Immunometric and functional measurement of endogenous vasoinhibin in human sera.

Introduction: Circulating levels of the antiangiogenic protein vasoinhibin, a fragment of prolactin, are of interest in vasoproliferative retinopathies, preeclampsia, and peripartum cardiomyopathy; however, it is difficult to determine the circulating levels of vasoinhibin due to the lack of quantitative assays. Methods: This study used human serum samples to assess the concentration and bioactivity of vasoinhibin using a novel enzyme-linked immunosorbent assay (ELISA) for human vasoinhibin, which employs an anti-vasoinhibin monoclonal antibody, a human umbilical vein endothelial cell (HUVEC) proliferation assay, and a chick chorioallantoic membrane (CAM) angiogenesis assay. Results: Serum samples from 17 pregnant women without (one group) and with preeclampsia and pregnancy induced hypertension (another group) demonstrated endogenous vasoinhibin concentrations in the range of 5-340 ng/ml. Immunoactive vasoinhibin levels were significantly higher in preeclampsia serum compared to healthy pregnancy serum (mean 63.09 ± 22.15 SD vs. 19.67 ± 13.34 ng/ml, p = 0.0003), as was the bioactive vasoinhibin level as determined by the HUVEC proliferation assay (56.12 ± 19.83 vs. 13.38 ± 4.88 ng/ml, p < 0.0001). There was a correlation between the concentration of vasoinhibin measured by ELISA and the HUVEC proliferation assay (Pearson r = 0.95, p < 0.0001). Healthy serum demonstrated a proangiogenic effect in the CAM assay (p < 0.05, compared to control), while serum from preeclamptic patients demonstrated an antiangiogenic effect (p < 0.05 vs. control), as did recombinant human vasoinhibin and a synthetic circular retro-inverse vasoinhibin analogue (CRIVi45-51). The antiangiogenic effects in the CAM assay and the inhibition of HUVEC proliferation were abolished by addition of the ELISA anti-vasoinhibin monoclonal antibody, but not by mouse IgG. Discussion: These results demonstrate the first quantitation of endogenous vasoinhibin in human sera and the elevation of it levels and antiangiogenic activity in sera from women with preeclampsia. The development and implementation of a quantitative assay for vasoinhibin overcomes a long-standing barrier and suggests the thorough clinical verification of vasoinhibin as a relevant biomarker.

Neuroblastoma suppressor of tumorigenicity 1 is a circulating protein associated with progression to end-stage kidney disease in diabetes.

Circulating proteins associated with transforming growth factor-ß (TGF-ß) signaling are implicated in the development of diabetic kidney disease (DKD). It remains to be comprehensively examined which of these proteins are involved in the pathogenesis of DKD and its progression to end-stage kidney disease (ESKD) in humans. Using the SOMAscan proteomic platform, we measured concentrations of 25 TGF-ß signaling family proteins in four different cohorts composed in total of 754 Caucasian or Pima Indian individuals with type 1 or type 2 diabetes. Of these 25 circulating proteins, we identified neuroblastoma suppressor of tumorigenicity 1 (NBL1, aliases DAN and DAND1), a small secreted protein known to inhibit members of the bone morphogenic protein family, to be most strongly and independently associated with progression to ESKD during 10-year follow-up in all cohorts. The extent of damage to podocytes and other glomerular structures measured morphometrically in 105 research kidney biopsies correlated strongly with circulating NBL1 concentrations. Also, in vitro exposure to NBL1 induced apoptosis in podocytes. In conclusion, circulating NBL1 may be involved in the disease process underlying progression to ESKD, and its concentration in circulation may identify subjects with diabetes at increased risk of progression to ESKD.

Characterization of Opa interacting protein 5 as a new biomarker and therapeutic target for oral cancer.

Oral cancer is a leading cause of cancer­related death worldwide. Current treatment for oral cancer includes surgery, radiotherapy, and chemotherapy; however, their effectiveness is still limited. To identify a new prognostic biomarker and therapeutic target for oral cancer, the Opa interacting protein 5 (OIP5), which plays an essential role in the proper segregation of chromosomes, was examined. Immunohistochemical staining using tissue microarrays indicated that OIP5 was expressed in 120 of 164 (73.2%) oral cancers but was minimally expressed in normal oral tissues. OIP5 expression was significantly associated with poor prognosis in patients with oral cancer. Overexpression of OIP5 enhanced the growth of oral cancer cells, whereas OIP5 knockdown using small interfering RNAs (siRNAs) significantly inhibited cell growth through cell cycle arrest at the G2/M phase. Suppression of OIP5 expression also induced senescence of oral cancer cells. Overall, the findings of the present study suggest that OIP5 may be a candidate prognostic biomarker and therapeutic target in oral cancer.

Serum levels of vasohibin-1 in type 2 diabetes mellitus patients with diabetic retinopathy.

CLINICAL RELEVANCE: To determine whether Vasohibin-1 which is a potential clinical biomarker is an independent risk factor in patients with diabetic retinopathy. BACKGROUND: Diabetic retinopathy (DR) is a common chronic microangiopathy in type 2 diabetes mellitus (T2DM). Vasohibin-1 (VASH-1) is an angiogenesis regulator that is closely related to pathological vascularization in DM. This study aimed to determine whether the serum levels of VASH-1 were related to the occurrence of DR in T2DM patients. METHODS: T2DM patients were divided into three groups: the nondiabetic retinopathy (NDR) group (n = 41), the nonproliferative diabetic retinopathy (NPRD) group (n = 40), and the proliferative diabetic retinopathy (PDR) group (n = 41). A control (CON) group consisting of 40 healthy subjects was also recruited. The serum levels of VASH-1 were measured by enzyme-linked immunosorbent assay kits. RESULTS: The concentration of VASH-1 in the CON groups was less significantly than that of the NDR, NPDR and PDR groups. (P < 0.05). Body mass index, fasting plasma glucose (FPG), hemoglobina1c (HbA1C), blood urea nitrogen (BUN) and diabetic durations were positively correlated with the serum concentration of VASH-1 (all P < 0.05). In univariate logistic regression analyses, the HbA1C, diabetic durations, HDL-c, eGFR and VASH1 were associated with the presence of diabetic retinopathy. Multivariate logistic regression analysis showed that duration of diabetes were significantly associated with diabetic retinopathy. CONCLUSION: We have shown that VASH-1 is associated with an increased risk of developing diabetic retinopathy. But the serum levels of VASH-1 are not independent risk factors for DR in T2DM.

Phosphorylation of CAP1 regulates lung cancer proliferation, migration, and invasion.

PURPOSE: Cyclase-associated protein 1 (CAP1) is a ubiquitous protein which regulates actin dynamics. Previous studies have shown that S308 and S310 are the two major phosphorylated sites in human CAP1. In the present study, we aimed to investigate the role of CAP1 phosphorylation in lung cancer. METHODS: Massive bioinformatics analysis was applied to determine CAP1's role in different cancers and especially in lung cancer. Lung cancer patients' serum and tissue were collected and analyzed in consideration of clinical background. CAP1 shRNA-lentivirus and siRNA were applied to CAP1 gene knockdown, and plasmids were constructed for CAP1 phosphorylation and de-phosphorylation. Microarray analysis was used for CAP1-associated difference analysis. Both in vitro and in vivo experiments were performed to investigate the roles of CAP1 phosphorylation and de-phosphorylation in lung cancer A549 cells. RESULTS: CAP1 is a kind of cancer-related protein. Its mRNA was overexpressed in most types of cancer tissues when compared with normal tissues. CAP1 high expression correlated with poor prognosis. Our results showed that serum CAP1 protein concentrations were significantly upregulated in non-small cell lung cancer (NSCLC) patients when compared with the healthy control group, higher serum CAP1 protein concentration correlated with shorter overall survival (OS) in NSCLC patients, and higher pCAP1 and CAP1 protein level were observed in lung cancer patients' tumor tissue compared with adjacent normal tissue. Knockdown CAP1 in A549 cells can inhibit proliferation and migration, and the effect is validated in H1975 cells. It can also lead to an increase ratio of F-actin/G-actin. In addition, phosphorylated S308 and S310 in CAP1 promoted lung cancer cell proliferation, migration, and metastasis both in vitro and in vivo. When de-phosphorylated, these two sites in CAP1 showed the opposite effect. Phosphorylation of CAP1 can promote epithelial-mesenchymal transition (EMT). CONCLUSION: These findings indicated that CAP1 phosphorylation can promote lung cancer proliferation, migration, and invasion. Phosphorylation sites of CAP1 might be a novel target for lung cancer treatment.

Effects of an acute exercise bout in hypoxia on extracellular vesicle release in healthy and prediabetic subjects.

The purpose of this study is to investigate exosome-like vesicle (ELV) plasma concentrations and markers of multivesicular body (MVB) biogenesis in skeletal muscle in response to acute exercise. Seventeen healthy [body mass index (BMI): 23.5 ± 0.5 kg·m-2] and 15 prediabetic (BMI: 27.3 ± 1.2 kg·m-2) men were randomly assigned to two groups performing an acute cycling bout in normoxia or hypoxia ([Formula: see text] 14.0%). Venous blood samples were taken before (T0), during (T30), and after (T60) exercise, and biopsies from m. vastus lateralis were collected before and after exercise. Plasma ELVs were isolated by size exclusion chromatography, counted by nanoparticle tracking analysis (NTA), and characterized according to international standards, followed by expression analyses of canonical ELV markers in skeletal muscle. In the healthy normoxic group, the total number of particles in the plasma increased during exercise from T0 to T30 (+313%) followed by a decrease from T30 to T60 (-53%). In the same group, an increase in TSG101, CD81, and HSP60 protein expression was measured after exercise in plasma ELVs; however, in the prediabetic group, the total number of particles in the plasma was not affected by exercise. The mRNA content of TSG101, ALIX, and CD9 was upregulated in skeletal muscle after exercise in normoxia, whereas CD9 and CD81 were downregulated in hypoxia. ELV plasma abundance increased in response to acute aerobic exercise in healthy subjects in normoxia, but not in prediabetic subjects, nor in hypoxia. Skeletal muscle analyses suggested that this tissue did not likely play a major role of the exercise-induced increase in circulating ELVs.

Circulating TRB3 and GRP78 levels in type 2 diabetes patients: crosstalk between glucose homeostasis and endoplasmic reticulum stress.

PURPOSE: Endoplasmic reticulum (ER) stress is implicated in the development of type 2 diabetes mellitus (T2DM) and insulin resistance. Tribbles homolog 3 (TRB3) is a pseudokinase upregulated by ER stress and hyperglycemia. Glucose-regulated protein 78 (GRP78) is an ER stress protein that is overexpressed under ER stress conditions. The current study aimed to investigate serum levels of TRB3 and GRP78, as an ER stress marker, in T2DM patients and their correlations with the metabolic profile. METHODS: Fifty-seven patients with type 2 diabetes and 23 healthy control subjects were evaluated for serum concentrations of TRB3, GRP78, and AGEs by enzyme-linked immunosorbent assay (ELISA). Fasting plasma glucose (FPG), HbA1c, lipid profile, TNF-α and insulin were also measured, and insulin resistance was calculated using a homeostasis model assessment of insulin resistance (HOMA-IR). RESULTS: Serum concentrations of TRB3, GRP78, AGEs, and TNF-α were significantly higher in T2DM patients compared to the healthy controls. Moreover, a statistically significant positive correlation was observed between plasma concentrations of TRB3 and FPG, HbA1c, HOMA-IR, and AGE. GRP78 levels were positively correlated with HbA1c and AGEs. There was also a positive correlation between GRP78 and TRB3. AGEs levels were positively correlated with the levels of FPG, HbA1c, HOMA-IR, and TNF-α. CONCLUSION: The current findings suggest that TRB3 and GRP78 may contribute to the pathogenesis of T2DM and might be considered as a therapeutic targets for the treatment of this disease.

Upregulation of miR-29c-3p Hinders Melanoma Progression by Inhibiting CDCA4 Expression.

OBJECTIVE: To investigate the expression and regulation mechanism of miR-29c-3p and cell division cycle associated 4 (CDCA4) in melanoma (MM). Data and Methods. Fifty-nine patients with MM admitted to our hospital were enrolled as the MM group. They were followed up for 3 years to analyze the prognostic factors; meanwhile, 51 healthy subjects were allocated into a normal group. MM cell lines (M21 and C8161) were transfected with miR-29c-3p-mimics, miR-29c-3p-inhibitor, miR-NC, si-CDCA4, and sh-CDCA4. The expression of miR-29c-3p, CDCA4, Bax, Caspase3, Bcl-2, N-cadherin, vimentin, and E-cadherin was quantified, and cell proliferation, migration, invasion, and apoptosis, as well as epithelial-mesenchymal transition (EMT), were determined. RESULTS: Serum miR-29c-3p was lowly expressed and CDCA4 was highly expressed in the MM group. The area under the curve (AUC) of both for diagnosing MM was greater than 0.9. miR-29c-3p and CDCA4 were related to regional lymph node staging (N staging), distant metastasis (M staging), tumor diameter, and pathological differentiation. Low miR-29c-3p and high CDCA4 were associated with poor prognosis of MM. Overexpression of miR-29c-3p and suppression of CDCA4 hindered cell proliferation, migration, invasion, and expression of Bax, Caspase3, N-cadherin, and vimentin, but cell apoptosis and expression of Bcl-2 and E-cadherin were enhanced. Dual-luciferase reporter (DLR) assay confirmed the targeted relationship between miR-29c-3p and CDCA4. After miR-29c-3p-mimics+sh-CDCA4 was transfected into M21 and C8161 cells, the proliferation, invasion, and apoptosis were not different from those in the miR-NC group transfected with unrelated sequences. CONCLUSION: Overexpression of miR-29c-3p suppresses CDCA4 expression and decreases proliferation, migration, invasion, apoptosis, and EMT of MM cells, thus hindering MM progression.

Clinically Applicable Serum Biomarkers Among 14 Candidates Associated With Recurrence of Stage II and III Colorectal Cancer.

BACKGROUND/AIM: We evaluated the predictive value of candidate serum biomarkers for recurrence in stage II and III colorectal cancer (CRC) after curative surgery. PATIENTS AND METHODS: A total of 33 and 120 patients with CRC with or without recurrence at 5 years after curative surgery were included in the training set and the validation set, respectively. Possible serum biomarkers were examined for associations with CRC recurrence using receiver operating characteristics (ROC) curve analysis. RESULTS: In the training set, the expression levels of the 14 biomarkers were compared according to recurrence. Among them, five biomarkers that had significantly different expression levels were validated in 60 patients with recurrence at 5 years after curative surgery and 60 patients without. Multivariate analysis showed that natural log-transformed values of carcinoembryonic antigen (CEA), cyclin-dependent kinase regulatory subunit 2 (CKS2), 2'-5'-oligoadenylate synthetase 2 (OAS2), and autophagy-related gene 5 (ATG5) in preoperative serum were significantly related to recurrence. ROC analysis showed that these biomarkers were able to discriminate patients with recurrence from those without (area under the curve=0.828, 95% confidence interval=0.755-0.990). CONCLUSION: Preoperative serum levels of CEA, CKS2, OAS2 and ATG5 were independent risk factors for recurrence. A combination of serum CEA, CKS2, OAS2 and ATG5 predicted tumor recurrence well in patients with stage II and III CRC.

Assessing acute myeloid leukemia susceptibility in rearrangement-driven patients by DNA breakage at topoisomerase II and CCCTC-binding factor/cohesin binding sites.

An initiating DNA double strand break (DSB) event precedes the formation of cancer-driven chromosomal abnormalities, such as gene rearrangements. Therefore, measuring DNA breaks at rearrangement-participating regions can provide a unique tool to identify and characterize susceptible individuals. Here, we developed a highly sensitive and low-input DNA break mapping method, the first of its kind for patient samples. We then measured genome-wide DNA breakage in normal cells of acute myeloid leukemia (AML) patients with KMT2A (previously MLL) rearrangements, compared to that of nonfusion AML individuals, as a means to evaluate individual susceptibility to gene rearrangements. DNA breakage at the KMT2A gene region was significantly greater in fusion-driven remission individuals, as compared to nonfusion individuals. Moreover, we identified select topoisomerase II (TOP2)-sensitive and CCCTC-binding factor (CTCF)/cohesin-binding sites with preferential DNA breakage in fusion-driven patients. Importantly, measuring DSBs at these sites, in addition to the KMT2A gene region, provided greater predictive power when assessing individual break susceptibility. We also demonstrated that low-dose etoposide exposure further elevated DNA breakage at these regions in fusion-driven AML patients, but not in nonfusion patients, indicating that these sites are preferentially sensitive to TOP2 activity in fusion-driven AML patients. These results support that mapping of DSBs in patients enables discovery of novel break-prone regions and monitoring of individuals susceptible to chromosomal abnormalities, and thus cancer. This will build the foundation for early detection of cancer-susceptible individuals, as well as those preferentially susceptible to therapy-related malignancies caused by treatment with TOP2 poisons.

Serum levels of cytoskeleton remodeling proteins and their mRNA expression in tumor tissue of metastatic laryngeal and hypopharyngeal cancers.

Actin-binding proteins (ABPs) and various signaling systems are involved in the process of squamous cell carcinoma of the larynx and hypopharynx (SCCLH) metastasis. The clinical significance of these proteins has not yet been determined. We analyzed the relationship between the mRNA levels of cofilin 1 (CFL1), profilin 1 (PFN1), adenylyl cyclase-associated protein 1 (CAP1), SNAI1 and RND3 and SCCLH metastasis. The serum levels of the above ABPs were estimated and the relationship between them and their mRNA expressions was analyzed. The expression levels of ABP mRNAs were measured by real-time RT-PCR in paired tissue samples taken from 54 patients with SCCLH (T1-4N0-1M0). Expression analysis was performed using the 2-ΔΔCT method. The levels of ABPs in the blood serum were measured by ELISA. Statistical analysis was carried out using the SPSS Statistica 20.0 software package. No significant difference in the mRNA gene expression in tumor tissue of patients with T1-3N0M0 SCCLH and patients with T2-4N1-2M0 SCCLH was found. High expression of RND3 mRNA was accompanied by an increase in mRNA expression of all studied ABPs. In the blood serum of T2-4N1-2M0 patients, the level of PFN1 was lower by 21% and the level of CAP1 was higher by 75% than those observed in T1-4N0M0 patients. The data obtained showed that RND3 is involved in the regulation of molecular cascades of SCCLH metastasis. PFN1 and CAP1 serum levels can be good classifiers of metastases in patients with SCCLH.

Development of Vasoinhibin-Specific Monoclonal Antibodies.

Vasoinhibin is a protein hormone with antiangiogenic, antivasodilatatory, and antivasopermeability effects generated by the proteolytic cleavage of prolactin. The discovery of its role in diabetic retinopathy and peripartum cardiomyopathy led to the evaluation of new pharmacological treatments in clinical interventional trials. However, the quantitative evaluation of vasoinhibin in biological samples from patients has not been possible due to the lack of vasoinhibin-specific antibodies. Recently, loop 1 of vasoinhibin was identified to have a different three-dimensional structure compared to PRL, and thus to contain vasoinhibin-specific epitopes. Here, we report the development of two sets of vasoinhibin-specific monoclonal antibodies against two neighboring regions of the vasoinhibin loop 1. An experimental sandwich ELISA with two monoclonal anti-vasoinhibin antibodies was developed, which had no cross-reactivity to recombinant human full-length prolactin. The ELISA had a quantitation limit of 100 ng/ml, and intra-assay- and inter-assay coefficients of variation of 12.5% and 14%, respectively. The evaluation of 15 human serum samples demonstrated concentrations of below limit of detection (n=3), below limit of quantitation (n=1) and between 0.23 µg/ml (230 ng/ml) to 605 µg/ml (n=12) in the quantifiable range. Despite the high specificity of the monoclonal-monoclonal antibody sandwiches which discriminate vasoinhibin from PRL, there might be cross-reactivities by serum proteins other than vasoinhibin. A fully established vasoinhibin ELISA may support diagnostic and therapeutic measures in vascular diseases.

Serum vasohibin-1 levels: A potential marker of dermal and pulmonary fibrosis in systemic sclerosis.

Vasohibin-1 (VASH-1) is a potent anti-angiogenic factor mainly produced by endothelial cells. In addition, VASH-1 prevents TGF-ß-dependent activation of renal fibroblasts. Since systemic sclerosis (SSc) is an autoimmune disease characterized by vasculopathy and fibrosis of multiple organs, VASH-1 may be involved in the development of this disease. In this study, we investigated the potential role of VASH-1 in SSc by evaluating the clinical correlation between serum VASH-1 levels and the expression of VASH-1 in SSc-involved skin. Serum VASH-1 levels were higher in SSc patients, especially those with diffuse cutaneous involvement, than in healthy controls and positively correlated with skin score. Furthermore, SSc patients with interstitial lung disease had significantly elevated levels of serum VASH-1 as compared to those without. Importantly, serum VASH-1 levels correlated inversely with both the percentage of predicted vital capacity and the percentage of predicted diffusion lung capacity for carbon monoxide and positively with serum KL-6 levels, but not serum surfactant protein D levels. In SSc-involved skin, VASH1 mRNA was remarkably upregulated compared with healthy control skin, but the major source of VASH-1 was not clear. Fli1 deficiency, a predisposing factor inducing SSc-like endothelial properties, did not affect VASH-1 expression in human dermal microvascular endothelial cells. Collectively, these results suggest that VASH-1 upregulation in the skin and sera is linked to dermal and pulmonary fibrotic changes in SSc, while the contribution of VASH-1 to SSc vasculopathy seems to be limited.

Low G0S2 gene expression levels in peripheral blood may be a genetic marker of acute myocardial infarction in patients with stable coronary atherosclerotic disease: A retrospective clinical study.

BACKGROUND: The G0/G1 switch 2 (G0S2) gene is closely related to lipolysis, cell proliferation, apoptosis, oxidative phosphorylation, and the development of a variety of tumors. The aim of the present study was to expand the sample size to confirm the relationship between the expression of the G0S2 gene in peripheral blood and acute myocardial infarction (AMI) based on previous gene chip results. METHODS: Three hundred patients were initially selected, of which 133 were excluded in accordance with the exclusion criteria. Peripheral blood leukocytes were collected from 92 patients with AMI and 75 patients with stable coronary atherosclerotic disease (CAD). mRNA expression levels of G0S2 in peripheral blood leukocytes was measured by RT-PCR, and protein expression levels by Western blot analysis. The results of these assays in the 2 groups were compared. RESULTS: mRNA expression levels of GOS2 in the peripheral blood leukocytes of patients with AMI were 0.41-fold lower than those of patients with stable CAD (P < .05), and GOS2 protein expression levels were 0.45-fold lower. Multivariate logistic regression analysis indicated that low expression levels of the G0S2 gene increased the risk of AMI by 2.08-fold in stable CAD patients. CONCLUSIONS: G0S2 gene expression in the peripheral blood leukocytes of AMI patients was lower than that of stable CAD patients. Low G0S2 gene expression in peripheral blood leukocytes is an independent risk factor for AMI in stable CAD patients.

Intra-platelet serotonin and YAP contributed to poor prognosis of hepatocellular carcinoma.

AIMS: Intra-platelet 5-HT (IP 5-HT) and YAP exhibit an important role in hepatocellular carcinoma (HCC). The aim of the study was to investigate whether IP 5-HT and YAP could affect the progression and prognosis of HCC. METHODS: 5-HT level and YAP expression were measured and were compared between HCC patients and control patients. By grouping HCC patients, we analyzed clinical indicators and survival. The predictive nomogram was established by R software according to the risk factors obtained from multivariate analysis. RESULTS: Higher IP 5-HT level and higher YAP expression were associated with poorer prognosis. In addition, they were also associated with BCLC stages. Higher IP 5-HT was found to be related with higher international normalized ratio (INR) (p = 0.040), more death (p = 0.015) and higher YAP expression (p < 0.001). Similarly, higher YAP expression was proved to be associated with lower platelet counts (PLT) (p = 0.032), smaller tumor size (p = 0.017), more death (p < 0.001) and higher IP 5-HT (p < 0.001). In addition, alkaline phosphatase (ALP), YAP and tumor size were proved to be independent risk factors. By using risk factors, we have established a prognostic prediction nomogram for HCC patients. In the prognostic prediction nomogram, patients with higher scores would have poorer prognosis. CONCLUSIONS: IP 5-HT and YAP might affect the progression and prognosis of HCC through synergistic effect. Moreover, IP 5-HT might affect HCC by regulating YAP expression. Thus, both of them might be potential therapeutic targets. By establishing the prognostic prediction nomogram, we could improve the prediction system.

Evaluation of BRD4 levels in patients with early-onset preeclampsia.

OBJECTIVE: This study aimed to detect Bromodomain Containing Protein 4 (BRD4) concentrations in the serum of early-onset preeclamptic patients and compare them with the healthy control group. MATERIAL AND METHODS: This prospective case-control study was performed from June 2019 to December 2019. Of the 80 pregnant patients included in the study, we enrolled 40 patients with early-onset preeclampsia as the study group, and 40 normotensive healthy gestational age- and gravidity-matched patients with normal blood pressure without proteinuria as the control group. Demographic characteristics, amount of proteinuria, and serum BRD4 concentrations were recorded. RESULTS: Maternal serum BRD4 concentrations were significantly higher in patients with preeclampsia (39.10 ± 42.14 ng/mL) compared to the participants in the control group (13.64 ± 7.24 ng/mL, p < 0.001). There was a positive intermediate correlation between serum BRD4 levels and the amount of proteinuria (r = 0.447, p = 0.006). CONCLUSION: Maternal serum BRD4 levels were significantly higher in preeclamptic patients compared to healthy pregnant women. Also, the amount of proteinuria was positively correlated with serum BRD4 levels. Although this preliminary study shows increased BRD4 levels in preeclampsia, its utility as a biomarker must be clarified.

circRNA circ-MYBL2 is a novel tumor suppressor and potential biomarker in multiple myeloma.

Currently, multiple myeloma (MM) is still an incurable disease. Deciphering its pathogenesis will bring new targets for clinical diagnosis and treatment. In the present study, we identified a MM-associated circular RNA (circRNA), circ-MYBL2, which was dramatically decreased in MM tissue and serum samples in comparison to normal samples. Low circ-MYBL2 level was closely correlated with high clinical stage and unfavorable outcome, and serum circ-MYBL2 had excellent accuracy in diagnosing MM. Exogenous circ-MYBL2 expression notably repressed MM cell viability, DNA synthesis and cell cycle progression. Further exploration revealed that circ-MYBL2 exerted the tumor-inhibiting effect by affecting the phosphorylation level of its linear isoform, in which circ-MYBL2 facilitated the binding of Cyclin F to MYBL2, dampening MYBL2 phosphorylation and activation, thereby inhibiting the transcription of a number of well-known proliferation-related oncogenes. Importantly, overexpression of circ-MYBL2 significantly reduced the tumor size of subcutaneous xenografts in nude mice. Taken together, our data unveil a regulatory mechanism linking circ-MYBL2 and its host gene mediated by Cyclin F, providing a potential diagnostic, prognostic and therapeutic target for MM patients.

Establishment of international autoantibody reference standards for the detection of autoantibodies directed against PML bodies, GW bodies, and NuMA protein.

Objectives: Reference materials are important in the standardization of autoantibody testing and only a few are freely available for many known autoantibodies. Our goal was to develop three reference materials for antibodies to PML bodies/multiple nuclear dots (MND), antibodies to GW bodies (GWB), and antibodies to the nuclear mitotic apparatus (NuMA). Methods: Reference materials for identifying autoantibodies to MND (MND-REF), GWB (GWB-REF), and NuMA (NuMA-REF) were obtained from three donors and validated independently by seven laboratories. The sera were characterized using indirect immunofluorescence assay (IFA) on HEp-2 cell substrates including two-color immunofluorescence using antigen-specific markers, western blot (WB), immunoprecipitation (IP), line immunoassay (LIA), addressable laser bead immunoassay (ALBIA), enzyme-linked immunosorbent assay (ELISA), and immunoprecipitation-mass spectrometry (IP-MS). Results: MND-REF stained 6-20 discrete nuclear dots that colocalized with PML bodies. Antibodies to Sp100 and PML were detected by LIA and antibodies to Sp100 were also detected by ELISA. GWB-REF stained discrete cytoplasmic dots in interphase cells, which were confirmed to be GWB using two-color immunofluorescence. Anti-Ge-1 antibodies were identified in GWB-REF by ALBIA, IP, and IP-MS. All reference materials produced patterns at dilutions of 1:160 or greater. NuMA-REF produced fine speckled nuclear staining in interphase cells and staining of spindle fibers and spindle poles. The presence of antibodies to NuMA was verified by IP, WB, ALBIA, and IP-MS. Conclusions: MND-REF, GWB-REF, and NuMA-REF are suitable reference materials for the corresponding antinuclear antibodies staining patterns and will be accessible to qualified laboratories.

Paraclinical serum markers as aids in the diagnosis of autoimmune encephalitis.

Expert opinion suggests the presence ANA and thyroid antibodies may be helpful to diagnosis autoimmune encephalitis (AE). This study investigates the sensitivity of these serum markers in a cohort of 26 patients with AE. TPO-Ab, TG-Ab and ANA (titer ≥1:320) were present in 45%, 35% and 32% of patients tested, respectively. The prevalence of TPO-Ab (11.3%), TG-Ab (10.4%) and ANA ≥1:320 (3.3%) has been previously reported in disease-free populations. Although these antibodies represent non-specific markers of autoimmunity, this study demonstrated that TPO-Ab, TG-Ab and ANA were significantly elevated in AE compared to disease-free populations (p < .001, p = .003, p < .001, respectively).

Decreased Peripheral Blood ALKBH5 Correlates with Markers of Autoimmune Response in Systemic Lupus Erythematosus.

Although it has been proved that the epigenetic modification of DNA and histones is involved in the pathogenesis of systemic lupus erythematosus (SLE), there is no study to explore whether the modification of N6-methyladenosine (m6A) in RNA is involved. In this study, the mRNA levels of m6A "writers" (METTL3, MTEEL14, and WTAP), "erasers" (FTO and ALKBH5), and "readers" (YTHDF2) in peripheral blood were determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The results demonstrated that the mRNA levels of METTL3, WTAP, FTO, ALKBH5, and YTHDF2 in peripheral blood from SLE patients were significantly decreased. The levels of ALKBH5 mRNA in SLE patients were associated with anti-dsDNA, antinucleosome, rash, and ulceration. Multivariate logistic regression analysis showed that the level of ALKBH5 mRNA in peripheral blood is a risk factor of SLE (P < 0.001). Moreover, our results suggested that there was a positive correlation between m6A"writers" (METTL3 and WTAP), "erasers" (FTO and ALKBH5), and "readers" (YTHDF2) in SLE patients. This study suggests that the mRNA level of ALKBH5 in peripheral blood may be involved in the pathogenesis of SLE.