New insight into social relationships in dairy cows and how time of birth, parity, and relatedness affect spatial interactions later in life.

Social interactions between cows play a fundamental role in the daily activities of dairy cattle. Real-time location systems provide on a continuous and automated basis information about the position of individual cows inside barns, offering a valuable opportunity to monitor dyadic social contacts. Understanding dyadic social interactions could be applied to enhance the stability of the social structure promoting animal welfare and to model disease transmission in dairy cattle. This study aimed to identify the effect of different cow characteristics on the likelihood of the formation and persistence of social contacts in dairy cattle. The individual position of the lactating cows was automatically collected once per second for 2 wk, using an ultra-wideband system on a Swedish commercial farm consisting of almost 200 dairy cows inside a freestall barn. Social networks were constructed using the position data of 149 cows with available information on all characteristics during the study period. Social contacts were considered as a binary variable indicating whether a cow pair was within 2.5 m of each other for at least 10 min per day. The role of cow characteristics in social networks was studied by applying separable temporal exponential random graph models. Our results revealed that cows of the same parity interacted more consistently, as well as those born within 7 d of each other or closely related by pedigree. The repeatability of the topological parameters indicated a consistent short-term stability of the individual animal roles within the social network structure. Additional research is required to elucidate the underlying mechanisms governing the long-term evolution of social contacts among dairy cattle and to investigate the relationship between these networks and the transmission of diseases in the dairy cattle population.

Message in a bubble: the translational potential of extracellular vesicles.

Extracellular vesicles (EVs) are small, membrane-enclosed vesicles released by cells into the extracellular milieu. They are found in all body fluids and contain a variety of functional cargo including DNA, RNA, proteins, glycoproteins and lipids, able to provoke phenotypic responses in cells, both locally and at distant sites. They are implicated in a wide array of physiological and pathological processes and hence have attracted considerable attention in recent years as potential therapeutic targets, drug delivery vehicles and biomarkers of disease. In this review we summarise the major functions of EVs in health and disease and discuss their translational potential, highlighting opportunities of - and challenges to - capitalising on our rapidly increasing understanding of EV biology for patient benefit.

Cow characteristics associated with the variation in number of contacts between dairy cows.

In modern freestall barns where large groups of cows are housed together, the behavior displayed by herd mates can influence the welfare and production of other individuals. Therefore, understanding social interactions in groups of dairy cows is important to enhance herd management and optimize the outcomes of both animal health and welfare in the future. Many factors can affect the number of social contacts in a group. This study aimed to identify which characteristics of a cow are associated with the number of contacts it has with other group members in 2 different functional areas (feeding and resting area) to increase our understanding of the social behavior of dairy cows. Inside 2 herds housed in freestall barns with around 200 lactating cows each, cow positions were recorded with an ultra-wideband real-time location system collecting all cows' positions every second over 2 wk. Using the positioning data of the cows, we quantified the number of contacts between them, assuming that cows spending time in proximity to one another (within a distance of 2.5 m for at least 10 min per day) were interacting socially. We documented in which barn areas these interactions occurred and used linear mixed models to investigate if lactation stage, parity, breed, pregnancy status, estrus, udder health, and claw health affect the number of contacts. We found variation in the number of contacts a cow had between individuals in both functional areas. Cows in later lactation had more contacts in the feeding area than cows in early lactation. Furthermore, in one herd, higher parity cows had fewer contacts in the feeding area than first parity cows, and in the other herd, cows in third parity or higher had more contacts in the resting area. This study indicates that cow characteristics such as parity and days in milk are associated with the number of contacts a cow has daily to its herd mates and provides useful information for further research on social interactions of dairy cows.

Identification of potential crucial genes associated with vasculogenic mimicry in human osteosarcoma based on gene expression profile.

We previously reported the presence of vasculogenic mimicry (VM) in human osteosarcoma. However, the mechanistic basis of osteosarcoma VM remains unclear. Three hundred eighty-one upregulated differentially expressed genes (DEGs) and 526 downregulated DEGs between human osteosarcoma cell line 143B and HOS cell exposed to Matrigel were screened out by microarray. GO categories such as "cell adhesion", "angiogenesis" were enriched in 143B group. PATHWAY analysis showed enriched TGF-beta, Wnt and VEGF signaling pathway in 143B group. The hub gene ITGA2 in signal-network of DEGs exhibited pro-VM and pro-metastasis effect. Our study provides fundamental data for further studies regarding molecules involved in osteosarcoma VM.

[Establishment of clinical features and prognostic scoring model in early-stage hepatitis B-related acute-on-chronic liver failure].

Objective: To explore the clinical characteristics and establish a corresponding prognostic scoring model in patients with early-stage clinical features of hepatitis B-induced acute-on-chronic liver failure (HBV-ACLF). Methods: Clinical characteristics of 725 cases with hepatitis B-related acute-on-chronic hepatic dysfunction (HBV-ACHD) were retrospectively analyzed using Chinese group on the study of severe hepatitis B (COSSH). The independent risk factors associated with 90-day prognosis to establish a prognostic scoring model was analyzed by multivariate Cox regression, and was validated by 500 internal and 390 external HBV-ACHD patients. Results: Among 725 cases with HBV-ACHD, 76.8% were male, 96.8% had cirrhosis base,66.5% had complications of ascites, 4.1% had coagulation failure in respect to organ failure, and 9.2% had 90-day mortality rate. Multivariate Cox regression analysis showed that TBil, WBC and ALP were the best predictors of 90-day mortality rate in HBV-ACHD patients. The established scoring model was COSS-HACHADs = 0.75 × ln(WBC) + 0.57 × ln(TBil)-0.94 × ln(ALP) +10. The area under the receiver operating characteristic curve (AUROC) of subjects was significantly higher than MELD, MELD-Na, CTP and CLIF-C ADs(P < 0.05). An analysis of 500 and 390 cases of internal random selection group and external group had similar verified results. Conclusion: HBV-ACHD patients are a group of people with decompensated cirrhosis combined with small number of organ failure, and the 90-day mortality rate is 9.2%. COSSH-ACHDs have a higher predictive effect on HBV-ACHD patients' 90-day prognosis, and thus provide evidence-based medicine for early clinical diagnosis and treatment.

Increasing the autotrophic growth of Chlorella USTB-01 via the control of bacterial contamination by Bdellovibrio USTB-06.

AIMS: (i) To obtain and identify the predatory bacteria for the control of contaminated bacteria and to promote the autotrophic growth of Chlorella USTB-01. (ii) To identify and measure the different cell numbers in microalgal culture using flow cytometer. METHODS AND RESULTS: A predatory bacterial strain was isolated using Escherichia coli BL21 as a sole prey host, which was identified as Bdellovibrio USTB-06 by the analysis of 16S rDNA sequence. A flow cytometer was successfully used to identify and measure the cell numbers of Chlorella USTB-01, the contaminated bacteria and Bdellovibrio USTB-06 simultaneously in the autotrophic culture of Chlorella USTB-01 according to the identification of the different cell sizes. With the addition of Bdellovibrio USTB-06 at initial 104 plaque-forming units per ml, the contaminated bacteria severely decreased by about five counts (in log10  CFU per ml) and the growth of Chlorella USTB-01 was greatly increased by 37·0% compared with those of control respectively. CONCLUSIONS: Bdellovibrio USTB-06 could effectively promote the growth of Chlorella USTB-01 via the killing of the contaminated bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study reveals a good biotechnology method to increase the growth of Chlorella USTB-01 which is very important in the industry of microalgal culture.

[Clinical significance of NS1-BP expression in esophageal squamous cell carcinoma].

Objective: To investigate the clinical significance of NS1-BP expression in patients with esophageal squamous cell carcinoma (ESCC), and to study the roles of NS1-BP in proliferation and apoptosis of ESCC cells. Methods: A total of 98 tumor tissues and 30 adjacent normal tissues from 98 ESCC patients were used as study group and control group, and these samples were collected in Sun Yat-Sen University Cancer Center between 2002 and 2008. In addition, 46 ESCC tissues which were collected in Cancer Institute and Hospital of Tianjin Medical University were used as validation group. Expression of mucosal NS1-BP was detected by immunohistochemistry. Kaplan-Meier curve and log-rank test were used to analyze the survival rate. Multivariate Cox proportional hazard model was used to analyze the prognostic factors. Furthermore, NS1-BP was over expressed or knocked down in ESCC cells by transient transfection. Protein levels of c-Myc were detected by western blot. Cell viability and apoptosis was analyzed by MTT assay and flow cytometry. Results: Among all of tested samples, NS1-BP were down-regulated in 9 out of 30 non-tumorous normal esophageal tissues (30.0%) and 85 out of 144 ESCC tissues (59.0%), respectively, showing a statistically significant difference (P=0.012). In the study group, three-year disease-free survival rate of NS1-BP high expression group (53.2%) was significantly higher than that of NS1-BP low expression group (27.6%; P=0.009). In the validation group, the three-year disease-free survival rates were 57.8% and 25.5% in NS1-BP high and low levels groups, respectively, showing a similar results (P=0.016). Importantly, multivariate analyses showed that low expression of NS1-BP was an independent predictor for chemoradiotherapy sensitivity and shorter disease-free survival time in ESCC patients(P<0.05 for all). Furthermore, overexpressed NS1-BP in TE-1 cells repressed c-Myc expression, inhibited cell proliferation and promoted apoptosis. In contrast, knockdown NS1-BP in KYSE510 cells induced c-Myc expression, increased cell proliferation and repressed apoptosis. Conclusions: NS1-BP is an independent favorable prognostic factor in ESCC. It inhibits cell proliferation and enhances cell apoptosis via repressing c-Myc. Targeting NS1-BP may be a new therapeutic strategy for ESCC patients.

Thoracic stomach-right main bronchus fistula treated with dual Y-shaped covered airway stents.

AIM: To determine the efficacy of dual Y-shaped covered airway stents to treat thoracic stomach-right main bronchus fistulae. MATERIAL AND METHODS: Fifteen patients who developed thoracic stomach-right main bronchus fistula after oesophageal cancer resection and postoperative irradiation were retrospectively analysed. All fistulae were close to the right upper lobe bronchus. Two Y-shaped covered airway stents were designed for each patient. Under radiographic guidance, one stent was placed from the right main bronchus into the bifurcation of upper lobe and intermediate bronchus, the other was placed from the trachea into both main bronchi. RESULTS: All fistulae were closed immediately after stenting. All patients could eat a semi-solid diet. The symptom of coughing while lying down resolved in all patients, and no complications, such as airway bleeding or pneumothorax, occurred. The average survival time was 26.65 months (range 2-40 months, 11 patients were still alive at the study end). Two patients died of tumour recurrence. Another two patients died of pulmonary infections. In one of these patients, there was a long delay between symptom onset and stenting. In the other patient, a small rupture occurred in the silicone membrane covering the stent, which allowed the leakage of gastric contents into the lung. CONCLUSION: Dual Y-shaped covered airway stent placement is feasible and safe to treat thoracic stomach-right main bronchus fistulae. Improvements to the material covering the stents is required.

WITHDRAWN: Expression and clinicopathological significance of Mel-18 and Bmi-1 in oesophageal squamous cell carcinoma.

This article has been withdrawn at the request authors.

[Investigation of renal corticomedullary differentiation with age-related change on non-contrast-enhanced MRI].

OBJECTIVE: To evaluate the relationship between renal corticomedullary differentiation, renal cortical thickness and age-related changes with non-contrast-enhanced steady-state free precession(SSFP) magnetic resonance imaging (MRI) and spatially selective inversion recovery(IR) pulse technology as well as its applied value . METHODS: A total of 76 healthy volunteers had been recruited from August 2014 to June 2015 in First Hospital of China Medical University.All volunteers were divided into three groups: 2-40 years old, 41-60 years old, 61-80 years old. All 76 volunteers underwent non-contrast-enhanced steady-state free precession(SSFP) 3.0 T MRI scan using variable inversion times (TIs)(TI=1 000, 1 100, 1 200, 1 300, 1 400, 1 500, 1 600, 1 700 ms). The renal corticomedullary differentiation was observed and the signal intensity of renal cortex and medulla were measured respectively as well in order to calculate renal corticomedullary contrast ratio. Besides, renal cortical thickness and renal size were measured. RESULTS: All 76 volunteers were successfully performed all the sequences of MRI scan, including 152 useful imaging of kidney in total. The renal corticomedullary differentiation was clearly shown in all subjects. There was negative correlation between the optimal inversion time(TI) and age(r=-0.65, P<0.01). Similarly, negative correlation was observed between renal corticomedullary contrast ratio and age(r=-0.35, P<0.01). The mean renal cortical thickness of all subjects was (5.33±0.71)mm and there were statistically significant difference among those different groups, which was negative-related with age(r=-0.79, P<0.01). There was no statistically significant difference between sexuality and renal cortical thickness.Additionally, renal cortical thickness had no statistically significant difference in both sides of kidneys. CONCLUSION: The renal corticomedullary differentiation is depicted clearly by means of non-contrast-enhanced steady-state free precession MRI with spatially selective inversion recovery pulse technology. The optimal inversion time decreases along with the increase of age. In the meanwhile, the renal cortical thickness could be measured truthfully and accurately.

Treatment of vertebral body compression fractures using percutaneous kyphoplasty guided by a combination of computed tomography and C-arm fluoroscopy with finger-touch guidance to determine the needle entry point.

This study aimed to evaluate the results and complications of image-guided percutaneous kyphoplasty (PKP) using computed tomography (CT) and C-arm fluoroscopy, with finger-touch guidance to determine the needle entry point. Of the 86 patients (106 PKP) examined, 56 were treated for osteoporotic vertebral compression fractures and 30 for vertebral tumors. All patients underwent image-guided treatment using CT and conventional fluoroscopy, with finger-touch identification of a puncture point within a small incision (1.5 to 2 cm). Partial or complete pain relief was achieved in 98% of patients within 24 h of treatment. Moreover, a significant improvement in functional mobility and reduction in analgesic use was observed. CT allowed the detection of cement leakage in 20.7% of the interventions. No bone cement leakages with neurologic symptoms were noted. All work channels were made only once, and bone cement was distributed near the center of the vertebral body. Our study confirms the efficacy of PKP treatment in osteoporotic and oncological patients. The combination of CT and C-arm fluoroscopy with finger-touch guidance reduces the risk of complications compared with conventional fluoroscopy alone, facilitates the detection of minor cement leakage, improves the operative procedure, and results in a favorable bone cement distribution.

Protective Effects of Liquiritin on the Brain of Rats with Alzheimer's Disease.

Background: Alzheimer's disease (AD) is a sort of nerve degenerative disease with clinical manifestation of memory damage and cognitive dysfunction. Its typical pathological change is the abnormal deposition of amyloid-beta (Aß). Method: In this study, a rat AD model with liquiritin (LQ) interference was established to observe the effects of LQ on the AD rats' behavioural memory and primary hippocampus cells. Results: Liquiritin had the effect of improving the rats' learning and memory ability, enhancing the activity of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in rats' brain tissues, increasing the antioxidant ability, protecting the primary cultured hippocampal neurons and inhibiting the apoptosis induced by Aß25-35. Conclusion: The protective effects of LQ can be related to the enhancement of antioxidase activity and clearance of oxygen radicals.

An updated meta-analysis of the signal transducer and activator of transcription 4 (STAT4) rs7574865 G/T polymorphism and rheumatoid arthritis risk in an Asian population.

OBJECTIVES: Signal transducer and activator of transcription 4 (STAT4) transmits signals induced by the cytokines interleukin (IL)-12, IL-23, and interferon (IFN)-γ, which play an important role in the development of rheumatoid arthritis (RA). Studies have shown conflicting results concerning the association between the rs7574865 G/T polymorphism in the STAT4 gene and RA in an Asian population. We have performed a meta-analysis to examine this relationship. METHOD: We searched PubMed and WanFang databases for all papers published up to 5 October 2013. Eight case-control studies with 6029 cases and 4685 controls were retrieved based on the search criteria for RA susceptibility related to the STAT4 rs7574865 G/T polymorphism. Risk ratios (RRs) and 95% confidence intervals (CIs) were used to assess the strength of this association. Publication bias was assessed using Begg's test. RESULTS: A significant association was found between the STAT4 rs7574865 G/T polymorphism and RA risk (e.g. GG+GT vs. TT: RR 0.96, 95% CI 0.95-0.97; GG+TT vs. GT: RR 0.94, 95% CI 0.91-0.97). Subgroup analysis of rheumatoid factor (RF) status revealed a protective relationship between the STAT4 rs7574865 G/T polymorphism and RF(+)/RF(-) RA risk. A similar relationship was detected in the anti-cyclic citrullinated peptide (CCP) status subgroup. No clear evidence of publication bias was detected in the overall analysis. CONCLUSIONS: Our study indicates that the STAT4 rs7574865 G/T polymorphism was significantly associated with a decreased RA risk in an Asian population.

Percutaneous transhepatic cholangiobiopsy to determine the pathological cause of anastomotic stenosis after cholangiojejunostomy for malignant obstructive jaundice.

AIM: To investigate the feasibility and advantages of cholangiobiopsy during percutaneous transhepatic cholangiography in the histopathological diagnosis of anastomotic stenosis after cholangiojejunostomy for malignant obstructive jaundice. MATERIALS AND METHODS: Using biopsy forceps, specimens were collected from the site of stenosis in patients with recurrent jaundice (n = 24) who had previously undergone cholangiojejunostomy for malignant obstructive jaundice. RESULTS: Stenosis occurred in all patients at the biliary-enteric anastomosis based on percutaneous transhepatic cholangiography, and was the location of the biopsy. Satisfactory specimens were obtained from 22 out of 24 patients. The sensitivity was 91.7% (22/24). Tumour tissue was obtained in 18 cases, confirming disease recurrence. Histopathological changes in four patients were diagnosed as fibroplasia and/or inflammation. These were considered cicatricial stenoses based on histopathological, imaging, and laboratory findings. The remaining two histopathology-negative patients were proven to have recurrent tumour based on imaging, laboratory, and follow-up data. No complications occurred during biopsy, including gastrointestinal haemorrhage or perforation. Either cholangial drainage and/or an inner stent was used following biopsy, which resulted in a noticeable decrease in jaundice postoperatively (p < 0.05). CONCLUSION: Percutaneous transhepatic cholangiobiopsy using biopsy forceps for the diagnosis of anastomotic stenosis after cholangiojejunostomy for malignant biliary obstructive jaundice is easy to perform and safe, with a high level of sensitivity. Interventional therapies, such as percutaneous transhepatic cholangial drainage and stent placement, can be performed concurrently, markedly improving the symptoms of patients with obstructive jaundice.

Multi-phosphine-chelated iron-carbide clusters via redox-promoted ligand exchange on an inert hexa-iron-carbide carbonyl cluster, [Fe6(µ6-C)(µ2-CO)4(CO)12]2.

We report the reactivity, structures and spectroscopic characterization of reactions of phosphine-based ligands (mono-, di- and tri-dentate) with iron-carbide carbonyl clusters. Historically, the archetype of this cluster class, namely [Fe6(µ6-C)(µ2-CO)4(CO)12]2-, can be prepared on a gram-scale but is resistant to simple ligand substitution reactions. This limitation has precluded the relevance of iron-carbide clusters relating to organometallics, catalysis and the nitrogenase active site cluster. Herein, we aimed to derive a simple and reliable method to accomplish CO → L (where L = phosphine or other general ligands) substitution reactions without harsh reagents or multi-step synthetic strategies. Ultimately, our goal was ligand-based chelation of an Fe n (µ n -C) core to achieve more synthetic control over multi-iron-carbide motifs relevant to the nitrogenase active site. We report that the key intermediate is the PSEPT-non-conforming cluster [Fe6(µ6-C)(CO)16] (2: 84 electrons), which can be generated in situ by the outer-sphere oxidation of [Fe6(µ6-C)(CO)16]2- (1: closo, 86 electrons) with 2 equiv. of [Fc]PF6. The reaction of 2 with excess PPh3 generates a singly substituted neutral cluster [Fe5(µ5-C)(CO)14PPh3] (4), similar to the reported reactivity of the substitutionally active cluster [Fe5(µ5-C)(CO)15] with monodentate phosphines (Cooke & Mays, 1990). In contrast, the reaction of 2 with flexible, bidentate phosphines (DPPE and DPPP) generates a wide range of unisolable products. However, the rigid bidentate phosphine bis(diphenylphosphino)benzene (bdpb) disproportionates the cluster into non-ligated Fe3-carbide anions paired with a bdpb-supported Fe(ii) cation, which co-crystallize in [Fe3(µ3-CH)(µ3-CO)(CO)9]2[Fe(MeCN)2(bdpb)2] (6). A successful reaction of 2 with the tripodal ligand Triphos generates the first multi-iron-chelated, authentic carbide cluster of the formula [Fe4(µ4-C)(κ3-Triphos)(CO)10] (9). DFT analysis of the key (oxidized) intermediate 2 suggests that its (µ6-C)Fe6 framework remains fully intact but is distorted into an axially compressed, 'ruffled' octahedron distinct from the parent closo cluster 1. Oxidation of the cluster in non-coordinating solvent allows for the isolation and crystallization of the CO-saturated, intact closo-analogue [Fe6(µ6-C)(CO)17] (3), indicating that the intact (µ6-C)Fe6 motif is retained during initial oxidation with [Fc]PF6. Overall, we demonstrate that redox modulation beneficially 'bends' Wade-Mingo's rules via the generation of electron-starved (non-PSEPT) intermediates, which are the key intermediates in promoting facile CO → L substitution reactions in iron-carbide-carbonyl clusters.

Osteoarthritis pain mechanisms: basic studies in animal models.

OBJECTIVE: Osteoarthritis (OA) is a complex and painful disease of the whole joint. At present there are no satisfying agents for treating OA. To promote OA research and improved treatment, this review summarizes current preclinical evidence on the development of OA. METHODS: Preclinical OA research was searched and key findings are summarized and commented. RESULTS: Mechanisms of OA-associated pain have been studied in rodent knee OA models produced by intra-knee injection of the chondrocyte glycolytic inhibitor mono-iodoacetate (MIA), surgery, or spontaneous development in some species. These models are clinically relevant in terms of histological damage and functional changes, and are used to study mechanisms underlying mechanical, thermal, ambulatory, body weight supporting-evoked, and ongoing OA pain. Recent peripheral, spinal, and supraspinal biochemical and electrophysiological studies in these models suggest that peripheral pro-inflammatory mediators and neuropeptides sensitize knee nociceptors. Spinal cytokines and neuropeptides promote OA pain, and peripheral and spinal cannabinoids inhibit OA pain respectively through cannabinoid-1 (CB1) and CB1/CB2 receptors. TRPV1 and metalloproteinases contribute and supraspinal descending facilitation of 5-hydroxytryptamine (5-HT)/5-HT 3 receptors may also contribute to OA pain. Conditioned place preference tests demonstrate that OA pain induces aversive behaviors, suggesting the involvement of brain. During OA, brain functional connectivity is enhanced, but at present it is unclear how this change is related to OA pain. CONCLUSION: Animal studies demonstrate that peripheral and central sensitization contributes to OA pain, involving inflammatory cytokines, neuropeptides, and a variety of chemical mediators. Interestingly, brainstem descending facilitation of 5-HT/5-HT3 receptors plays a role OA pain.

An updated meta-analysis of the Fc receptor-like 3 -169T/C polymorphism and rheumatoid arthritis risk.

OBJECTIVES: Published studies have shown conflicting results concerning the association between the -169T/C promoter polymorphism in the Fc receptor-like 3 (FCRL3) gene and rheumatoid arthritis (RA). In this study we conducted an up-to-date meta-analysis to examine the relationship. METHOD: We searched the PubMed database for all papers published up to 20 April 2012. Overall, 18 case-control studies with 12 620 cases and 12 613 controls were retrieved based on the search criteria for RA susceptibility related to the FCRL3 -169T/C polymorphism. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the strength of this association. Publication bias was assessed using the Egger test. RESULTS: We found that the FCRL3 -169T/C polymorphism increased the risk for RA overall in genetic models (allelic contrast: OR 1.09, 95% CI 1.03-1.14, p = 0.001; homozygote comparison: OR 1.20, 95% CI 1.08-1.34, p = 0.001; dominant genetic model: OR 1.03, 95% CI 1.01-1.05, p = 0.001). Stratified analysis by race also showed a significant positive association with Asians and Caucasians. Subgroup analysis of rheumatoid factor (RF) revealed a slightly positive relationship between the FCRL3 -169T/C polymorphism and RF-positive RA risk. No obvious evidence of publication bias was detected in the overall analysis. CONCLUSION: Our study indicates that the FCRL3 -169T/C polymorphism is significantly associated with increased RA risk.