Passive DNA demethylation preferentially up-regulates pluripotency-related genes and facilitates the generation of induced pluripotent stem cells.

A high proliferation rate has been observed to facilitate somatic cell reprogramming, but the pathways that connect proliferation and reprogramming have not been reported. DNA methyltransferase 1 (DNMT1) methylates hemimethylated CpG sites produced during S phase and maintains stable inheritance of DNA methylation. Impairing this process results in passive DNA demethylation. In this study, we show that the cell proliferation rate positively correlated with the expression of Dnmt1 in G1 phase. In addition, as determined by whole-genome bisulfate sequencing and high-performance liquid chromatography, global DNA methylation of mouse embryonic fibroblasts was significantly higher in G1 phase than in G2/M phase. Thus, we suspected that high cellular proliferation requires more Dnmt1 expression in G1 phase to prevent passive DNA demethylation. The methylation differences of individual CpG sites between G1 and G2/M phase were related to the methylation status and the positions of their surrounding CpG sites. In addition, larger methylation differences were observed on the promoters of pluripotency-related genes; for example, Oct4, Nanog, Sox2, Esrrb, Cdh1, and Epcam When such methylation differences or passive DNA demethylation accumulated with Dnmt1 suppression and proliferation acceleration, DNA methylation on pluripotency-related genes was decreased, and their expression was up-regulated, which subsequently promoted pluripotency and mesenchymal-epithelial transition, a necessary step for reprogramming. We infer that high cellular proliferation rates promote generation of induced pluripotent stem cells at least partially by inducing passive DNA demethylation and up-regulating pluripotency-related genes. Therefore, these results uncover a connection between cell reprogramming and DNA methylation.

Opioid doses required for pain management in lung cancer patients with different cholesterol levels: negative correlation between opioid doses and cholesterol levels.

BACKGROUND: Pain management has been considered as significant contributor to broad quality-of-life improvement for cancer patients. Modulating serum cholesterol levels affects analgesia abilities of opioids, important pain killer for cancer patients, in mice system. Thus the correlation between opioids usages and cholesterol levels were investigated in human patients with lung cancer. METHODS: Medical records of 282 patients were selected with following criteria, 1) signed inform consent, 2) full medical records on total serum cholesterol levels and opioid administration, 3) opioid-naïve, 4) not received/receiving cancer-related or cholesterol lowering treatment, 5) pain level at level 5-8. The patients were divided into different groups basing on their gender and cholesterol levels. Since different opioids, morphine, oxycodone, and fentanyl, were all administrated at fixed low dose initially and increased gradually only if pain was not controlled, the percentages of patients in each group who did not respond to the initial doses of opioids and required higher doses for pain management were determined and compared. RESULTS: Patients with relative low cholesterol levels have larger percentage (11 out of 28 in female and 31 out of 71 in male) to not respond to the initial dose of opioids than those with high cholesterol levels (0 out of 258 in female and 8 out of 74 in male). Similar differences were obtained when patients with different opioids were analyzed separately. After converting the doses of different opioids to equivalent doses of oxycodone, significant correlation between opioid usages and cholesterol levels was also observed. CONCLUSIONS: Therefore, more attention should be taken to those cancer patients with low cholesterol levels because they may require higher doses of opioids as pain killer.

Transcriptomics reveals amygdala neuron regulation by fasting and ghrelin thereby promoting feeding.

The central amygdala (CeA) consists of numerous genetically defined inhibitory neurons that control defensive and appetitive behaviors including feeding. Transcriptomic signatures of cell types and their links to function remain poorly understood. Using single-nucleus RNA sequencing, we describe nine CeA cell clusters, of which four are mostly associated with appetitive and two with aversive behaviors. To analyze the activation mechanism of appetitive CeA neurons, we characterized serotonin receptor 2a (Htr2a)-expressing neurons (CeAHtr2a) that comprise three appetitive clusters and were previously shown to promote feeding. In vivo calcium imaging revealed that CeAHtr2a neurons are activated by fasting, the hormone ghrelin, and the presence of food. Moreover, these neurons are required for the orexigenic effects of ghrelin. Appetitive CeA neurons responsive to fasting and ghrelin project to the parabrachial nucleus (PBN) causing inhibition of target PBN neurons. These results illustrate how the transcriptomic diversification of CeA neurons relates to fasting and hormone-regulated feeding behavior.

Juvenile primary Sjogren's syndrome with cutaneous involvement.

OBJECTIVE: This study aims to analyze the clinical characteristics of juvenile primary Sjogren's syndrome (pSS) with cutaneous involvement. METHODS: We investigated the clinical and immunological characteristics of 37 children with pSS. All the patients met the 2012 American College of Rheumatology Classification Criteria for Sjogren's syndrome. RESULTS: The cutaneous involvement presented in 19 children (excluding drug eruption), 16 (84.2%) female patients, and 3 (15.8%) male patients, with a mean age of 11 ± 2.68 years, 17 of whom (89.5%) had cutaneous lesions as the first symptom, with a median time of 12 months (1 day to 4 years) before the diagnosis of pSS. The cutaneous lesions included 12 cases of palpable purpura (63.2%), 5 cases of urticaria (26.3%), 2 cases of xeroderma (10.5%), 1 case of skin ulcer, 1 case of erythema nodosum, 1 case of livedo reticularis, 1 case of Raynaud's phenomenon, and 1 case of hard erythema. Children with cutaneous lesions had a higher prevalence of articular involvement (42.1% vs. 11.1%, P = 0.016), fever (47.4% vs. 5.6%, P = 0.004), ESR > 50 mm/h (47.4% vs. 11.1%, P = 0.016), and a lower prevalence of thrombocytopenia (0% vs. 27.8%, P = 0.013) and methylprednisolone pulse treatment (0% vs. 13.5%, P = 0.013), compared with pSS without cutaneous involvement. CONCLUSION: More than half (51.3%) of the children with juvenile pSS presented with cutaneous lesions; the main cutaneous involvement was palpable purpura. Children with cutaneous lesions were more likely to have fever and arthritis, were more likely to have stronger inflammatory response, and were less likely to have serious complications. In many cases, the cutaneous lesion could be the first symptom of juvenile pSS, which could easily lead to a misdiagnosis. The possibility of pSS should be considered for children with skin lesions such as palpable purpura and urticaria, and further examinations should be carried out. Key Points • Cutaneous lesions of juvenile pSS are not uncommon and often present as the first symptom. • Palpable purpura was the most common skin lesion in juvenile pSS, followed by urticaria. • Juvenile pSS with skin lesions does not increase the risk of serious complications such as blood system damage. • Skin lesions in juvenile pSS patients may easily cause misdiagnosis.

Risk factors for intussusception in children with Henoch-Schönlein purpura: A case-control study.

BACKGROUND: The etiology of Henoch-Schönlein purpura (HSP) with intussusception remains undefined. AIM: To investigate the risk factors for intussusception in children with HSP and gastrointestinal (GI) involvement. METHODS: Sixty children with HSP and concomitant intussusception admitted to the Beijing Children's Hospital of Capital Medical University between January 2006 and December 2018 were enrolled in this study. One hundred pediatric patients with HSP and GI involvement but without intussusception, admitted to the same hospital during the same period, were randomly selected as a control group. The baseline clinical characteristics of all patients, including sex, age of onset, duration of disease, clinical manifestations, laboratory test results, and treatments provided, were assessed. Univariate and multiple logistic regression analyses were performed to identify possible risk factors. RESULTS: The 60 children in the intussusception group comprised 27 girls (45%) and 33 boys (55%) and the 100 children in the non-intussusception group comprised 62 girls (62%) and 38 boys (38%). The median age of all patients were 6 years and 5 mo. Univariate and multiple regression analyses revealed age at onset, not receiving glucocorticoid therapy within 72 h of emergence of GI symptoms, hematochezia, and D-dimer levels as independent risk factors for intussusception in children with HSP (P < 0.05). CONCLUSION: The four independent risk factors for intussusception in pediatric HSP with GI involvement would be a reference for early prevention and treatment of this potentially fatal disease.

Hemi-methylated CpG sites connect Dnmt1-knockdown-induced and Tet1-induced DNA demethylation during somatic cell reprogramming.

The relationship between active DNA demethylation induced by overexpressing Tet1 and passive DNA demethylation induced by suppressing Dnmt1 remains unclear. Here, we found that DNMT1 preferentially methylated, but TET1 preferentially demethylated, hemi-methylated CpG sites. These phenomena resulted in a significant overlap in the targets of these two types of DNA demethylation and the counteractions of Dnmt1 and Tet1 during somatic cell reprogramming. Since the hemi-methylated CpG sites generated during cell proliferation were enriched at core pluripotency loci, DNA demethylation induced by Tet1 or sh-RNA against Dnmt1 (sh-Dnmt1) was enriched in these loci, which, in combination with Yamanaka factors, led to the up-regulation of these genes and promoted somatic cell reprogramming. In addition, since sh-Dnmt1 induces DNA demethylation by impairing the further methylation of hemi-methylated CpG sites generated during cell proliferation, while Tet1 induced DNA demethylation by demethylating these hemi-methylated CpG sites, Tet1-induced DNA demethylation, compared with sh-Dnmt1-induced DNA demethylation, exhibited a higher ability to open the chromatin structure and up-regulate gene expression. Thus, Tet1-induced but not sh-Dnmt1-induced DNA demethylation led to the up-regulation of an additional set of genes that can promote the epithelial-mesenchymal transition and impair reprogramming. When vitamin C was used to further increase the demethylation ability of TET1 during reprogramming, Tet1 induced a larger up-regulation of these genes and significantly impaired reprogramming. Therefore, the current studies provide additional information regarding DNA demethylation during somatic cell reprogramming.

Sequential EMT-MET induces neuronal conversion through Sox2.

Direct neuronal conversion can be achieved with combinations of small-molecule compounds and growth factors. Here, by studying the first or induction phase of the neuronal conversion induced by defined 5C medium, we show that the Sox2-mediated switch from early epithelial-mesenchymal transition (EMT) to late mesenchymal-epithelial transition (MET) within a high proliferation context is essential and sufficient for the conversion from mouse embryonic fibroblasts (MEFs) to TuJ+ cells. At the early stage, insulin and basic fibroblast growth factor (bFGF)-induced cell proliferation, early EMT, the up-regulation of Stat3 and Sox2, and the subsequent activation of neuron projection. Up-regulated Sox2 then induced MET and directed cells towards a neuronal fate at the late stage. Inhibiting either stage of this sequential EMT-MET impaired the conversion. In addition, Sox2 could replace sequential EMT-MET to induce a similar conversion within a high proliferation context, and its functions were confirmed with other neuronal conversion protocols and MEFs reprogramming. Therefore, the critical roles of the sequential EMT-MET were implicated in direct cell fate conversion in addition to reprogramming, embryonic development and cancer progression.

Lysine-specific histone demethylase 1 inhibition promotes reprogramming by facilitating the expression of exogenous transcriptional factors and metabolic switch.

Lysine-specific histone demethylase 1 (LSD1) regulates histone methylation and influences the epigenetic state of cells during the generation of induced pluripotent stem cells (iPSCs). Here we reported that LSD1 inhibition via shRNA or specific inhibitor, tranylcypromine, promoted reprogramming at early stage via two mechanisms. At early stage of reprogramming, LSD1 inhibition increased the retrovirus-mediated exogenous expression of Oct4, Klf4, and Sox2 by blocking related H3K4 demethylation. Since LSD1 inhibition still promoted reprogramming even when iPSCs were induced with small-molecule compounds in a virus-free system, additional mechanisms should be involved. When RNA-seq was used for analysis, it was found that LSD1 inhibition reversed some gene expression changes induced by OKS, which subsequently promoted reprogramming. For example, by partially rescuing the decreased expression of Hif1α, LSD1 inhibition reversed the up-regulation of genes in oxidative phosphorylation pathway and the down-regulation of genes in glycolysis pathway. Such effects facilitated the metabolic switch from oxidative phosphorylation to glycolysis and subsequently promoted iPSCs induction. In addition, LSD1 inhibition also promoted the conversion from pre-iPSCs to iPSCs by facilitating the similar metabolic switch. Therefore, LSD1 inhibition promotes reprogramming by facilitating the expression of exogenous transcriptional factors and metabolic switch.

MicroRNA-128-3p impaired water maze learning by suppressing Doublecortin expression in both wild type and Aß-42 infused mice.

MicroRNA-128-3p (miR-128) is a brain-enriched microRNA reported to target Doublecortin (Dcx), a key transcriptional factor during adult neurogenesis. However, the downstream physiological effects of this miR-128-DCX axis remain unclear. Here we demonstrated that miR-128 could suppress Dcx expression by complementally binding to the -849 to -856 region of the 3'UTR of mouse Dcx. During differentiation of neural stem cells, over-expressing miR-128 with a lentivirus system inhibited the up-regulation of Dcx on Day 5, subsequently decreasing the percentage of TuJ+ cells on Day 16. Administration of the lentivirus encoding miR-128 into mouse hippocampi significantly impaired water maze learning after 14days, which could be attenuated when the Dcx-encoding virus was delivered simultaneously. In addition, similar changes including miR-128 up-regulation, Dcx down-regulation and learning defects were observed after a 14-day infusion of Aß-42, which were also partially reversed by over-expressing Dcx. Collectively, the regulation axis from miR-128 to Dcx is critical for hippocampus-related contextual learning not only in wild type, but also in mice infused with Aß-42.

Psora-4, a Kv1.3 Blocker, Enhances Differentiation and Maturation in Neural Progenitor Cells.

AIM: The self-repair ability of neural progenitor cells (NPCs) has been found to be activated and protected in several therapies helpful in multiple sclerosis (MS), an inflammatory demyelinating disease of the CNS. As a potential therapeutic target in MS, the role of the ion channel Kv1.3 in NPC self-repair has received limited attention. The aim of this study was to explore the effects of a selective Kv1.3 blocker on NPC neuronal differentiation and maturation. METHODS: A small-molecule selective blocker for Kv1.3, Psora-4, was added to the differentiation medium of cultured mouse NPCs to assess its effect on NPC differentiation efficiency. Both a polypeptide Kv1.3 blocker and Kv1.3-specific RNA interference were used in parallel experiments. Further, the maturity of newborn neurons in the presence of Psora-4 was measured both by morphological analysis and by whole-cell patch clamping. RESULTS: Psora-4 induced a significant increase in the percentage of neurons. Knockdown of Kv1.3 in NPCs also promoted neuronal differentiation. Both morphological and electrophysiological analyses suggested that NPC-derived neurons in the presence of Psora-4 were more mature. CONCLUSION: Our studies reveal a crucial role for the ion channel Kv1.3 in the regulation of NPC differentiation and maturation, making Psora-4 a promising candidate molecule for MS treatment.

Reprogramming somatic cells to cells with neuronal characteristics by defined medium both in vitro and in vivo.

BACKGROUND: Currently, direct conversion from somatic cells to neurons requires virus-mediated delivery of at least one transcriptional factor or a combination of several small-molecule compounds. Delivery of transcriptional factors may affect genome stability, while small-molecule compounds may require more evaluations when applied in vivo. Thus, a defined medium with only conventional growth factors or additives for cell culture is desirable for inducing neuronal trans-differentiation. RESULTS: Here, we report that a defined medium (5C) consisting of basic fibroblast growth factor (bFGF), N2 supplement, leukemia inhibitory factor, vitamin C (Vc), and ß-mercaptoethanol (ßMe) induces the direct conversion of somatic cells to cells with neuronal characteristics. Application of 5C medium converted mouse embryonic fibroblasts (MEFs) into TuJ+ neuronal-like cells, which were capable of survival after being transplanted into the mouse brain. The same 5C medium could convert primary rat astrocytes into neuronal-like cells with mature electrophysiology characteristics in vitro and facilitated the recovery of brain injury, possibly by inducing similar conversions, when infused into the mouse brain in vivo. Crucially, 5C medium could also induce neuronal characteristics in several human cell types. CONCLUSIONS: In summary, this 5C medium not only provides a means to derive cells with neuronal characteristics without viral transfection in vitro but might also be useful to produce neurons in vivo for neurodegenerative disease treatment.

Neurod1 modulates opioid antinociceptive tolerance via two distinct mechanisms.

BACKGROUND: The activity of neurogenic differentiation 1 (Neurod1) decreases after morphine administration, which leads to impairments of the stability of dendritic spines in primary hippocampal neurons, adult neurogenesis in mouse hippocampi, and drug-associated contextual memory. The current study examined whether Neurod1 could affect the development of opioid tolerance. METHODS: Lentivirus encoding Neurod1, microRNA-190 (miR-190), or short hairpin RNA against Neurod1 was injected into mouse hippocampi separately or combined (more than eight mice for each treatment) to modulate NeuroD1 activity. The antinociceptive median effective dose values of morphine and fentanyl were determined with tail-flick assay and used to calculate development of tolerance. Contextual learning and memory were assayed using the Morris water maze. RESULTS: Decrease in NeuroD1 activity increased the initial antinociceptive median effective dose values of both morphine and fentanyl, which was reversed by restoring NeuroD1 activity. In contrast, decrease in NeuroD1 activity inhibited development of tolerance in a time-dependent manner, paralleling its effects on the acquisition and extinction of contextual memory. In addition, only development of tolerance, but not antinociceptive median effective dose values, was modulated by the expression of miR-190 and Neurod1 driven by Nestin promoter. CONCLUSIONS: Neurod1 regulates the developments of opioid tolerance via a time-dependent pathway through contextual learning and a short-response pathway through antinociception.

Sequential introduction of reprogramming factors reveals a time-sensitive requirement for individual factors and a sequential EMT-MET mechanism for optimal reprogramming.

Present practices for reprogramming somatic cells to induced pluripotent stem cells involve simultaneous introduction of reprogramming factors. Here we report that a sequential introduction protocol (Oct4-Klf4 first, then c-Myc and finally Sox2) outperforms the simultaneous one. Surprisingly, the sequential protocol activates an early epithelial-to-mesenchymal transition (EMT) as indicated by the upregulation of Slug and N-cadherin followed by a delayed mesenchymal-to-epithelial transition (MET). An early EMT induced by 1.5-day TGF-ß treatment enhances reprogramming with the simultaneous protocol, whereas 12-day treatment blocks reprogramming. Consistent results were obtained when the TGF-ß antagonist Repsox was applied in the sequential protocol. These results reveal a time-sensitive role of individual factors for optimal reprogramming and a sequential EMT-MET mechanism at the start of reprogramming. Our studies provide a rationale for further optimizing reprogramming, and introduce the concept of a sequential EMT-MET mechanism for cell fate decision that should be investigated further in other systems, both in vitro and in vivo.