Acidic Environment Induces Pyroptosis of NLRP3 Inflammatory Vesicle-Mediated Nucleus Pulposus Cells Through Up-Regulation of POSTN Expression

SUMMARY: Intervertebral disc degeneration (IVDD) is induced by nucleus pulposus (NP) dysfunction as a result of massive loss of NP cells. It has been reported that the acidic microenvironment of the intervertebral disc (IVD) can induce NP cell pyroptosis, and that up-regulation of periostin (POSTN) expression has a negative effect on NP cell survival. However, the relationship between the acidic environment, POSTN expression level and NP cell pyroptosis is unclear. Therefore, the aim of this study was to explore the relationship between acidic environment and POSTN expression level in NP cells, as well as the effect of POSTN in acidic environment on NP cell pyroptosis. NP cells were obtained from the lumbar vertebrae of Sprague Dawley (SD) male rats. These cells were divided into normal and acidic groups according to whether they were exposed to 6 mM lactic acid solution. And NP cells in the acidic group were additionally divided into three groups: (1) Blank group: no transfection; (2) NC group: cells transfected with empty vector plasmid; (3) sh-POSTN group: cells transfected with sh-POSTN plasmid to knock down the expression level of POSTN. Quantitative real-time PCR (qRT-PCR) and western blot was performed to assess the expression of POSTN at the mRNAand protein levels. CCK8 was used to evaluate cell survival. Western blot, in addition, was performed to examine acid-sensing ion channels (ASIC)-related proteins. And pyroptosis was detected by ELISA and western blot. The expression level of POSTN was significantly increased in NP cells in acidic environment. Knockdown of POSTN expression promoted the survival of NP cells in acidic environment and reduced the protein levels of ASIC3 and ASIC1a in NP cells. Moreover, knockdown of POSTN expression decreased the pyroptosis proportion of NP cells and the levels of pro-inflammatory cytokines interleukin (IL)-1β and IL-18. The levels of pyroptosis-related proteins NLRP3, ASC, cleaved-Caspase-1, and cleaved-GSDMD were also affected by the decreased POSTN expression. The extracellular acidic environment created by lactic acid solution activated NLRP3 inflammatory vesicle-induced caspase-1 to get involved in NP cell pyroptosis by up-regulating POSTN expression.

La degeneración del disco intervertebral (DDIV) es inducida por una disfunción del núcleo pulposo (NP) como resultado de una pérdida masiva de células NP. Se ha informado que el microambiente ácido del disco intervertebral (DIV) puede inducir la piroptosis de las células NP y que la regulación positiva de la expresión de periostina (POSTN) tiene un efecto negativo en la supervivencia de las células NP. Sin embargo, la relación entre el ambiente ácido, el nivel de expresión de POSTN y la piroptosis de las células NP es poco clara. Por lo tanto, el objetivo de este estudio fue explorar la relación entre el ambiente ácido y el nivel de expresión de POSTN en células NP, así como el efecto de POSTN en ambiente ácido sobre la piroptosis de las células NP. Las células NP se obtuvieron de las vertebras lumbares de ratas macho Sprague Dawley (SD). Estas células se dividieron en grupos normales y ácidos según se expusieron a una solución de ácido láctico 6 mM. Las células NP en el grupo ácido se dividieron adicionalmente en tres grupos: (1) Grupo en blanco: sin transfección; (2) grupo NC: células transfectadas con plásmido vector vacío; (3) grupo sh-POSTN: células transfectadas con plásmido sh-POSTN para reducir el nivel de expresión de POSTN. Se realizó una PCR cuantitativa en tiempo real (qRT-PCR) y una transferencia Western para evaluar la expresión de POSTN en los niveles de ARNm y proteína. Se utilizó CCK8 para evaluar la supervivencia celular. Además, se realizó una transferencia Western para examinar las proteínas relacionadas con los canales iónicos sensibles al ácido (ASIC). La piroptosis se detectó mediante ELISA y Western blot. El nivel de expresión de POSTN aumentó significativamente en células NP en ambiente ácido. La eliminación de la expresión de POSTN promovió la supervivencia de las células NP en un ambiente ácido y redujo los niveles de proteína de ASIC3 y ASIC1a en las células NP. Además, la eliminación de la expresión de POSTN disminuyó la proporción de piroptosis de las células NP y los niveles de citocinas proinflamatorias interleucina (IL) - 1β e IL-18. Los niveles de proteínas relacionadas con la piroptosis NLRP3, ASC, Caspasa-1 escindida y GSDMD escindida también se vieron afectados por la disminución de la expresión de POSTN. El ambiente ácido extracelular creado por la solución de ácido láctico activó la caspasa-1 inducida por vesículas inflamatorias NLRP3 para involucrarse en la piroptosis de las células NP mediante la regulación positiva de la expresión de POSTN.

The weight-adjusted-waist index predicts all-cause and cardiovascular mortality in general US adults.

BACKGROUND: The authors examined the relationship between Weight-adjusted Waist Index (WWI) and all-cause and cardiovascular mortality among adults in the US. METHODS: This prospective cohort study included 26,882 individuals who participated in the National Health and Nutrition Examination Survey (NHANES) from 2005 through 2014. WWI was calculated as waist circumference divided by the square root of weight. The main outcomes of this study were all-cause mortality and cardiovascular mortality. Mortality status and cause of death were determined by NHANES-linked National Death Index records through December 31, 2015. Cox proportional hazard models and Kaplan-Meier analysis were used to estimate Hazard Ratios (HR) and 95% CIs for mortality for all causes and cardiovascular diseases. RESULTS: A total of 26,882 participants with a mean WWI of 10.89 ± 0.01, of whom 49.23% were male. The average follow-up time was 68.95 ± 1.07 months, and 1870 participants were determined as deceased (4.99%), including 349 cardiovascular death (0.88%). The Kaplan-Meier analysis demonstrated a significant difference in all-cause and cardiovascular mortality between patients with WWI <11.33 and ≥11.33 (both log-rank test p < 0.0001). The fully adjusted Cox proportional hazard model indicated that a higher WWI level (≥ 11.33) was associated with an increased 95% risk for cardiovascular mortality (HR = 1.95, 95% CI 1.30‒2.93) and 68% risk for all-cause death (HR = 1.68, 95% CI 1.41‒2.00) compared with the counterparts. CONCLUSIONS: Elevated WWI levels were associated with a higher risk of cardiovascular mortality and all-cause mortality independently.

A novel nomogram integrated with PDL1 and CEA to predict the prognosis of patients with gastric cancer.

INTRODUCTION: This study aimed to develop a prognostic nomogram for patients with gastric cancer (GC) based on the levels of programmed death 1 ligand 1 (PDL1) and carcinoembryonic antigen (CEA). METHODS: The nomogram was developed using data from a primary cohort of 247 patients who had been clinicopathologically diagnosed with GC, as well as a validation cohort of 63 patients. Furthermore, the nomogram divided the patients into three different risk groups for overall survival (OS)-the low-risk, middle-risk, and high-risk groups. Univariate and multivariate Cox hazard analyses were used to determine all of the factors included in the model. Decision curve analysis and receiver operating characteristic (ROC) curves were used to assess the accuracy of the nomogram. RESULTS: The Kaplan-Meier survival analysis revealed that metastasis stage, clinical stage, and CEA and PDL1 levels were predictors for progress-free survival (PFS) and OS of patients with GC. Metastasis stage, clinical stage, and CEA and PDL1 levels were found to be independent risk factors for the PFS and OS of patients with GC in a multivariate analysis, and the nomogram was based on these factors. The concordance index of the nomogram was 0.763 [95% confidence interval (CI) 0.740-0.787]. The area under the concentration-time curve of the nomogram model was 0.81 (95% CI 0.780-0.900). According to the decision curve analysis and ROC curves, the nomogram model had a higher overall net efficiency in forecasting OS than clinical stage, CEA and PDL1 levels. CONCLUSION: In conclusion, we proposed a novel nomogram that integrated PDL1 and CEA, and the proposed nomogram provided more accurate and useful prognostic predictions for patients with GC.

The weight-adjusted-waist index predicts all-cause and cardiovascular mortality in general US adults

Abstract Background The authors examined the relationship between Weight-adjusted Waist Index (WWI) and all-cause and cardiovascular mortality among adults in the US. Methods This prospective cohort study included 26,882 individuals who participated in the National Health and Nutrition Examination Survey (NHANES) from 2005 through 2014. WWI was calculated as waist circumference divided by the square root of weight. The main outcomes of this study were all-cause mortality and cardiovascular mortality. Mortality status and cause of death were determined by NHANES-linked National Death Index records through December 31, 2015. Cox proportional hazard models and Kaplan-Meier analysis were used to estimate Hazard Ratios (HR) and 95% CIs for mortality for all causes and cardiovascular diseases. Results A total of 26,882 participants with a mean WWI of 10.89 ± 0.01, of whom 49.23% were male. The average follow-up time was 68.95 ± 1.07 months, and 1870 participants were determined as deceased (4.99%), including 349 cardiovascular death (0.88%). The Kaplan-Meier analysis demonstrated a significant difference in all-cause and cardiovascular mortality between patients with WWI <11.33 and ≥11.33 (both log-rank testp < 0.0001). The fully adjusted Cox proportional hazard model indicated that a higher WWI level (≥ 11.33) was associated with an increased 95% risk for cardiovascular mortality (HR = 1.95, 95% CI 1.30‒2.93) and 68% risk for all-cause death (HR = 1.68, 95% CI 1.41‒2.00) compared with the counterparts. Conclusions Elevated WWI levels were associated with a higher risk of cardiovascular mortality and all-cause mortality independently.

Atmospheric organophosphate esters in the Western Antarctic Peninsula over 2014-2018: Occurrence, temporal trend and source implication.

Organophosphate esters (OPEs) were comprehensively investigated in the air samples collected using high-volume samplers near the Chinese Great Wall Station in the Western Antarctic Peninsula over the period of 2014-2018. The concentrations of ∑8OPEs (gaseous + particle phases) ranged from 33.9 to 404 pg/m3 with a geometric mean of 119 ± 12.0 pg/m3. Tris [(2R)-1-chloro-2-propyl] phosphate (TCIPP) and tris(2-chloroethyl) phosphate (TCEP) dominated in the gaseous phase, while tris-n-butyl phosphate (TnBP) was the most abundant OPEs in the particle phase, followed by TCIPP and TCEP. An apparently temporal trend was observed for atmospheric ∑8OPEs over the five years, with a doubling time of about 3.8 years, which indicated continuous inputs of OPEs into the sampling area. The particle-bound ∑8OPEs accounted for 45% of the total, generally lower than that reported in the Arctic. Gas-particle partitioning modeling suggested that the partitioning of OPEs with higher logKOA values approached the steady state in the Antarctic air. The back-trajectory modeling showed that high levels of OPEs were usually associated with air inputs from the northwest of the peninsula. This suggested that long-range transport from South America, which was confirmed by the no temperature dependencies of OPEs concentrations (excluding TnBP). Nevertheless, a steady high level of particle-bound TnBP implied local sources in the Western Antarctic Peninsula, which required further investigation in future works.

Endometrial thickness is associated with incidence of small-for-gestational-age infants in fresh in vitro fertilization-intracytoplasmic sperm injection and embryo transfer cycles.

OBJECTIVE: To investigate whether endometrial thickness (EMT) is associated with adverse obstetric and neonatal outcomes in fresh in vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET) cycles. DESIGN: Retrospective cohort study. SETTING: University-based reproductive medical center. PATIENT(S): Women under the age of 42 years who underwent IVF/ICSI treatment and received fresh ET in our unit from January 2017 to December 2018, resulting in a live singleton birth. INTERVENTION(S): Controlled ovarian hyperstimulation and IVF/ICSI; fresh ET. MAIN OUTCOME MEASURE(S): Birth weight, gestational age, small for gestational age (SGA), large for gestational age (LGA), placenta previa, placental abruption, hypertensive disorders, and gestational diabetes mellitus. RESULT(S): The risk of being born SGA was statistically significantly increased in the EMT ≤7.5 mm group compared with those from the EMT >12 mm group (adjusted odds ratio [aOR] 2.391; 95% confidence interval [CI], 1.155-4.950). Moreover, maternal body mass index, secondary infertility, preterm delivery, and hypertensive disorders were all independent predictors for SGA. The mean birth weights of singletons in women with EMT ≤7.5 mm were lower than in the groups with EMT >7.5-12 mm and EMT >12 mm (3.25 ± 0.56 kg vs. 3.38 ± 0.51 kg and 3.39 ± 0.53 kg, respectively). CONCLUSION(S): After fresh IVF/ICSI-ET, the risk of SGA was increased twofold in women with EMT ≤7.5 mm compared with women with EMT >12 mm. We suggest that women with a thin EMT after obtaining a pregnancy by IVF should receive improved prenatal care to reduce the risk of delivering a SGA infant.

High-throughput sleep phenotyping produces robust and heritable traits in Diversity Outbred mice and their founder strains.

STUDY OBJECTIVES: This study describes high-throughput phenotyping strategies for sleep and circadian behavior in mice, including examinations of robustness, reliability, and heritability among Diversity Outbred (DO) mice and their eight founder strains. METHODS: We performed high-throughput sleep and circadian phenotyping in male mice from the DO population (n = 338) and their eight founder strains: A/J (n = 6), C57BL/6J (n = 14), 129S1/SvlmJ (n = 6), NOD/LtJ (n = 6), NZO/H1LtJ (n = 6), CAST/EiJ (n = 8), PWK/PhJ (n = 8), and WSB/EiJ (n = 6). Using infrared beam break systems, we defined sleep as at least 40 s of continuous inactivity and quantified sleep-wake amounts and bout characteristics. We developed assays to measure sleep latency in a new environment and during a modified Murine Multiple Sleep Latency Test, and estimated circadian period from wheel-running experiments. For each trait, broad-sense heritability (proportion of variability explained by all genetic factors) was derived in founder strains, while narrow-sense heritability (proportion of variability explained by additive genetic effects) was calculated in DO mice. RESULTS: Phenotypes were robust to different inactivity durations to define sleep. Differences across founder strains and moderate/high broad-sense heritability were observed for most traits. There was large phenotypic variability among DO mice, and phenotypes were reliable, although estimates of heritability were lower than in founder mice. This likely reflects important nonadditive genetic effects. CONCLUSIONS: A high-throughput phenotyping strategy in mice, based primarily on monitoring of activity patterns, provides reliable and heritable estimates of sleep and circadian traits. This approach is suitable for discovery analyses in DO mice, where genetic factors explain some proportion of phenotypic variation.

p53 Up-regulated Modulator of Apoptosis Induction Mediates Acetaminophen-Induced Necrosis and Liver Injury in Mice.

Acetaminophen (APAP) overdose is one of the leading causes of hepatotoxicity and acute liver failure in the United States. Accumulating evidence suggests that hepatocyte necrosis plays a critical role in APAP-induced liver injury (AILI). However, the mechanisms of APAP-induced necrosis and liver injury are not fully understood. In this study, we found that p53 up-regulated modulator of apoptosis (PUMA), a B-cell lymphoma-2 (Bcl-2) homology domain 3 (BH3)-only Bcl-2 family member, was markedly induced by APAP in mouse livers and in isolated human and mouse hepatocytes. PUMA deficiency suppressed APAP-induced mitochondrial dysfunction and release of cell death factors from mitochondria, and protected against APAP-induced hepatocyte necrosis and liver injury in mice. PUMA induction by APAP was p53 independent, and required receptor-interacting protein kinase 1 (RIP1) and c-Jun N-terminal kinase (JNK) by transcriptional activation. Furthermore, a small-molecule PUMA inhibitor, administered after APAP treatment, mitigated APAP-induced hepatocyte necrosis and liver injury. Conclusion: Our results demonstrate that RIP1/JNK-dependent PUMA induction mediates AILI by promoting hepatocyte mitochondrial dysfunction and necrosis, and suggest that PUMA inhibition is useful for alleviating acute hepatotoxicity attributed to APAP overdose.

Comparative proteomics analysis of chronic atrophic gastritis: changes of protein expression in chronic atrophic gastritis without Helicobacter pylori infection

Chronic atrophic gastritis (CAG) is a very common gastritis and one of the major precursor lesions of gastric cancer, one of the most common cancers worldwide. The molecular mechanism underlying CAG is unclear, but its elucidation is essential for the prevention and early detection of gastric cancer and appropriate intervention. A combination of two-dimensional gel electrophoresis and mass spectrometry was used in the present study to analyze the differentially expressed proteins. Samples from 21 patients (9 females and 12 males; mean age: 61.8 years) were used. We identified 18 differentially expressed proteins in CAG compared with matched normal mucosa. Eight proteins were up-regulated and 10 down-regulated in CAG when compared with the same amounts of proteins in individually matched normal gastric mucosa. Two novel proteins, proteasome activator subunit 1 (PSME1), which was down-regulated in CAG, and ribosomal protein S12 (RPS12), which was up-regulated in CAG, were further investigated. Their expression was validated by Western blot and RT-PCR in 15 CAG samples matched with normal mucosa. The expression level of RPS12 was significantly higher in CAG than in matched normal gastric mucosa (P < 0.05). In contrast, the expression level of PSME1 in CAG was significantly lower than in matched normal gastric mucosa (P < 0.05). This study clearly demonstrated that there are some changes in protein expression between CAG and normal mucosa. In these changes, down-regulation of PSME1 and up-regulation of RPS12 could be involved in the development of CAG. Thus, the differentially expressed proteins might play important roles in CAG as functional molecules.

Comparative proteomics analysis of chronic atrophic gastritis: changes of protein expression in chronic atrophic gastritis without Helicobacter pylori infection.

Chronic atrophic gastritis (CAG) is a very common gastritis and one of the major precursor lesions of gastric cancer, one of the most common cancers worldwide. The molecular mechanism underlying CAG is unclear, but its elucidation is essential for the prevention and early detection of gastric cancer and appropriate intervention. A combination of two-dimensional gel electrophoresis and mass spectrometry was used in the present study to analyze the differentially expressed proteins. Samples from 21 patients (9 females and 12 males; mean age: 61.8 years) were used. We identified 18 differentially expressed proteins in CAG compared with matched normal mucosa. Eight proteins were up-regulated and 10 down-regulated in CAG when compared with the same amounts of proteins in individually matched normal gastric mucosa. Two novel proteins, proteasome activator subunit 1 (PSME1), which was down-regulated in CAG, and ribosomal protein S12 (RPS12), which was up-regulated in CAG, were further investigated. Their expression was validated by Western blot and RT-PCR in 15 CAG samples matched with normal mucosa. The expression level of RPS12 was significantly higher in CAG than in matched normal gastric mucosa (P < 0.05). In contrast, the expression level of PSME1 in CAG was significantly lower than in matched normal gastric mucosa (P < 0.05). This study clearly demonstrated that there are some changes in protein expression between CAG and normal mucosa. In these changes, down-regulation of PSME1 and up-regulation of RPS12 could be involved in the development of CAG. Thus, the differentially expressed proteins might play important roles in CAG as functional molecules.

A functional ABCA1 gene variant is associated with low HDL-cholesterol levels and shows evidence of positive selection in Native Americans.

It has been suggested that the higher susceptibility of Hispanics to metabolic disease is related to their Native American heritage. A frequent cholesterol transporter ABCA1 (ATP-binding cassette transporter A1) gene variant (R230C, rs9282541) apparently exclusive to Native American individuals was associated with low high-density lipoprotein cholesterol (HDL-C) levels, obesity and type 2 diabetes in Mexican Mestizos. We performed a more extensive analysis of this variant in 4405 Native Americans and 863 individuals from other ethnic groups to investigate genetic evidence of positive selection, to assess its functional effect in vitro and to explore associations with HDL-C levels and other metabolic traits. The C230 allele was found in 29 of 36 Native American groups, but not in European, Asian or African individuals. C230 was observed on a single haplotype, and C230-bearing chromosomes showed longer relative haplotype extension compared with other haplotypes in the Americas. Additionally, single-nucleotide polymorphism data from the Human Genome Diversity Panel Native American populations were enriched in significant integrated haplotype score values in the region upstream of the ABCA1 gene. Cells expressing the C230 allele showed a 27% cholesterol efflux reduction (P< 0.001), confirming this variant has a functional effect in vitro. Moreover, the C230 allele was associated with lower HDL-C levels (P = 1.77 x 10(-11)) and with higher body mass index (P = 0.0001) in the combined analysis of Native American populations. This is the first report of a common functional variant exclusive to Native American and descent populations, which is a major determinant of HDL-C levels and may have contributed to the adaptive evolution of Native American populations.