Delayed Accumulation of H3K27me3 on Nascent DNA Is Essential for Recruitment of Transcription Factors at Early Stages of Stem Cell Differentiation.

Recruitment of transcription factors (TFs) to repressed genes in euchromatin is essential to activate new transcriptional programs during cell differentiation. However, recruitment of all TFs, including pioneer factors, is impeded by condensed H3K27me3-containing chromatin. Single-cell and gene-specific analyses revealed that, during the first hours of induction of differentiation of mammalian embryonic stem cells (ESCs), accumulation of the repressive histone mark H3K27me3 is delayed after DNA replication, indicative of a decondensed chromatin structure in all regions of the replicating genome. This delay provides a critical "window of opportunity" for recruitment of lineage-specific TFs to DNA. Increasing the levels of post-replicative H3K27me3 or preventing S phase entry inhibited recruitment of new TFs to DNA and significantly blocked cell differentiation. These findings suggest that recruitment of lineage-specifying TFs occurs soon after replication and is facilitated by a decondensed chromatin structure. This insight may explain the developmental plasticity of stem cells and facilitate their exploitation for therapeutic purposes.

Age and incidence of occult pancreaticobiliary reflux in patients with benign gallbladder diseases.

BACKGROUND: Occult pancreaticobiliary reflux (OPBR) has a significant correlation with diseases of the gallbladder and biliary system. This study examined the incidence of OPBR by age in patients with benign gallbladder diseases. METHODS: We assessed 475 patients with benign gallbladder diseases who underwent surgery at Shanghai East Hospital from December 2020 to December 2021. Bile samples collected during surgery were tested for amylase. Patients with bile amylase >110 U/L (n = 64) were classified as the OPBR group; the rest (n = 411) as controls. RESULTS: Of the participants, 375 had gallbladder stone (GS), 170 had gallbladder polyp (GP), and 49 had gallbladder adenomyomatosis (GA). The OPBR group was generally older, with OPBR incidence increasing with age, peaking post-45. Rates by age were: 4.9% (<35), 5.2% (35-44), 20.7% (45-54), 22.5% (55-64) and 17.6% (≥65), mainly in GS patients. ROC analysis for predicting OPBR by age yielded an area under the curve of 0.656, optimal cut-off at 45 years. Logistic regression indicated age > 45, GP, male gender, and BMI ≥ 24 kg*m-2 as independent OPBR predictors in GS patients. Based on these variables, a predictive nomogram was constructed, and its effectiveness was validated using the ROC curve, calibration curve and decision curve analysis (DCA). Further stratification revealed that among GS patients ≤ 45, concurrent GA was an OPBR risk; for > 45, it was GP and male gender. CONCLUSIONS: The incidence of OPBR in GS patients is notably influenced by age, with those over 45, especially males without GP, being at heightened risk.

Identification of changes in bile composition in pancreaticobiliary reflux based on liquid chromatography/mass spectrometry metabolomics.

INTRODUCTION: Pancreaticobiliary reflux (PBR) can induce gallstone formation; however, its pathogenic mechanism remains unclear. In this study, we explored the mechanism of PBR by the non-targeted metabolomic analysis of bile in patients with PBR. OBJECTIVE: The aim of this study was to investigate the pathogenic mechanism in PBR by the non-targeted metabolomic analysis of bile collected during surgery. METHODS: Sixty patients who underwent gallstone surgery at our center from December 2020 to May 2021 were enrolled in the study. According to the level of bile amylase, 30 patients with increased bile amylase ( > 110 U/L) were classified into the PBR group, and the remaining 30 patients were classified into the control group (≤ 110 U/L). The metabolomic analysis of bile was performed. RESULTS: The orthogonal projections to latent structure-discriminant analysis of liquid chromatography mass spectrometry showed significant differences in bile components between the PBR and control groups, and 40 metabolites were screened by variable importance for the projection value (VIP > 1). The levels of phosphatidylcholine (PC) and PC (20:3(8Z,11Z,14Z)/14:0) decreased significantly, whereas the levels of lysoPC (16:1(9z)/0:0), lysoPC (15:0), lysoPC (16:0), palmitic acid, arachidonic acid, leucine, methionine, L-tyrosine, and phenylalanine increased. CONCLUSIONS: Significant differences in bile metabolites were observed between the PBR and control groups. Changes in amino acids and lipid metabolites may be related to stone formation and mucosal inflammation.

Pilocytic astrocytoma harboring a novel GNAI3-BRAF fusion.

Pilocytic astrocytoma (PA), a central nervous system (CNS) World Health Organization grade 1 tumor, is mainly seen in children or young adults aged 5-19. Surgical resection often provides excellent outcomes, but residual tumors may still remain. This low-grade tumor is well recognized for its classic radiological and morphological features; however, some unique molecular findings have been unveiled by the application of next-generation sequencing (NGS). Among the genetic abnormalities identified in this low-grade tumor, increasing evidence indicates that BRAF alterations, especially BRAF fusions, play an essential role in PA tumorigenesis. Among the several fusion partner genes identified in PAs, KIAA1549-BRAF fusion is notably the most common detectable genetic alteration, especially in the cerebellar PAs. Here, we report a case of a young adult patient with a large, right-sided posterior fossa cerebellar and cerebellopontine angle region mass consistent with a PA. Of note, NGS detected a novel GNAI3-BRAF fusion, which results in an in-frame fusion protein containing the kinase domain of BRAF. This finding expands the knowledge of BRAF fusions in the tumorigenesis of PAs, provides an additional molecular signature for diagnosis, and a target for future therapy.

METTL3 promotes glycolysis and cholangiocarcinoma progression by mediating the m6A modification of AKR1B10.

OBJECTIVE: N6-methyladenosine (m6A) RNA methylation is involved in governing the mechanism of tumor progression. We aimed to excavate the biological role and mechanism of the m6A methyltransferase METTL3 in cholangiocarcinoma (CCA). METHODS: METTL3 expression was determined by database and tissue microarray analyses. The role of METTL3 in CCA was explored by loss- and gain-of-function experiments. The m6A target of METTL3 was detected by RNA sequencing. The role of AKR1B10 in CCA was explored, and the association between METTL3 and AKR1B10 was confirmed by rescue experiments. RESULT: METTL3 expression was upregulated in CCA tissue, and higher METTL3 expression was implicated in poor prognoses in CCA patients. Overexpression of METTL3 facilitated proliferation, migration, invasion, glucose uptake, and lactate production in CCA cells, whereas knockdown of METTL3 had the opposite effects. We further found that METTL3 deficiency inhibited CCA tumor growth in vivo. RNA sequencing and MeRIP-qPCR confirmed that METTL3 enhanced AKR1B10 expression and m6A modification levels. Furthermore, METTL3 directly binds with AKR1B10 at an m6A modification site. A CCA tissue microarray showed that AKR1B10 expression was upregulated in CCA tissue and that silencing AKR1B10 suppressed the malignant phenotype mentioned above in CCA. Notably, knockdown of AKR1B10 rescued the tumor-promoting effects induced by METTL3 overexpression. CONCLUSION: Elevated METTL3 expression promotes tumor growth and glycolysis in CCA through m6A modification of AKR1B10, indicating that METTL3 is a potential target for blocking glycolysis for application in CCA therapy.

Free fatty acids and triglyceride change in the gallbladder bile of gallstone patients with pancreaticobiliary reflux.

BACKGROUND: Pancreaticobiliary reflux (PBR) causes chronic inflammation of the gallbladder mucosa and changes in the bile components, which are known to promote gallstone formation. This study aimed to investigate the bile biochemistry changes in gallstone patients with PBR and provide new clues for research on the involvement of PBR in gallstone formation. METHODS: Patients undergoing surgery for gallstones between December 2020 and May 2021 were eligible for inclusion. The bile biochemistry (including amylase, lipase, triglyceride, cholesterol, free fatty acids [FFAs], alanine aminotransferase [ALT], aspartate aminotransferase [AST], alkaline phosphatase [ALP], and γ-glutamyl transferase [γ-GT]) of the included gallstone patients was analysed to determine correlations with PBR. RESULTS: In this study, 144 gallstone patients who underwent surgery were enrolled. Overall, 15.97 % of the patients had an increased bile amylase level, which was associated with older age and significantly higher bile levels of ALP, lipase, triglyceride, and FFAs. Positive correlations were observed between amylase and lipase, triglyceride, FFAs levels in the gallbladder bile. However, the bile levels of triglyceride, FFAs, and lipase were positively correlated with each other only in the PBR group and showed no significant correlation in the control (N) group. In addition, elevated bile FFAs levels were found to be an independent risk factor for gallbladder wall thickening. CONCLUSIONS: In conclusion, PBR-induced increase in FFAs and triglyceride in the gallbladder bile is a cause of gallstone formation, and an increase in bile ALP suggests the presence of cholestasis in PBR.

Reabsorption of bile acids regulated by FXR-OATP1A2 is the main factor for the formation of cholesterol gallstone.

The purpose of this study was to demonstrate the aberrant metabolism of bile acids in patients with cholesterol gallstone and explore for its underlying mechanisms. The composition of bile acids collected from the patients with cholelithiasis and the control individuals was analyzed by LC-MS. The expression of genes regulating the metabolism of bile acids was quantitatively determined by real-time PCR or Western blot analysis. Cholesterol saturation index of patients with gallstone was significantly higher than that of the controls. The concentrations of taurodeoxycholic acid and taurolithocholic acid in the bile of patients were significantly higher than that of the controls. When compared with the controls, it was remarkable in the patients that the mRNA expression of farnesoid X receptor (FXR) was lower, whereas that of organic anion transporting polypeptide (OATP1A2) was higher. However, the expressions of both mRNA and protein of cytochrome P-450 family 8 subfamily B member 1 (CYP8B1) did not differ between the patients and the controls. Although the protein level of CYP8B1 was significantly lower in the subjects with single nucleotide polymorphism (SNP) rs3732860(G), the composition of bile acids and the ratio of CA to CDCA remained unaltered in the patients with different SNP genotype of CYP8B1. In conclusion, the axis of FXR-OATP1A2 that physiologically regulated the reabsorption of bile acids might play an important role in the composition of bile acids and the development of gallstone. CYP8B1 gene was irrelevant to the altered composition of bile acids in patients with gallstone.NEW & NOTEWORTHY For the first time, our results indicate that the axis of farnesoid X receptor-organic anion transporter polypeptide 1A2 that physiologically regulates the reabsorption of bile acids might play an important role in the regulation of the composition of bile acids and make contribution to the development of cholelithiasis.

Systemic Factors Trigger Vasculature Cells to Drive Notch Signaling and Neurogenesis in Neural Stem Cells in the Adult Brain.

It is well documented that adult neural stem cells (NSCs) residing in the subventricular zone (SVZ) and the subgranular zone (SGZ) are induced to proliferate and differentiate into new neurons after injury such as stroke and hypoxia. However, the role of injury-related cues in driving this process and the means by which they communicate with NSCs remains largely unknown. Recently, the coupling of neurogenesis and angiogenesis and the extensive close contact between vascular cells and other niche cells, known as the neurovascular unit (NVU), has attracted interest. Further facilitating communication between blood and NSCs is a permeable blood-brain-barrier (BBB) present in most niches, making vascular cells a potential conduit between systemic signals, such as vascular endothelial growth factor (VEGF), and NSCs in the niche, which could play an important role in regulating neurogenesis. We show that the leaky BBB in stem cell niches of the intact and stroke brain can respond to circulating VEGF165 to drive induction of the Notch ligand DLL4 (one of the most important cues in angiogenesis) in endothelial cells (ECs), pericytes, and further induce significant proliferation and neurogenesis of stem cells. Stem Cells 2019;37:395-406.

Subregional differences in astrocytes underlie selective neurodegeneration or protection in Parkinson's disease models in culture.

Parkinson's disease (PD) is characterized by the selective degeneration of dopamine (DA) neurons of the substantia nigra pars compacta (SN), while the neighboring ventral tegmental area (VTA) is relatively spared. The mechanisms underlying this selectivity are not fully understood. Here, we demonstrate a vital role for subregional astrocytes in the protection of VTA DA neurons. We found that elimination of astrocytes in vitro exposes a novel vulnerability of presumably protected VTA DA neurons to the PD mimetic toxin MPP+ , as well as exacerbation of SN DA neuron vulnerability. Conversely, VTA astrocytes protected both VTA and SN DA neurons from MPP+ toxicity in a dose dependent manner, and this protection was mediated via a secreted molecule. RNAseq analysis of isolated VTA and SN astrocytes demonstrated a vast array of transcriptional differences between these two closely related populations demonstrating regional heterogeneity of midbrain astrocytes. We found that GDF15, a member of the TGFß superfamily which is expressed 230-fold higher in VTA astrocytes than SN, recapitulates neuroprotection of both rat midbrain and iPSC-derived DA neurons, whereas its knockdown conversely diminished this effect. Neuroprotection was likely mediated through the GRFAL receptor expressed on DA neurons. Together; these results suggest that subregional differences in astrocytes underlie the selective degeneration or protection of DA neurons in PD.

Long noncoding RNA HOTAIRM1 inhibits cell progression by regulating miR-17-5p/ PTEN axis in gastric cancer.

OBJECTIVES: This study was conducted to identify the significantly altered long noncoding RNAs (lncRNAs), messenger RNA (mRNA) and pathways in gastric cancer (GC). METHODS: We used microarray analysis to identify differentially expressed lncRNAs and mRNAs, whereas the obviously changed pathways were found by gene set enrichment analysis. The coexpression network of lncRNA and mRNA was constructed by Cytoscape, and their target relationships with miRNAs were predicted by miRcode and TargetScan. qRT-PCR and Western blot were performed to determine the expression levels of mRNAs and proteins in tissues and cell lines. Dual-luciferase reporter assay was applied to achieve the determination of the specific target relationships. Cell viability, migration, and apoptosis were detected by MTT assay, wound healing assay and flow cytometry, respectively. Through the xenograft assay, the gastric tumor was implanted into nude mice to investigate the influence of HOTAIRM1 in vivo. RESULTS: HOTAIRM1 and phosphatase and tensin homolog (PTEN) were both downregulated in GC, whereas miR-17-5p was upregulated. Moreover, the PI3K/AKT pathway was found activated in GC. HOTAIRM1 targeted miR-17-5p, whereas PTEN was the downstream target gene of miR-17-5p. HOTAIRM1 suppressed proliferation and migration of GC cell line and induced their apoptosis, whereas miR-17-5p played the opposite role on GC cell line. HOTAIRM1 also postponed tumor growth in vivo and inhibited the PI3K/AKT pathway in GC. CONCLUSIONS: LncRNA HORAIRM1 suppressed the PI3K/AKT pathway in GC and inhibited the progression of GC by serving as a competing endogenous RNA of miR-17-5p, mediating the expression of PTEN.

Regional microglia are transcriptionally distinct but similarly exacerbate neurodegeneration in a culture model of Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is characterized by selective degeneration of dopaminergic (DA) neurons of the substantia nigra pars compacta (SN) while neighboring ventral tegmental area (VTA) DA neurons are relatively spared. Mechanisms underlying the selective protection of the VTA and susceptibility of the SN are still mostly unknown. Here, we demonstrate the importance of balance between astrocytes and microglia in the susceptibility of SN DA neurons to the PD mimetic toxin 1-methyl-4-phenylpyridinium (MPP+). METHODS: Previously established methods were used to isolate astrocytes and microglia from the cortex (CTX), SN, and VTA, as well as embryonic midbrain DA neurons from the SN and VTA. The transcriptional profile of isolated microglia was examined for 21 canonical pro- and anti-inflammatory cytokines by qRT-PCR with and without MPP+ exposure. Homo- and heterotypic co-cultures of neurons and astrocytes were established, and the effect of altering the ratio of astrocytes and microglia in vitro on the susceptibility of midbrain DA neurons to the PD mimetic toxin MPP+ was investigated. RESULTS: We found that regionally isolated microglia (SN, VTA, CTX) exhibit basal differences in their cytokine profiles and that activation of these microglia with MPP+ results in differential cytokine upregulation. The addition of microglia to cultures of SN neurons and astrocytes was not sufficient to cause neurodegeneration; however, when challenged with MPP+, all regionally isolated microglia resulted in exacerbation of MPP+ toxicity which was alleviated by inhibition of microglial activation. Furthermore, we demonstrated that isolated VTA, but not SN, astrocytes were able to mediate protection of both SN and VTA DA neurons even in the presence of exacerbatory microglia; however, this protection could be reversed by increasing the numbers of microglia present. CONCLUSION: These results suggest that the balance of astrocytes and microglia within the midbrain is a key factor underlying the selective vulnerability of SN DA neurons seen in PD pathogenesis and that VTA astrocytes mediate protection of DA neurons which can be countered by greater numbers of deleterious microglia.

Fumarate modulates the immune/inflammatory response and rescues nerve cells and neurological function after stroke in rats.

BACKGROUND: Dimethyl fumarate (DMF), working via its metabolite monomethylfumarate (MMF), acts as a potent antioxidant and immunomodulator in animal models of neurologic disease and in patients with multiple sclerosis. These properties and their translational potential led us to investigate whether DMF/MMF could also protect at-risk and/or dying neurons in models of ischemic stroke in vitro and in vivo. Although the antioxidant effects have been partially addressed, the benefits of DMF immunomodulation after ischemic stroke still need to be explored. METHODS: In vitro neuronal culture with oxygen-glucose deprivation and rats with middle cerebral artery occlusion were subjected to DMF/MMF treatment. Live/dead cell counting and LDH assay, as well as behavioral deficits, plasma cytokine assay, western blots, real-time PCR (Q-PCR) and immunofluorescence staining, were used to evaluate the mechanisms and neurological outcomes. RESULTS: We found that MMF significantly rescued cortical neurons from oxygen-glucose deprivation (OGD) in culture and suppressed pro-inflammatory cytokines produced by primary mixed neuron/glia cultures subjected to OGD. In rats, DMF treatment significantly decreased infarction volume by nearly 40 % and significantly improved neurobehavioral deficits after middle cerebral artery occlusion (MCAO). In the acute early phase (72 h after MCAO), DMF induced the expression of transcription factor Nrf2 and its downstream mediator HO-1, important for the protection of infarcted cells against oxidative stress. In addition to its antioxidant role, DMF also acted as a potent immunomodulator, reducing the infiltration of neutrophils and T cells and the number of activated microglia/macrophages in the infarct region by more than 50 % by 7-14 days after MCAO. Concomitantly, the levels of potentially harmful pro-inflammatory cytokines were greatly reduced in the plasma and brain and in OGD neuron/glia cultures. CONCLUSIONS: We conclude that DMF is neuroprotective in experimental stroke because of its potent immunomodulatory and antioxidant effects and thus may be useful as a novel therapeutic agent to treat stroke in patients.

Neurogenesis is enhanced by stroke in multiple new stem cell niches along the ventricular system at sites of high BBB permeability.

Previous studies have established the subventricular (SVZ) and subgranular (SGZ) zones as sites of neurogenesis in the adult forebrain (Doetsch et al., 1999a; Doetsch, 2003a). Work from our laboratory further indicated that midline structures known as circumventricular organs (CVOs) also serve as adult neural stem cell (NSC) niches (Bennett et al., 2009, 2010). In the quiescent rat brain, NSC proliferation remains low in all of these sites. Therefore, we recently examined whether ischemic stroke injury (MCAO) or sustained intraventricular infusion of the mitogen bFGF could trigger an up-regulation in NSC proliferation, inducing neurogenesis and gliogenesis. Our data show that both stroke and bFGF induce a dramatic and long-lasting (14day) rise in the proliferation (BrdU+) of nestin+Sox2+GFAP+ NSCs capable of differentiating into Olig2+ glial progenitors, GFAP+nestin-astrocyte progenitors and Dcx+ neurons in the SVZ and CVOs. Moreover, because of the upsurge in NSC number, it was possible to detect for the first time several novel stem cell niches along the third (3V) and fourth (4V) ventricles. Importantly, a common feature of all brain niches was a rich vasculature with a blood-brain-barrier (BBB) that was highly permeable to systemically injected sodium fluorescein. These data indicate that stem cell niches are more extensive than once believed and exist at multiple sites along the entire ventricular system, consistent with the potential for widespread neurogenesis and gliogenesis in the adult brain, particularly after injury. We further suggest that because of their leaky BBB, stem cell niches are well-positioned to respond to systemic injury-related cues which may be important for stem-cell mediated brain repair.

BMP and TGF-ß pathway mediators are critical upstream regulators of Wnt signaling during midbrain dopamine differentiation in human pluripotent stem cells.

Although many laboratories currently use small molecule inhibitors of the BMP (Dorsomorphin/DM) and TGF-ß (SB431542/SB) signaling pathways in protocols to generate midbrain dopamine (mDA) neurons from hES and hiPS cells, until now, these substances have not been thought to play a role in the mDA differentiation process. We report here that the transient inhibition of constitutive BMP (pSMADs 1, 5, 8) signaling, either alone or in combination with TGF-ß inhibition (pSMADs 2, 3), is critically important in the upstream regulation of Wnt1-Lmx1a signaling in mDA progenitors. We postulate that the mechanism via which DM or DM/SB mediates these effects involves the up-regulation in SMAD-interacting protein 1 (SIP1), which results in greater repression of the Wnt antagonist, secreted frizzled related protein 1 (Sfrp1) in stem cells. Accordingly, knockdown of SIP1 reverses the inductive effects of DM/SB on mDA differentiation while Sfrp1 knockdown/inhibition mimics DM/SB. The rise in Wnt1-Lmx1a levels in SMAD-inhibited cultures is, however, accompanied by a reciprocal down-regulation in SHH-Foxa2 levels leading to the generation of few TH+ neurons that co-express Foxa2. If however, exogenous SHH/FGF8 is added along with SMAD inhibitors, equilibrium in these two important pathways is achieved such that authentic (Lmx1a+Foxa2+TH+) mDA neuron differentiation is promoted while alternate cell fates are suppressed in stem cell cultures. These data indicate that activators/inhibitors of BMP and TGF-ß signaling play a critical upstream regulatory role in the mDA differentiation process in human pluripotent stem cells.

Latexin inhibits the proliferation of CD133+ miapaca-2 pancreatic cancer stem-like cells.

BACKGROUND: An increasing number of evidence suggests that pancreatic cancer contains cancer stem cells (CSCs), which may be relevant to the resistance of chemotherapy. Latexin (Lxn) is a negative regulator of stem cell proliferation and we investigate the effects of Lxn on CD133+ pancreatic cancer stem-like cells. METHODS: CD133+ miapaca-2 cells, a human pancreatic carcinoma cell line, were isolated and sorted by magnetic activated cell sorting and flow cytometry. The capacity for self-renewal, proliferation, and tumorigenicity of CD133+ miapaca-2 cells was determined by the floating spheres test and tumor xenograft assays. Protein and mRNA expression of Lxn in CD133+ and CD133- miapaca-2 cells were detected by Western blotting and qRT-PCR, respectively. After CD133+ miapaca-2 cells were treated with Lxn in serum-free medium (SFM), cell proliferation was assayed with a Cell Counting Kit 8 (CCK-8) and apoptosis was analyzed by flow cytometry. The protein and mRNA expression levels of Bcl-2, bax, and c-myc were also analyzed. RESULTS: We successfully isolated CD133+ miapaca-2 cells that exhibited the capacity for self-renewal in SFM, a proliferation potential in DMEM supplemented with FBS, and high tumorigenicity in nude mice. Lxn protein and mRNA expression levels in CD133+ miapaca-2 cells were significantly lower than those in CD133- cells. Lxn-treated CD133+ miapaca-2 cells exhibited increased apoptosis and low proliferation activity, down-regulation of Bcl-2 and c-myc expression, and up-regulation of Bax expression in a dose-dependent manner. CONCLUSIONS: Lxn induces apoptosis and inhibits the proliferation of CD133+ miapaca-2 cells. These changes are associated with down-regulation of Bcl-2 and c-myc and up-regulation of Bax.

IGF2BP3 drives gallbladder cancer progression by m6A-modified CLDN4 and inducing macrophage immunosuppressive polarization.

INTRODUCTION: N6-methyladenosine (m6A) is an emerging epigenetic modification, which plays a crucial role in the development of cancer. Nevertheless, the underlying mechanism of m6A-associated proteins and m6A modification in gallbladder cancer remains largely unknown. MATERIALS AND METHODS: The Gene Expression Omnibus database and tissue microarray were used to identify the key m6A-related gene in gallbladder cancer. The function and mechanism of IGF2BP3 were further investigated by knockdown and overexpression techniques in vitro and in vivo. RESULTS: We found that IGF2BP3 was elevated and correlated with poor prognosis in gallbladder cancer, which can be used as an independent prognostic factor for gallbladder cancer. IGF2BP3 accelerated the proliferation, invasion and migration of gallbladder cancer cells in vitro and in vivo. Mechanistically, IGF2BP3 interacted with and augmented the stability of CLDN4 mRNA by m6A modification. Enhancement of CLDN4 reversed the inhibitory effect of IGF2BP3 deficiency on gallbladder cancer. Furthermore, we demonstrated that IGF2BP3 promotes the activation of NF-κB signaling pathway by up-regulation of CLDN4. Overexpression of IGF2BP3 in gallbladder cancer cells obviously promoted the polarization of immunosuppressive phenotype in macrophages. Besides, Gallbladder cancer cells-derived IGF2BP3 up-regulated the levels of STAT3 in M2 macrophages, and promoted M2 polarization. CONCLUSIONS: We manifested IGF2BP3 promotes the aggressive phenotype of gallbladder cancer by stabilizing CLDN4 mRNA in an m6A-dependent manner and induces macrophage immunosuppressive polarization, which might offer a new theoretical basis for against gallbladder cancer.

A stress-free strategy to correct point mutations in patient iPS cells.

When studying patient specific induced pluripotent stem cells (iPS cells) as a disease model, the ideal control is an isogenic line that has corrected the point mutation, instead of iPS cells from siblings or other healthy subjects. However, repairing a point mutation in iPS cells even with the newly developed CRISPR-Cas9 technique remains difficult and time-consuming. Here we report a strategy that makes the Cas9 "knock-in" methodology both hassle-free and error-free. Instead of selecting a Cas9 recognition site close to the point mutation, we chose a site located in the nearest intron. We constructed a donor template with the fragment containing the corrected point mutation as one of the homologous recombination arms flanking a PGK-PuroR cassette. After selection with puromycin, positive clones were identified and further transfected with a CRE vector to remove the PGK-PuroR cassette. Using this methodology, we successfully repaired the point mutation G2019S of the LRRK2 gene in a Parkinson Disease (PD) patient iPS line and the point mutation R329H of the AARS1 gene in a Charcot-Marie-Tooth disease (CMT) patient iPS line. These isogenic iPS lines are ideal as a control in future studies.

The role of Lmx1a in the differentiation of human embryonic stem cells into midbrain dopamine neurons in culture and after transplantation into a Parkinson's disease model.

Recent studies have provided important insight into the homeoprotein LIM homeobox transcription factor 1alpha (Lmx1a) and its role in the commitment of cells to a midbrain dopamine (mDA) fate in the developing mouse. We show here that Lmx1a also plays a pivotal role in the mDA differentiation of human embryonic stem (hES) cells. Thus, as indicated by small interfering RNA experiments, the transient early expression of Lmx1a is necessary for the coordinated expression of all other dopamine (DA)-specific phenotypic traits as hES cells move from multipotent human neural progenitor cells (hNPs) to more restricted precursor cells in vitro. Moreover, only Lmx1a-specified hNPs have the potential to differentiate into bona fide mDA neurons after transplantation into the 6-hydroxydopamine-treated rat striatum. In contrast, cortical human neuronal precursor cells (HNPCs) and mouse subventricular zone cells do not express Lmx1a or become mDA neurons even when placed in an environment that fosters their DA differentiation in vitro or in vivo. These findings suggest that Lmx1a may be critical to the development of mDA neurons from hES cells and that, along with other key early DA markers (i.e., Aldh1a1), may prove to be extremely useful for the selection of appropriately staged and suitably mDA-specified hES cells for cell replacement in Parkinson's disease.

Extraluminal laparoscopic wedge resection of gastric submucosal tumors: a retrospective review of 84 cases.

BACKGROUND: Laparoscopic resection of gastric stromal tumors is being performed with increased frequency. This study aims to evaluate the feasibility and safety of the extraluminal laparoscopic gastric wedge resection (ELWR) technique. METHODS: Clinical data of 84 patients who underwent ELWR for gastric submucosal tumors between September 2000 and December 2007 were reviewed and analyzed retrospectively. The operation includes: localization of the tumor, dissection of the omentum, mobilization of the upper stomach and the upper pole of the spleen, exposure of esophago-cardiac junction (ECJ), and wedge resection of the upper part of gastric body and/or the gastric fundus with endoscopic gastrointestinal anastomosis (Endo GIA) stapler. RESULTS: All of the procedures were performed successfully, with mean operation time of 62.6 +/- 8.9 min and mean intraoperative blood loss of 86.2 +/- 8.1 ml. Through extraluminal laparoscopic wedge resection, complete R0 resection was achieved for all tumors. All surgical margins were negative microscopically. No lesions were missed, nor were there any significant postoperative complications or intraoperative conversions to open surgery. A total of 78.6% of the patients recovered their gastrointestinal functions and began to eat and ambulate within 36 h of the operation. The smallest surgical margins were 0.7-2.5 cm, with a mean distance of 1.4 +/- 0.5 cm. Of the 84 cases of gastric submucosal tumors, 29 cases were leiomyomas, 51 cases were various types of stromal tumors, and 4 other cases were neurofibromas. Mean follow-up duration was 51 +/- 4.3 months (overall follow-up rate 73.8%, 62/84 cases), during which no recurrences or metastases were found. CONCLUSION: ELWR is a safe, simple, and effective procedure for treating submucosal tumors in the upper part of the stomach. It can avoid intraperitoneal contamination, possible tumor spillage, and postoperative esophageal stenosis, and provides unlimited scope for gastric resection.

The Clinical Application of a Self-developed Gasless Laparoendoscopic Operation Field Formation Device on Patients Undergoing Cholecystectomy.

BACKGROUND: We have designed a new gasless laparoscopic operation field formation (LOFF) device for cholecystectomy which was successfully tested on animal model. The goal of this study is to investigate the feasibility, safety and effectiveness of this LOFF device on patients undergoing cholecystectomy. METHODS: Patients with cholecystolithiasis or gallbladder polyps who underwent single port cholecystectomy from June 2015 to May 2016 were retrospectively reviewed. Either the LOFF-assisted laparoendoscopic single-port surgery (LESS) (LOFF-LESS) or the traditional LESS was performed. Operation time, intraoperative bleeding, postoperative hospital stay, surgical complications, incision pain score, shoulder and back pain and cosmetic satisfaction were compared. RESULTS: A total of 186 patients were included in this study, with 79 in the LOFF-LESS group and 107 in the LESS group. There was no significant difference between LOFF-LESS group and LESS group in operation field establishment time, cholecystectomy time, intraoperative bleeding, postoperative hospital stay, incision pain and cosmetic satisfaction. A lower intraoperative arterial carbon dioxide pressure was documented in the LOFF-LESS group (P<0.01). The incidence of postoperative shoulder and back pain was significantly lower in LOFF-LESS group (P<0.01). CONCLUSION: LOFF-LESS has comparable benefits of traditional LESS; it deceases incidence of pneumoperitoneum related complications as well.