LncRNA-mir3471-limd1 regulatory network plays critical roles in HIBD.

The purpose of this study was to identify the target genes of tcon_00044595, elucidate its activation site, and provide novel insights into the pathogenesis and treatment of neonatal hypoxic-ischemic brain damage (HIBD). Through homologous blast analysis, we identified predicted target sequences in the neighboring regions of the long non-coding RNA (lncRNA) tcon_00044595, suggesting that limd1 is its target gene. Starbase was utilized to identify potential candidate microRNAs associated with the lncRNA. The interaction between the candidate microRNAs and limd1 was investigated and validated using various experimental methods including in vitro cell culture, cell transfection, dual fluorescence reporter detection system, and real-time PCR. Homology alignment analysis revealed that the lncRNA tcon_00044595 exhibited a 246 bp homologous sequence at the 3' end of the adjacent limd1 gene, with a conservation rate of 68%. Analysis conducted on Starbase online identified three potential microRNA candidates: miR-3471, miR-883a-5p, and miR-214-3p. Intracellular expression of the limd1 gene was significantly down-regulated upon transfection with miR-3471, while the other two microRNAs did not produce noticeable effects. Luciferase reporter assays identified two interaction sites (UTR-1, UTR-2) between miR-3471 and the limd1 3'UTR, with UTR-1 exhibiting a strong influence. Further CCK8 assay indicated a protective role of miR-3471 during low oxygen stroke in HIBD. The potential regulatory relationship between lncRNA (tcon_00044595), miR-3471, and the target gene limd1 suggests their involvement in the occurrence and development of HIBD, providing new insights for investigating the underlying mechanisms and exploring targeted therapeutic approaches for HIBD.

PHLDA1 contributes to hypoxic ischemic brain injury in neonatal rats via inhibiting FUNDC1-mediated mitophagy.

Hypoxia-ischemia (HI) is one of the main causes of neonatal brain injury. Mitophagy has been implicated in the degradation of damaged mitochondria and cell survival following neonatal brain HI injury. Pleckstrin homology-like domain family A member 1 (PHLDA1) plays vital roles in the progression of various disorders including the regulation of oxidative stress, the immune responses and apoptosis. In the present study we investigated the role of PHLDA1 in HI-induced neuronal injury and further explored the mechanisms underlying PHLDA1-regulated mitophagy in vivo and in vitro. HI model was established in newborn rats by ligation of the left common carotid artery plus exposure to an oxygen-deficient chamber with 8% O2 and 92% N2. In vitro studies were conducted in primary hippocampal neurons subjected to oxygen and glucose deprivation/-reoxygenation (OGD/R). We showed that the expression of PHLDA1 was significantly upregulated in the hippocampus of HI newborn rats and in OGD/R-treated primary neurons. Knockdown of PHLDA1 in neonatal rats via lentiviral vector not only significantly ameliorated HI-induced hippocampal neuronal injury but also markedly improved long-term cognitive function outcomes, whereas overexpression of PHLDA1 in neonatal rats via lentiviral vector aggravated these outcomes. PHLDA1 knockdown in primary neurons significantly reversed the reduction of cell viability and increase in intracellular reactive oxygen species (ROS) levels, and attenuated OGD-induced mitochondrial dysfunction, whereas overexpression of PHLDA1 decreased these parameters. In OGD/R-treated primary hippocampal neurons, we revealed that PHLDA1 knockdown enhanced mitophagy by activating FUNDC1, which was abolished by FUNDC1 knockdown or pretreatment with mitophagy inhibitor Mdivi-1 (25 µM). Notably, pretreatment with Mdivi-1 or the knockdown of FUNDC1 not only increased brain infarct volume, but also abolished the neuroprotective effect of PHLDA1 knockdown in HI newborn rats. Together, these results demonstrate that PHLDA1 contributes to neonatal HI-induced brain injury via inhibition of FUNDC1-mediated neuronal mitophagy.

Predictive value of early amplitude integrated electroencephalogram (aEEG) in sleep related problems in children with perinatal hypoxic-ischemia (HIE).

BACKGROUND: While great attention has been paid to motor and cognitive impairments in children with neonatal Hypoxic-Ischemic Encephalopathy (HIE), sleep related circadian rhythm problems, although commonly present, are often neglected. Subsequently, no early clinical indicators have been reported to correlate with sleep-related circadian dysfunction during development. METHODS: In this study, we first analyzed patterns of the amplitude integrated electroencephalogram (aEEG) in a cohort of newborns with various degrees of HIE. Next, during follow-ups, we collected information of sleep and circadian related problems in these patients and performed correlation analysis between aEEG parameters and different sleep/circadian disorders. RESULTS: A total of 101 neonates were included. Our results demonstrated that abnormal aEEG background pattern is significantly correlated with circadian rhythmic (r = 0.289, P = 0.01) and breathing issues during sleep (r = 0.237, P = 0.037). In contrast, the establishment of sleep-wake cycle (SWC) showed no correlation with sleep/circadian problems. Detailed analysis showed that summation of aEEG score, along with low base voltage (r = 0.272, P = 0.017 and r = -0.228, P = 0.048, respectively), correlates with sleep circadian problems. In contrast, background pattern (BP) score highly correlates with sleep breathing problem (r = 0.319, P = 0.004). CONCLUSION: Abnormal neonatal aEEG pattern is correlated with circadian related sleep problems. Our study thus provides novel insights into predictive values of aEEG in sleep-related circadian problems in children with HIE.

The role of microglia mediated pyroptosis in neonatal hypoxic-ischemic brain damage.

Neonatal hypoxic-ischemic encephalopathy (HIE) often leads to neonatal death or severe, irreversible neurological deficits. Pathologically, the occurrence of massive cell death and subsequent inflammation suggested that pyroptosis, an inflammation associated programed cell death, might play a role in HIE. Here, by measuring changes of key molecules in pyroptosis pathway in HIE patients, we discovered that their elevation levels tightly correlate with the severity of HIE. Next, we demonstrated that application of MCC950, a small molecule to inhibit NLRP3 inflammasome and thus pyroptosis, substantially alleviated pyroptosis and the injury severity in rats with neonatal hypoxic-ischemic brain damage (HIBD). Mechanistically, we showed that NLRP-3/caspase-1/GSDMD axis is required for microglia pyroptosis and activation. Our data demonstrated that microglia mediated pyroptosis played a crucial role in neonatal HIE, which shed lights into the development of intervention avenues targeting pyroptosis to treat HIE and traumatic brain injuries.

The role of a lncRNA (TCONS_00044595) in regulating pineal CLOCK expression after neonatal hypoxia-ischemia brain injury.

A common, yet often neglectable, feature of neonatal hypoxic-ischemic brain damage (HIBD) is circadian rhythm disorders resulted from pineal gland dysfunction. Our previous work demonstrated that miRNAs play an important role in regulating key circadian genes in the pineal gland post HIBD [5,21]. In current study, we sought out to extend our investigation by profiling expression changes of pineal long non-coding RNAs (lncRNAs) upon neonatal HIBD using RNA-Seq. After validating lncRNA changes, we showed that one lncRNA: TCONS_00044595 is highly enriched in the pineal gland and exhibits a circadian expression pattern. Next, we performed bioinformatic analysis to predict the lncRNA-miRNA regulatory network and identified 168 miRNAs that potentially targetlncRNA TCONS_00044595. We further validated the bona fide interaction between one candidate miRNA: miR-182, a known factor to regulate pineal Clock expression, and lncRNA TCONS_00044595. Finally, we showed that suppression of lncRNA TCONS_00044595 alleviated the CLOCK activation both in the cultured pinealocytes under OGD conditions and in the pineal gland post HIBD in vivo. Our study thus shed light into novel mechanisms of pathophysiology of pineal dysfunction post neonatal HIBD.

[Role of microglial pyroptosis in hypoxic-ischemic brain damage].

OBJECTIVE: To investigate the role of microglial pyroptosis in hypoxic-ischemic brain damage. METHODS: An oxygen-glucose deprivation/reoxygenation (OGD/R) model of rat microglial cells were cultured in vitro. Western blot was used to measure the expression of the pyroptosis-related proteins caspase-1, interleukin-1ß (IL-1ß), and N-terminal gasdermin D (GSDMD-N) at 0, 1, 3, 6, 12, and 24 hours after OGD/R. After the microglial cells were transfected with lentivirus-mediated silenced gasdermin D (GSDMD), immunofluorescence assay and Western blot were used to measure the transfection rate of GSDMD. Microglial cell lines were divided into three groups: normal control, negative control, and LV-sh_GSDMD (lentivirus-mediated GSDMD silencing). CCK-8 assay and LDH kit were used to observe the effect of GSDMD silencing on the viability and toxicity of microglial cells at 24 hours after OGD/R. Western blot was used to observe the effect of GSDMD silencing on the levels of caspase-1, GSDMD-N, and IL-1ß in the microglial cells at 24 hours after OGD/R. RESULTS: The expression levels of the pyroptosis-related proteins caspase-1, GSDMD-N, and IL-1ß in microglial cells were upregulated since 0 hour after OGD/R and reached the peak levels at 24 hours. A microglial cell model of lentivirus-mediated GSDMD silencing was successfully constructed. At 24 hours after OGD/R, compared with the normal control group, the GSDMD silencing group had a significant increase in the cell viability and a significant reduction in the cytotoxicity (P<0.05), as well as significant reductions in the protein expression levels of caspase-1, GSDMD-N, and IL-1ß in microglial cells (P<0.05). CONCLUSIONS: Lentivirus silencing of the key substrate protein for pyroptosis GSDMD can alleviate hypoxic-ischemic brain damage, suggesting that microglial pyroptosis aggravates hypoxic-ischemic brain damage.

A meta-analysis of dietary carbohydrate intake and inflammatory bowel disease risk: evidence from 15 epidemiology studies.

BACKGROUND AND PURPOSE: epidemiological studies that assess the association of dietary total carbohydrate intake and inflammatory bowel disease risk (IBD) have yielded controversial results. Therefore, this study of various epidemiological studies was conducted in order to explore this relationship. METHODS: a systematic literature search of the PubMed, Embase, Web of Science and Medline databases was performed up to September 2017. Cohort, case-control or cross-sectional design studies were included that reported the association of dietary carbohydrate intake and IBD risk. Summary odds ratio (OR) and the corresponding 95% CI were calculated using the random effects model. RESULTS: a total of eight articles with 15 individual studies that included 1,361 cases were eligible according to the inclusion criteria. Dietary carbohydrate intake had a non-significant relationship with the risk of IBD (OR = 1.091, 95% CI = 0.817-1.455, I2 = 31.6%, pfor heterogeneity = 0.116). The pooled OR and 95% CI for ulcerative colitis (UC) and Crohn's disease (CD) with regard to dietary carbohydrate intake was 1.167 (0.777-1.752) and 1.010 (0.630-1.618), respectively. These associations were also non-significant in both European and Asia populations. CONCLUSIONS: a higher dietary total carbohydrate intake had a non-significant relationship with IBD risk. Further studies with large populations are needed to verify this relationship.

[Imaging Findings of Pulmonary Cryptococcosis].

Pulmonary cryptococcosis(PC)is a fungal infection that can be easily misdiagnosed due to its non-specific clinical features and imaging findings.This article reviews the imaging findings of PC,their relationships with pathology and immune status,and differential diagnosis of PC with other disease,so as to improve the clinical management of PC.

Molecular mechanism of G1 arrest and cellular senescence induced by LEE011, a novel CDK4/CDK6 inhibitor, in leukemia cells.

BACKGROUND: Overexpression of cyclin D1 dependent kinases 4 and 6 (CDK4/6) is a common feature of many human cancers including leukemia. LEE011 is a novel inhibitor of both CDK4 and 6. To date, the molecular function of LEE011 in leukemia remains unclear. METHODS: Leukemia cell growth and apoptosis following LEE011 treatment was assessed through CCK-8 and annexin V/propidium iodide staining assays. Cell senescence was assessed by ß-galactosidase staining and p16INK4a expression analysis. Gene expression profiles of LEE011 treated HL-60 cells were investigated using an Arraystar Human LncRNA array. Gene ontology and KEGG pathway analysis were then used to analyze the differentially expressed genes from the cluster analysis. RESULTS: Our studies demonstrated that LEE011 inhibited proliferation of leukemia cells and could induce apoptosis. Hoechst 33,342 staining analysis showed DNA fragmentation and distortion of nuclear structures following LEE011 treatment. Cell cycle analysis showed LEE011 significantly induced cell cycle G1 arrest in seven of eight acute leukemia cells lines, the exception being THP-1 cells. ß-Galactosidase staining analysis and p16INK4a expression analysis showed that LEE011 treatment can induce cell senescence of leukemia cells. LncRNA microarray analysis showed 2083 differentially expressed mRNAs and 3224 differentially expressed lncRNAs in LEE011-treated HL-60 cells compared with controls. Molecular function analysis showed that LEE011 induced senescence in leukemia cells partially through downregulation of the transcriptional expression of MYBL2. CONCLUSIONS: We demonstrate for the first time that LEE011 treatment results in inhibition of cell proliferation and induction of G1 arrest and cellular senescence in leukemia cells. LncRNA microarray analysis showed differentially expressed mRNAs and lncRNAs in LEE011-treated HL-60 cells and we demonstrated that LEE011 induces cellular senescence partially through downregulation of the expression of MYBL2. These results may open new lines of investigation regarding the molecular mechanism of LEE011 induced cellular senescence.

The direct analysis of drug distribution of rotigotine-loaded microspheres from tissue sections by LESA coupled with tandem mass spectrometry.

The direct analysis of drug distribution of rotigotine-loaded microspheres (RoMS) from tissue sections by liquid extraction surface analysis (LESA) coupled with tandem mass spectrometry (MS/MS) was demonstrated. The RoMS distribution in rat tissues assessed by the ambient LESA-MS/MS approach without extensive or tedious sample pretreatment was compared with that obtained by a conventional liquid chromatography tandem mass spectrometry (LC-MS/MS) method in which organ excision and subsequent solvent extraction were commonly employed before analysis. Results obtained from the two were well correlated for a majority of the organs, such as muscle, liver, stomach, and hippocampus. The distribution of RoMS in the brain, however, was found to be mainly focused in the hippocampus and striatum regions as shown by the LESA-imaged profiles. The LESA approach we developed is sensitive enough, with an estimated LLOQ at 0.05 ng/mL of rotigotine in brain tissue, and information-rich with minimal sample preparation, suitable, and promising in assisting the development of new drug delivery systems for controlled drug release and protection. Graphical abstract Workflow for the LESA-MS/MS imaging of brain tissue section after intramuscular RoMS administration.

Melatonin alleviates brain and peripheral tissue edema in a neonatal rat model of hypoxic-ischemic brain damage: the involvement of edema related proteins.

BACKGROUND: Previous studies have indicated edema may be involved in the pathophysiology following hypoxic-ischemic encephalopathy (HIE), and melatonin may exhibit neuro-protection against brain insults. However, little is known regarding the mechanisms that involve the protective effects of melatonin in the brain and peripheral tissues after HIE. The present study aimed to examine the effects of melatonin on multiple organs, and the expression of edema related proteins in a neonatal rat model of hypoxic-ischemic brain damage (HIBD). METHODS: One hundred ninety-two neonatal rats were randomly divided into three subgroups that underwent a sham surgery or HIBD. After the HIBD or sham-injury, the rats received an intraperitoneal injection of melatonin or an equal volume vehicle, respectively. We investigated the effects of melatonin on brain, kidney, and colon edema via histological examination and the expression of edema related proteins, including AQP-4, ZO-1 and occludin, via qPCR and western blot. RESULTS: Our data indicated (1) Melatonin reduced the histological injury in the brain and peripheral organs induced by HIBD as assessed via H-E staining and transmission electron microscopy. (2) Melatonin alleviated the HIBD-induced cerebral edema characterized by increased brain water content. (3) HIBD induced significant changes of edema related proteins, such as AQP-4, ZO-1 and occludin, and these changes were partially reversed by melatonin treatment. CONCLUSIONS: These findings provide substantial evidence that melatonin treatment has protective effects on the brain and peripheral organs after HIBD, and the edema related proteins, AQP4, ZO-1, and occludin, may indirectly contribute tothe mechanism of the edema protection by melatonin.

[Expression profiles of miRNA-182 and Clock mRNA in the pineal gland of neonatal rats with hypoxic-ischemic brain damage].

OBJECTIVE: To study the changes of miRNA expression in the pineal gland of neonatal rats with hypoxic-ischemic brain damage (HIBD) and the possible roles of miRNA in the pathogenesis of circadian rhythm disturbance after HIBD. METHODS: Seven-day-old Sprague-Dawley (SD) rats were randomly divided into 2 groups: HIBD and sham-operated. HIBD was induced according to the Rice-Vannucci method. The pineal glands were obtained 24 hours after the HIBD event. The expression profiles of miRNAs were determined using GeneChip technigue and quantitative real-time PCR (RT-PCR). Then the miRNA which was highly expressed was selected. The expression levels of the chosen miRNA were detected in different tissues (lungs, intestines, stomach, kidneys, cerebral cortex, pineal gland). RT-PCR analysis was performed to measure the expression profiles of the chosen miRNA and the targeted gene Clock mRNA in the pineal gland at 0, 24, 48 and 72 hours after HIBD. RESULTS: miRNA-182 that met the criteria was selected by GeneChip and RT-PCR. miRNA-182 was highly expressed in the pineal gland. Compared with the sham-operated group, the expression of miRNA-182 was significantly up-regulated in the pineal gland at 24 and 48 hours after HIBD (P<0.05). Compared with the sham-operated group, Clock mRNA expression in the HIBD group increased at 0 hour after HIBD, decreased at 48 hours after HIBD and increased at 72 hours after HIBD (P<0.05). CONCLUSIONS: miRNA-182 may be involved in the pathogenesis of circadian rhythm disturbance after HIBD.

Analyzing the gene expression profile of anaplastic histology Wilms' tumor with real-time polymerase chain reaction arrays.

BACKGROUND: Wilms' tumor (WT) is one of the most common malignant neoplasms of the urinary tract in children. Anaplastic histology (unfavorable histology) accounts for about 10% of whole WTs, and it is the single most important histologic predictor of treatment response and survival in patients with WT; however, until now the molecular basis of this phenotype is not very clearly. METHODS: A real-time polymerase chain reaction (PCR) array was designed and tested. Next, the gene expression profile of pediatric anaplastic histology WT and normal adjacent tissues were analyzed. These expression data were anlyzed with Multi Experiment View (MEV) cluster software further. Datasets representing genes with altered expression profiles derived from cluster analyses were imported into the Ingenuity Pathway Analysis Tool (IPA). RESULTS: 88 real-time PCR primer pairs for quantitative gene expression analysis of key genes involved in pediatric anaplastic histology WT were designed and tested. The gene expression profile of pediatric anaplastic histology WT is significantly different from adjacent normal controls; we identified 15 genes that are up-regulated and 16 genes that are down-regulated in the former. To investigate biological interactions of these differently regulated genes, datasets representing genes with altered expression profiles were imported into the IPA for further analysis, which revealed three significant networks: Cancer, Hematological Disease, and Gene Expression, which included 27 focus molecules and a significance score of 43. The IPA analysis also grouped the differentially expressed genes into biological mechanisms related to Cell Death and Survival 1.15E(-12), Cellular Development 2.84E(-11), Cellular Growth and Proliferation 2.84E(-11), Gene Expression 4.43E(-10), and DNA Replication, Recombination, and Repair 1.39E(-07). The important upstream regulators of pediatric anaplastic histology WT were TP53 and TGFß1 signaling (P = 1.15E(-14) and 3.79E(-13), respectively). CONCLUSIONS: Our study demonstrates that the gene expression profile of pediatric anaplastic histology WT is significantly different from adjacent normal tissues with real-time PCR array. We identified some genes that are dysregulated in pediatric anaplastic histology WT for the first time, such as HDAC7, and IPA analysis showed the most important pathways for pediatric anaplastic histology WT are TP53 and TGFß1 signaling. This work may provide new clues into the molecular mechanisms behind pediatric anaplastic histology WT.

Hypermethylation of the GATA binding protein 4 (GATA4) promoter in Chinese pediatric acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is the second-most common form of leukemia in children. Aberrant DNA methylation patterns are a characteristic feature of AML. GATA4 has been suggested to be a tumor suppressor gene regulated by promoter hypermethylation in various types of human cancers although the expression and promoter methylation of GATA4 in pediatric AML is still unclear. METHODS: Transcriptional expression levels of GATA4 were evaluated by semi-quantitative and real-time PCR. Methylation status was investigated by methylation-specific PCR (MSP) and bisulfate genomic sequencing (BGS). The prognostic significance of GATA4 expression and promoter methylation was assessed in 105 cases of Chinese pediatric acute myeloid leukemia patients with clinical follow-up records. RESULTS: MSP and BGS analysis showed that the GATA4 gene promoter is hypermethylated in AML cells, such as the HL-60 and MV4-11 human myeloid leukemia cell lines. 5-Aza treatment significantly upregulated GATA4 expression in HL-60 and MV4-11 cells. Aberrant methylation of GATA4 was observed in 15.0 % (3/20) of the normal bone marrow control samples compared to 56.2 % (59/105) of the pediatric AML samples. GATA4 transcript levels were significantly decreased in AML patients (33.06 ± 70.94; P = 0.011) compared to normal bone marrow/idiopathic thrombocytopenic purpura controls (116.76 ± 105.39). GATA4 promoter methylation was correlated with patient leukocyte counts (WBC, white blood cells) (P = 0.035) and minimal residual disease MRD (P = 0.031). Kaplan-Meier survival analysis revealed significantly shorter overall survival time in patients with GATA4 promoter methylation (P = 0.014). CONCLUSIONS: Epigenetic inactivation of GATA4 by promoter hypermethylation was observed in both AML cell lines and pediatric AML samples; our study implicates GATA4 as a putative tumor suppressor gene in pediatric AML. In addition, our findings imply that GATA4 promoter methylation is correlated with WBC and MRD. Kaplan-Meier survival analysis revealed significantly shorter overall survival in pediatric AML with GATA4 promoter methylation but multivariate analysis shows that it is not an independent factor. However, further research focusing on the mechanism of GATA4 in pediatric leukemia is required.

Molecular targeting of the oncoprotein PLK1 in pediatric acute myeloid leukemia: RO3280, a novel PLK1 inhibitor, induces apoptosis in leukemia cells.

Polo-like kinase 1 (PLK1) is highly expressed in many cancers and therefore a biomarker of transformation and potential target for the development of cancer-specific small molecule drugs. RO3280 was recently identified as a novel PLK1 inhibitor; however its therapeutic effects in leukemia treatment are still unknown. We found that the PLK1 protein was highly expressed in leukemia cell lines as well as 73.3% (11/15) of pediatric acute myeloid leukemia (AML) samples. PLK1 mRNA expression was significantly higher in AML samples compared with control samples (82.95 ± 110.28 vs. 6.36 ± 6.35; p < 0.001). Kaplan-Meier survival analysis revealed that shorter survival time correlated with high tumor PLK1 expression (p = 0.002). The 50% inhibitory concentration (IC50) of RO3280 for acute leukemia cells was between 74 and 797 nM. The IC50 of RO3280 in primary acute lymphocytic leukemia (ALL) and AML cells was between 35.49 and 110.76 nM and 52.80 and 147.50 nM, respectively. RO3280 induced apoptosis and cell cycle disorder in leukemia cells. RO3280 treatment regulated several apoptosis-associated genes. The regulation of DCC, CDKN1A, BTK, and SOCS2 was verified by western blot. These results provide insights into the potential use of RO3280 for AML therapy; however, the underlying mechanisms remain to be determined.

Metallothionein III (MT3) is a putative tumor suppressor gene that is frequently inactivated in pediatric acute myeloid leukemia by promoter hypermethylation.

BACKGROUND: Acute myeloid leukemia (AML) is the second most common form of leukemia in children. Aberrant DNA methylation patterns are a characteristic feature in various tumors, including AML. Metallothionein III (MT3) is a tumor suppresser reported to show promoter hypermethylated in various cancers. However, the expression and molecular function of MT3 in pediatric AML is unclear. METHODS: Eleven human leukemia cell lines and 41 pediatric AML samples and 20 NBM/ITP (Norma bone marrow/Idiopathic thrombocytopenic purpura) control samples were analyzed. Transcription levels of MT3 were evaluated by semi-quantitative and real-time PCR. MT3 methylation status was determined by methylation specific PCR (MSP) and bisulfite genomic sequencing (BSG). The molecular mechanism of MT3 was investigated by apoptosis assays and PCR array analysis. RESULTS: The MT3 promoter was hypermethylated in leukemia cell lines. More CpG's methylated of MT3 was observed 39.0% pediatric AML samples compared to 10.0% NBM controls. Transcription of MT3 was also significantly decreased in AML samples compared to NBM/ITP controls (P < 0.001); patients with methylated MT3 exhibited lower levels of MT3 expression compared to those with unmethylated MT3 (P = 0.049). After transfection with MT3 lentivirus, proliferation was significantly inhibited in AML cells in a dose-dependent manner (P < 0.05). Annexin V assay showed that apoptosis was significantly upregulated MT3-overexpressing AML cells compared to controls. Real-time PCR array analysis revealed 34 dysregulated genes that may be implicated in MT3 overexpression and apoptosis in AML, including FOXO1. CONCLUSION: MT3 may be a putative tumor suppressor gene in pediatric AML. Epigenetic inactivation of MT3 via promoter hypermethylation was observed in both AML cell lines and pediatric AML samples. Overexpression of MT3 may inhibit proliferation and induce apoptosis in AML cells. FOXO1 was dysregulated in MT3-overexpressing cells, offering an insight into the mechanism of MT3-induced apoptosis. However, further research is required to determine the underlying molecular details.

Suppression of Microglial ERO1a Alleviates Inflammation and Enhances the Efficacy of Rehabilitative Training After Ischemic Stroke.

Microglia mediated inflammation plays a crucial role in cellular events and functional recovery post ischemic stroke. In the current study, we profiled the proteome changes of microglia treated with oxygen and glucose deprivation (OGD). Bioinformatics analysis identified that differentially expressed proteins (DEPs) were enriched in pathways associated with oxidate phosphorylation and mitochondrial respiratory chain at both 6h and 24h post OGD. We next focused on one validated target named endoplasmic reticulum oxidoreductase 1 alpha (ERO1a) to study its role in stroke pathophysiology. We showed that over-expression of microglial ERO1a exacerbated inflammation, cell apoptosis and behavioral outcomes post middle cerebral artery occlusion (MCAO). In contrast, suppression of microglial ERO1a significantly reduced activation of both microglia and astrocyte, along with cell apoptosis. Furthermore, knocking down microglial ERO1a improved the efficacy of rehabilitative training and enhanced the mTOR activity in spared corticospinal neurons. Our study provided novel insights into the identification of therapeutic targets and the design of rehabilitative protocols to treat ischemic stroke and other traumatic CNS injuries.

A new method of spermatic cord mobilization in herniorrhaphy.

Spermatic cord mobilization is a routine part of inguinal hernia repair, but the method of cord mobilization varies among surgeons. This study establishes an anatomic plane for spermatic cord mobilization. We studied the anatomy of the superficial cremasteric fascia in 105 male patients during herniorrhaphy for primary inguinal hernias. The mean patient age was 44.8 (18-71) years and mean body mass index was 24.1 kg/m(2) (21.5-27.1 kg/m(2)). The two layers of the superficial cremasteric fascia between the spermatic cord and the inguinal falx were incised to mobilize the cord. We found that spermatic cord mobilization during herniorrhaphy can be easily approached through an anatomic plane between the spermatic cord and the conjoined tendon with subsequent division of the superficial cremasteric fascia. None of the patients experienced any hemorrhage or nerve injury during cord mobilization. We found this method to be both safe and easy to learn.

Adipose-derived mesenchymal stem cells combined with platinum nanoparticles accelerate fracture healing in a rat tibial fracture model.

Background: At present, bone union delay or failure remains challenging for clinicians. It has been reported that adipose-derived mesenchymal stem cells (ADMSCs) offer a promising way to promote bone fracture healing. In recent years, nanomaterials have been applied in regenerative medicine. This study aimed to investigate whether ADMSCs combined with platinum nanoparticles (PtNPs) could further improve fracture healing on the basis of ADMSCs. Methods: ADMSCs were co-cultured with PtNPs in vitro to investigate the effect of PtNPs on the differentiation of ADMSCs. Twenty Sprague-Dawley (SD) rats were randomly divided into four groups (with five rats in each group). The left tibias of all rats were fractured. Phosphate-buffered saline (PBS), PtNPs, ADMSC, and ADMSC mixed with PtNPs were then injected into the fracture sites based on the group classifications. The fracture was monitored by X-ray immediately after the fracture and on days 14 and 28 post-fracture. The tibias of the rats were subsequently harvested after the last X-ray and evaluated by micro computed tomography (micro-CT), histological analysis, and immunohistochemical detection. Results: PtNPs significantly enhanced the osteogenic differentiation of ADMSCs in vitro. On days 14 and 28 post-fracture, the radiographic score of the ADMSC + PtNPs group was higher than that of the ADMSC group, the score of the ADMSC group was higher than that of the PtNPs and control groups, and there was no significant difference between the PtNPs and control groups. Micro-CT confirmed that combined ADMSCs with PtNPs were more effective than using ADMSCs alone in promoting fracture healing. The histological and immunohistochemical results further supported this conclusion. Conclusions: Our findings demonstrated that PtNPs could promote osteogenic differentiation of ADMSC in vitro. ADMSCs combined with PtNPs could accelerate fracture healing further in vivo and are a promising a potential method for the treatment of fracture healing.

Profiling Temporal Changes of the Pineal Transcriptomes at Single Cell Level Upon Neonatal HIBD.

Neonatal hypoxic-ischemic brain damage (HIBD) often results in various neurological deficits. Among them, a common, yet often neglected, symptom is circadian rhythm disorders. Previous studies revealed that the occurrence of cysts in the pineal gland, an organ known to regulate circadian rhythm, is associated with circadian problems in children with HIBD. However, the underlying mechanisms of pineal dependent dysfunctions post HIBD remain largely elusive. Here, by performing 10x single cell RNA sequencing, we firstly molecularly identified distinct pineal cell types and explored their transcriptome changes at single cell level at 24 and 72 h post neonatal HIBD. Bioinformatic analysis of cell prioritization showed that both subtypes of pinealocytes, the predominant component of the pineal gland, were mostly affected. We then went further to investigate how distinct pineal cell types responded to neonatal HIBD. Within pinealocytes, we revealed a molecularly defined ß to α subtype conversion induced by neonatal HIBD. Within astrocytes, we discovered that all three subtypes responded to neonatal HIBD, with differential expression of reactive astrocytes markers. Two subtypes of microglia cells were both activated by HIBD, marked by up-regulation of Ccl3. Notably, microglia cells showed substantial reduction at 72 h post HIBD. Further investigation revealed that pyroptosis preferentially occurred in pineal microglia through NLRP3-Caspase-1-GSDMD signaling pathway. Taken together, our results delineated temporal changes of molecular and cellular events occurring in the pineal gland following neonatal HIBD. By revealing pyroptosis in the pineal gland, our study also provided potential therapeutic targets for preventing extravasation of pineal pathology and thus improving circadian rhythm dysfunction in neonates with HIBD.