal proB-type natriuretic peptide (NT-proBNP) has been demonstrated in subjects with and without heart failure. Obesity also has been linked with increased incidence of atrial fibrillation (AF), but its influence on NT-proBNP concentrations in AF patients remains unclear. This study aimed to investigate the effect of BMI on NT-proBNP levels in AF patients without heart failure.

Methods

A total of 239 consecutive patients with AF undergoing catheter ablation were evaluated. Levels of NT-proBNP and clinical characteristics were compared in overweight or obese (BMI≥25 kg/m²) and normal weight (BMI<25 kg/m²) patients.

Results

Of 239 patients, 129 (54%) were overweight or obese. Overweight or obese patients were younger, more likely to have a history of nonparoxysmal AF, hypertension, and diabetes mellitus. Levels of NT-proBNP were significantly lower in overweight or obese than in normal weight subjects (P<0.05). The relationship of obesity and decreased NT-proBNP levels persisted in subgroup of hypertension, both gender and both age levels (≥65 yrs and <65 yrs).Multivariate linear regression identified BMI as an independent negative correlate of LogNT-proBNP level.

Conclusions

An inverse relationship between BMI and plasma NT-proBNP concentrations have been demonstrated in AF patients without heart failure. Overweight or obese patients with AF appear to have lower NT-proBNP levels than normal weight patients.     

Immunization with Hypoallergens of Shrimp Allergen Tropomyosin Inhibits Shrimp Tropomyosin Specific IgE Reactivity

Designer proteins deprived of its IgE-binding reactivity are being sought as a regimen for allergen-specific immunotherapy. Although shrimp tropomyosin (Met e 1) has long been identified as the major shellfish allergen, no immunotherapy is currently available. In this study, we aim at identifying the Met e 1 IgE epitopes for construction of hypoallergens and to determine the IgE inhibitory capacity of the hypoallergens. IgE-binding epitopes were defined by three online computational models, ELISA and dot-blot using sera from shrimp allergy patients. Based on the epitope data, two hypoallergenic derivatives were constructed by site-directed mutagenesis (MEM49) and epitope deletion (MED171). Nine regions on Met e 1 were defined as the major IgE-binding epitopes. Both hypoallergens MEM49 and MED171 showed marked reduction in their in vitro reactivity towards IgE from shrimp allergy patients and Met e 1-sensitized mice, as well as considerable decrease in induction of mast cell degranulation as demonstrated in passive cutaneous anaphylaxis assay. Both hypoallergens were able to induce Met e 1-recognizing IgG antibodies in mice, specifically IgG_2a antibodies, that strongly inhibited IgE from shrimp allergy subjects and Met e 1-sensitized mice from binding to Met e 1. These results indicate that the two designer hypoallergenic molecules MEM49 and MED171 exhibit desirable preclinical characteristics, including marked reduction in IgE reactivity and allergenicity, as well as ability to induce blocking IgG antibodies. This approach therefore offers promises for development of immunotherapeutic regimen for shrimp tropomyosin allergy.     

Tissue Quality Assessment Using a Novel Direct Elasticity Assessment Device (The E-Finger): A Cadaveric Study of Prostatectomy Dissection

Introduction

Minimally invasive radical prostatectomy (RP) (robotic and laparoscopic), have brought improvements in the outcomes of RP due to improved views and increased degrees of freedom of surgical devices. Robotic and laparoscopic surgeries do not incorporate haptic feedback, which may result in complications secondary to inadequate tissue dissection (causing positive surgical margins, rhabdosphincter damage, etc). We developed a micro-engineered device (6 mm² sized) [E-finger]) capable of quantitative elasticity assessment, with amplitude ratio, mean ratio and phase lag representing this. The aim was to assess the utility of the device in differentiating peri-prostatic tissue types in order to guide prostate dissection.

Material and Methods

Two embalmed and 2 fresh frozen cadavers were used in the study. Baseline elasticity values were assessed in bladder, prostate and rhabdosphincter of pre-dissected embalmed cadavers using the micro-engineered device. A measurement grid was created to span from the bladder, across the prostate and onto the rhabdosphincter of fresh frozen cadavers to enable a systematic quantitative elasticity assessment of the entire area by 2 independent assessors. Tissue was sectioned along each row of elasticity measurement points, and stained with haematoxylin and eosin (H&E). Image analysis was performed with Image Pro Premier to determine the histology at each measurement point.

Results

Statistically significant differences in elasticity were identified between bladder, prostate and sphincter in both embalmed and fresh frozen cadavers (p = <0.001). Intra-class correlation (ICC) reliability tests showed good reliability (average ICC = 0.851). Sensitivity and specificity for tissue identification was 77% and 70% respectively to a resolution of 6 mm².

Conclusions

This cadaveric study has evaluated the ability of our elasticity assessment device to differentiate bladder, prostate and rhabdosphincter to a resolution of 6 mm². The results provide useful data for which to continue to examine the use of elasticity assessment devices for tissue quality assessment with the aim of giving haptic feedback to surgeons performing complex surgery.     

SH3BP2 Gain-Of-Function Mutation Exacerbates Inflammation and Bone Loss in a Murine Collagen-Induced Arthritis Model

Objective

SH3BP2 is a signaling adapter protein which regulates immune and skeletal systems. Gain-of-function mutations in SH3BP2 cause cherubism, characterized by jawbone destruction. This study was aimed to examine the role of SH3BP2 in inflammatory bone loss using a collagen-induced arthritis (CIA) model.

Methods

CIA was induced in wild-type (Sh3bp2^+/+) and heterozygous P416R SH3BP2 cherubism mutant knock-in (Sh3bp2^KI/+) mice, an SH3BP2 gain-of-function model. Severity of the arthritis was determined by assessing the paw swelling and histological analyses of the joints. Micro-CT analysis was used to determine the levels of bone loss. Inflammation and osteoclastogenesis in the joints were evaluated by quantitating the gene expression of inflammatory cytokines and osteoclast markers. Furthermore, involvement of the T- and B-cell responses was determined by draining lymph node cell culture and measurement of the serum anti-mouse type II collagen antibody levels, respectively. Finally, roles of the SH3BP2 mutation in macrophage activation and osteoclastogenesis were determined by evaluating the TNF-α production levels and osteoclast formation in bone marrow-derived M-CSF-dependent macrophage (BMM) cultures.

Results

Sh3bp2^KI/+ mice exhibited more severe inflammation and bone loss, accompanying an increased number of osteoclasts. The mRNA levels for TNF-α and osteoclast marker genes were higher in the joints of Sh3bp2^KI/+ mice. Lymph node cell culture showed that lymphocyte proliferation and IFN-γ and IL-17 production were comparable between Sh3bp2^+/+ and Sh3bp2^KI/+ cells. Serum anti-type II collagen antibody levels were comparable between Sh3bp2^+/+ and Sh3bp2^KI/+ mice. In vitro experiments showed that TNF-α production in Sh3bp2^KI/+ BMMs is elevated compared with Sh3bp2^+/+ BMMs and that RANKL-induced osteoclastogenesis is enhanced in Sh3bp2^KI/+ BMMs associated with increased NFATc1 nuclear localization.

Conclusion

Gain-of-function of SH3BP2 augments inflammation and bone loss in the CIA model through increased macrophage activation and osteoclast formation. Therefore, modulation of the SH3BP2 expression may have therapeutic potential for the treatment of rheumatoid arthritis.     

A New Model for Predicting Non-Sentinel Lymph Node Status in Chinese Sentinel Lymph Node Positive Breast Cancer Patients

Background

Our goal is to validate the Memorial Sloan-Kettering Cancer Center (MSKCC) nomogram and Stanford Online Calculator (SOC) for predicting non-sentinel lymph node (NSLN) metastasis in Chinese patients, and develop a new model for better prediction of NSLN metastasis.

Methods

The MSKCC nomogram and SOC were used to calculate the probability of NSLN metastasis in 120 breast cancer patients. Univariate and multivariate analyses were performed to evaluate the relationship between NSLN metastasis and clinicopathologic factors, using the medical records of the first 80 breast cancer patients. A new model predicting NSLN metastasis was developed from the 80 patients.

Results

The MSKCC and SOC predicted NSLN metastasis in a series of 120 patients with an area under the receiver operating characteristic curve (AUC) of 0.688 and 0.734, respectively. For predicted probability cut-off points of 10%, the false-negative (FN) rates of MSKCC and SOC were both 4.4%, and the negative predictive value (NPV) 75.0% and 90.0%, respectively. Tumor size, Kiss-1 expression in positive SLN and size of SLN metastasis were independently associated with NSLN metastasis (p<0.05). A new model (Peking University People''s Hospital, PKUPH) was developed using these three variables. The MSKCC, SOC and PKUPH predicted NSLN metastasis in the second 40 patients from the 120 patients with an AUC of 0.624, 0.679 and 0.795, respectively.

Conclusion

MSKCC nomogram and SOC did not perform as well as their original researches in Chinese patients. As a new predictor, Kiss-1 expression in positive SLN correlated independently with NSLN metastasis strongly. PKUPH model achieved higher accuracy than MSKCC and SOC in predicting NSLN metastasis in Chinese patients.     

The lock-washer: a reconciliation of the RIG-I activation models

RIG-I belongs to a type of intracellular pattern recognition receptors involved in the recognition of viral RNA by the innate immune system. A report by Peisley et al. published in Nature provides the crystal structure of human RIG-I revealing a tetrameric architecture of the RIG-I 2-CARD domain bound by three K63-linked ubiquitin chains, uncovering its activation mechanism for downstream signaling.The recognition of microbial-derived nucleic acids and the correct and specific activation of the molecular machinery governing the mammalian immune response are paramount to host survival during viral infection. Viral RNA represents a key trigger for the activation and mobilization of a series of pattern recognition receptors (PRRs) such as the Toll-like receptor (TLR) and retinoic acid-inducible gene 1 (RIG-I)-like receptor (RLR) families. While the TLRs are restricted to the cell surface or inside endosomal compartments, the RLRs are present in the cytosol and act as the key sentinels of actively invading and replicating viruses.The RLR family of receptors, RIG-I and Melanoma Differentiation-Associated protein 5 (MDA-5), are characterised by 3 distinct signaling domains critical for viral RNA recognition and response. The C-terminal repressor domain and the internal ATPase-containing DExD/H-box helicase domain of RIG-I function together to facilitate binding of viral dsRNA which contain either a 5′-ppp motif or 5′ blunt-end base-paired RNA with a triphosphate motif, moieties absent on self-nucleic acids¹. Upon viral RNA ligation, two N-terminal caspase activation and recruitment domains (CARD), known as 2-CARD, on RIG-I propagate signal transduction via interactions with mitochondrial antiviral signaling protein (MAVS)². Recent molecular and structural studies have elucidated the mechanisms by which RLR-activated MAVS mediates the antiviral response. During RIG-I signaling, MAVS forms large multimeric prion-like filaments on the mitochondrial membrane which are essential for RIG-I-mediated type I interferon (IFN) production³. Such functional aggregates are capable of recruiting key downstream signaling components such as members of TNF receptor associated factors (TRAF) family, resulting in the activation of the MAPKs, the NF-κB pathway and interferon regulatory factor 3/7 (IRF3/7) and consequently culminating in the upregulation of protective IFNs and pro-inflammatory cytokines. Viral infection is sufficient to convert nearly all endogenous detectable MAVS to functionally active aggregates, and interestingly this phenomenon can be recapitulated in vitro using only mitochondria, RIG-I and K63-linked ubiquitin chains, underscoring the functional importance of polyubiquitination events during RIG-I signaling⁴.In contrast to the well-documented and -accepted paradigm of MAVS activation, the model of RIG-I-mediated activation has remained incompletely understood. The classical model holds that RIG-I remains in an auto-repressed state in the absence of ligand. Upon viral recognition, the E3 ubiquitin ligase tripartite motif 25 (TRIM25) binds to the 2-CARD domain of RIG-I, resulting in the covalent conjugation of K63-linked polyubiquitin chains to induce a conformation change in the receptor and facilitate a “release” of the 2-CARD domain for MAVS interaction and activation⁵. However, this simple release model of the 2-CARD domain does not reconcile with recent compelling reports that RIG-I can act as a receptor for unanchored, non-covalently attached ubiquitin chains and that polyubiquitination of RIG-I induces the oligomerization of a heterotetrameric complex consisting of 4 RIG-I and 4 K63-ubiqutin chain molecules^6,7. In addition, although K63-ubiquitination is essential for the signaling potential of isolated 2-CARD molecules, full-length RIG-I can form filaments around the ends of dsRNA molecules, allowing 2-CARD regions of RIG-I molecules to come into close proximity to each other and facilitate MAVS aggregation in an ubiquitin-independent manner⁸.Although such conflicting reports seem to propose vastly different models of RIG-I activation, an elegent study published in Nature by Peisley et al.⁹ uses biochemical and structural studies to reconcile the different models and they finally offer a unified understanding of RIG-I receptor activation. They resolved the crystal structure of human RIG-I 2-CARD in complex with K63-ubiquitin at 3.7 Å. The structure revealed the tetrameric architecture of RIG-I 2-CARD bound by three K63 ubiquitin chains (Figure 1). Crystallization and structure determination reveal that four 2-CARD subunits form a tetrameric helical assembly, termed the “lock washer”, with the two ends displaced by half the thickness of 2-CARD.Open in a separate windowFigure 1A model of RIG-I-mediated antiviral response.Two key questions arise from the RIG-I 2-CARD structure. First, how does the tetrameric architecture of RIG-I serve as a platform to activate downstream signaling? The CARD domain belongs to the death domain (DD) superfamily, members of which have a similar three-dimensional fold. The structures of other DD oligomers such as Myddosome, PIDDosome, or FAS-FADD complex have recently been resolved. The assembly of DD oligomers is usually mediated at six surface areas, with the helical oligomeric structure of upstream signaling molecules serving as a scaffold to assemble the downstream DD oligomers through helical extension. In the current study, the authors show that the assembly and stability of the tetramer and its IFN-β signaling potential are dependent on several intermolecular and intramolecular CARD interactions by generating mutants on different interaction surfaces and analyzing their tetramer formation and IFN-β induction abilities. MAVS filament formation assays indicate that the helical tetrameric structure of RIG-I 2-CARD serves as the platform for MAVS-CARD filament assembly, with the top surface of the second CARD as the primary site for MAVS recruitment⁹.The second pertinent question addressed is how the interaction between ubiquitin and 2-CARD contributes to downstream signaling? Unlike other DD oligomers, tetramer formation of isolated RIG-I 2-CARD requires K63-linked ubiquitin chains. The structure predicts that longer ubiquitin chains might wrap around the 2-CARD tetramer at 1:4 or 2:4 molar ratios to stabilize the 2-CARD tetramerization. Another key problem addressed in this study is the relationship between the covalent conjugation and non-covalent binding of K63-ubiquitin in stabilizing 2-CARD tetramers during RIG-I signaling. The authors challenge previous publications on the significance of 6 lysine (K) residues in both covalent conjugation and non-covalent K63-ubiquitin binding.The authors show that only K6 is covalently conjugated with K63-ubiquitin chains and that non-covalent binding of K63-ubiquitin to 2-CARD can induce a further stabilization of the tetramer complex. RIG-I filament formation on dsRNA with appropriate length can also compensate for the requirement of both covalent and non-covalent K63-ubiquitin binding. Thus they arrive at the conclusion that these three mechanisms might act synergistically for signal activation. This compensatory mechanism could guarantee the detection of foreign pathogen RNA in case of the absence of one or two of the mechanisms or may allow an amplification of the signal potential. One could speculate that such functional redundancy in the initiation stage of signal activation may be a common theme in other innate immunity pathways.The significance of this study lies in the resolution of the structural basis of the activated RIG-I 2-CARD tetramer and its initiation of MAVS aggregation and filament formation — the first elements of the dsRNA sensing signaling cascade that lead to production of type I IFNs and pro-inflammatory cytokines. It provides another detailed example of DD oligomers and adds to the growing realization of a common role of oligomeric molecular scaffolds in mediating innate immune signaling. Such exciting findings will no doubt instigate further study into the exact molecular interactions and mechanisms controlling dsRNA sensing. For example, the authors use a crystallized K115A/R117A 2-CARD double mutant for structural analysis; although it retains the ability to tetramerize with K63-ubiquitin and activate type I IFNs, the structure might still not be consistent with the wild-type 2-CARD and this may warrant further investigation. Furthermore, whether the RIG-I signaling activation mechanism that derived from this structure could be generalized and applied to other CARD domain receptors such as MDA-5, NOD1, NOD2, IPAF and NLRP1 will require further investigation. By utilizing advanced structural determination techniques coupled with sophisticated in vitro assays such as those described in this study, these questions will no doubt be addressed in the near future.