Critical regulation of miR-200/ZEB2 pathway in Oct4/Sox2-induced mesenchymal-to-epithelial transition and induced pluripotent stem cell generation.

Fibroblasts can be reprogrammed to induced pluripotent stem cells (iPSCs) by application of transcription factors octamer-binding protein 4 (Oct4), SRY-box containing gene 2 (Sox2), Kruppel-like factor 4 (Klf4), and c-Myelocytomatosis oncogene (c-Myc) (OSKM), but the underlying mechanisms remain unclear. Here, we report that exogenous Oct4 and Sox2 can bind at the promoter regions of mir-141/200c and mir-200a/b/429 cluster, respectively, and induce the transcription activation of miR-200 family during the OSKM-induced reprogramming. Functional suppression of miR-200s with specific inhibitors significantly represses the OSKM-caused mesenchymal-to-epithelial transition (MET, an early event in reprogramming of fibroblasts to iPSCs) and iPSC generation, whereas overexpression of miR-200s promotes the MET and iPSC generation. Mechanistic studies showed that miR-200s significantly repress the expression of zinc finger E-box binding homeobox 2 (ZEB2) through directly targeting its 3' UTR and direct inhibition of ZEB2 can mimic the effects of miR-200s on iPSC generation and MET process. Moreover, the effects of miR-200s during iPSC generation can be blocked by ZEB2 overexpression. Collectively, our findings not only reveal that members of the miR-200 family are unique mediators of the reprogramming factors Oct4/Sox2, but also demonstrate that the miR-200/ZEB2 pathway as one critical mechanism of Oct4/Sox2 to induce somatic cell reprogramming at the early stage.

Dietary calories and lipids synergistically shape adipose tissue cellularity during postnatal growth.

OBJECTIVE: The susceptibility to abdominal obesity and the metabolic syndrome is determined to a substantial extent during childhood and adolescence, when key adipose tissue characteristics are established. Although the general impact of postnatal nutrition is well known, it is not clear how specific dietary components drive adipose tissue growth and how this relates to the risk of metabolic dysfunction in adulthood. METHODS: Adipose tissue growth including cell proliferation was analyzed in juvenile mice upon dietary manipulation with in vivo nucleotide labeling. The proliferative response of progenitors to specific fatty acids was assayed in primary cultures. Long-term metabolic consequences were assessed through transient dietary manipulation post-weaning with a second obesogenic challenge in adulthood. RESULTS: Dietary lipids stimulated adipose tissue progenitor cell proliferation in juvenile mice independently of excess caloric intake and calorie-dependent adipocyte hypertrophy. Excess calories increased mitogenic IGF-1 levels systemically, whereas palmitoleic acid was able to enhance the sensitivity of progenitors to IGF-1, resulting in synergistic stimulation of proliferation. Early transient consumption of excess lipids promoted hyperplastic adipose tissue expansion in response to a second dietary challenge in adulthood and this correlated with abdominal obesity and hyperinsulinemia. CONCLUSIONS: Dietary lipids and calories differentially and synergistically drive adipose tissue proliferative growth and the programming of the metabolic syndrome in childhood.

[Relationship between measurements of tidal volume and thoracic inflating volume by pressure plethysmography in mice].

OBJECTIVE: To evaluate the accordance of tidal volume (TV) with thoracic inflating volume (TIV) measured by pressure whole-body or double-chamber plethysmography in mice. METHODS: TV and TIV by double-chamber plethysmography were simultaneously measured in 6 mice with spontaneous respiratory frequencies 90 - 120/min; TV and TIV by whole-body plethysmography were measured in 8 paralyzed mice ventilated by fixed frequency. RESULT: TIV value by double-chamber plethysmography was significantly higher than TV [(0.369 +/- 0.014) ml vs (0.356 +/- 0.012) ml, P < 0.01)] in 6 spontaneously breathing mice. TIV value by whole-body plethysmography was significantly lower than TV[(0.233 +/- 0.003) ml vs 0.3 ml, P < 0.001] in 8 paralyzed mice. CONCLUSION: The TV value assessed by TIV measurement can not be accurate, because of the humidifying and heating of inhaled air and negative thoracic pressure during the measurement.

LincRNA-1614 coordinates Sox2/PRC2-mediated repression of developmental genes in pluripotency maintenance.

Large-intergenic noncoding RNAs (lincRNAs) cooperate with core transcription factors to coordinate the pluripotency network of embryonic stem cells. The mechanisms by which lincRNAs affect chromatin structure and gene transcription remain mostly unknown. Here, we identified that a lincRNA (linc1614), occupied by pluripotency factors at its promoter, was indispensable for both maintenance and acquisition of pluripotency. Linc1614 served as a specific partner of core factor Sox2 in maintaining pluripotency, primarily by mediating the function of Sox2 in the repression of developmental genes. Moreover, Ezh2, an essential subunit of polycomb repressive complex 2 (PRC2), physically interacted with linc1614 and contributed to lincRNA-mediated transcriptional silencing. Thus, we propose that the interplay of linc1614 with Sox2 implicates this lincRNA as a recruitment platform that mediates transcriptional silencing by guiding the PRC2 complex to the loci of developmental genes.

Comparison of low-molecular-weight-heparin and unfractionated heparin for acute PTE.

OBJECTIVE: Acute pulmonary thromboembolism (PTE) is a serious high mortality pulmonary vascular disease whose effective treatment decreases morbidity and mortality. To determine if low-molecular-weight-heparin (LMWH) is clinically as efficient and safe as unfractionated heparin (UH) in patients with diagnosis of acute non-massive PTE, our study compares the efficacy, adverse effects and costs of LMWH and UH. METHODS: One hundred and fourteen patients with non-massive acute PTE were randomly divided into LMWH (nadroparin calcium) and UH groups. Oxygenation index, D-dimer, fibrinogen (FG), lung ventilation/perfusion (V/Q) scan and computed tomography pulmonary angiography (CTPA) were observed before anticoagulation and on day 14 after anticoagulation. RESULTS: In both groups, the ABG (arterial blood gas) analysis showed PaO(2) and PaCO(2) were elevated, P(A-a)O(2) was decreased and oxygenation index (PaO(2)/FIO(2)) was elevated, D-dimer and fibrinogen were decreased, lung V/Q and CTPA showed embolized segments reduced (P<0.05). Hemorrhage and thrombocytopenia occurred in 3.5% of the LMWH group. Hemorrhage occurred in 5.3% and thrombocytopenia occurred in 7.0% of the UH group. The average cost in the LMWH group was RMB 1218.60 Yuan and RMB 1541.40 Yuan in the UH group. CONCLUSION: LMWH and UH are equally effective for treatment of non-massive acute PTE, but LMWH may have a lower prevalence of complications and is less expensive.

[Pulmonary cryptococcosis: analysis of nine cases].

OBJECTIVE: To investigate the diagnosis and treatment of pulmonary cryptococcosis. METHODS: A total of 9 cases of pulmonary cryptococcosis, diagnosed at Sir Run Run Shaw Hospital of Zhejiang University from January 2002 to August 2004, identified by pathological examinations, were retrospectively studied. RESULTS: The patients consisted of 7 males and 2 females aged from 28 to 69 years. Pulmonary nodules, either solitary or multiple, present in 8 of the 9 cases, were the most common CT finding. The diagnosis was confirmed in all cases by pathological study. The lung specimens of 5 cases were obtained by CT guided transthoracic needle aspiration lung biopsy, and these 5 cases were treated with fluconazole, after 0.5 - 1 year of follow-up, the pulmonary lesion essentially vanished. The other 4 cases were confirmed after surgery. CONCLUSIONS: Most pulmonary cryptococcosis presented as pulmonary nodules or masses on CT, either solitary or multiple. Pathology was essential to the diagnosis. Fluconazole is active against cryptococcus neoformans, and appears to be effective in the treatment.

[Large-volume whole lung lavage in the treatment of severe pulmonary alveolar proteinosis].

OBJECTIVE: To evaluate the efficacy and safety of large-volume whole lung lavage in the treatment of severe pulmonary alveolar proteinosis (PAP). METHODS: Five severe PAP patients underwent large-volume whole lung lavage under general anaesthesia using a double lumen endotracheal tube. Suction was used for returning fluid. The volume of lavage and return volume and the return rate were calculated. The efficacy was evaluated according to the improvement of symptoms, amelioration of chest radiograph or chest CT scan and the changes of pulmonary function and artery blood gas analysis. RESULTS: After lavage, the symptoms, pulmonary function and artery blood gas were markedly improved, and the chest radiographs showed dissipation of lung consolidation. No severe complications were observed in the 5 cases. CONCLUSION: Large-volume whole lung lavage is an effective and safe therapeutic method in the treatment of PAP.

microRNA-29b is a novel mediator of Sox2 function in the regulation of somatic cell reprogramming.

Fibroblasts can be reprogrammed into induced pluripotent stem cells (iPSCs) by the application of Yamanaka factors (OSKM), but the mechanisms underlying this reprogramming remain poorly understood. Here, we report that Sox2 directly regulates endogenous microRNA-29b (miR-29b) expression during iPSC generation and that miR-29b expression is required for OSKM- and OSK-mediated reprogramming. Mechanistic studies show that Dnmt3a and Dnmt3b are in vivo targets of miR-29b and that Dnmt3a and Dnmt3b expression is inversely correlated with miR-29b expression during reprogramming. Moreover, the effect of miR-29b on reprogramming can be blocked by Dnmt3a or Dnmt3b overexpression. Further experiments indicate that miR-29b-DNMT signaling is significantly involved in the regulation of DNA methylation-related reprogramming events, such as mesenchymal-to-epithelial transition (MET) and Dlk1-Dio3 region transcription. Thus, our studies not only reveal that miR-29b is a novel mediator of reprogramming factor Sox2 but also provide evidence for a multistep mechanism in which Sox2 drives a miR-29b-DNMT signaling axis that regulates DNA methylation-related events during reprogramming.

Synergetic cooperation of microRNAs with transcription factors in iPS cell generation.

Induced pluripotent stem (iPS) cells were first generated by forced expression of transcription factors (TFs) in fibroblasts. Recently, iPS cells have been generated more rapidly and efficiently using miRNAs with or without other transcription factors. However, the specific and collaborative roles of miRNAs and transcription factors in pluripotency acquisition and maintenance remain to be further investigated. Here, based on the miRNA profiling in mouse embryonic fibroblasts (MEFs), MEFs infected with Oct3/4, Sox2, Klf4 and c-Myc (OSKM) for 1, 2, 4, or 8 day, two iPS cell lines and ES cells, representing iPS activation and maintenance steps, we found that two unique miRNA sets are responsible for different steps of iPS generation, and the miRNA expression profiles of iPS cells are very similar to that of ES cells. Furthermore, we searched for transcription factors binding sites at the promoter regions of up-regulated miRNAs, and found that up-regulated miRNAs such as the miR-429-200 and miR-17 clusters are directly activated by exogenous TFs. The GO and pathway enrichment for candidate target gene sets of miRNAs or OSKM provided a clear picture of division and collaboration between miRNAs and OSKM during completion of the iPS process. Compared with the pathways regulated by OSKM, we found that miRNAs play critical roles in regulating iPS-specific pathways, such as the adherens junction and Wnt signaling pathways. Furthermore, we blocked miRNA expression using Dicer knockdown, and found that the level of miRNAs was decreased following this treatment, and the efficiency of iPS generation was significantly repressed. By combining high-throughput analysis, biostatistical analysis and functional experiments, this study provides new ideas for investigating the important roles of miRNAs, the mechanisms of miRNAs and related signaling pathways, and the potential for many more applications of miRNAs in somatic cell reprogramming.