Recent progress in metabolic reprogramming in gestational diabetes mellitus: a review.

Gestational diabetes mellitus is a prevalent metabolic disease that can impact the normal course of pregnancy and delivery, leading to adverse outcomes for both mother and child. Its pathogenesis is complex and involves various factors, such as insulin resistance and ß-cell dysfunction. Metabolic reprogramming, which involves mitochondrial oxidative phosphorylation and glycolysis, is crucial for maintaining human metabolic balance and is involved in the pathogenesis and progression of gestational diabetes mellitus. However, research on the link and metabolic pathways between metabolic reprogramming and gestational diabetes mellitus is limited. Therefore, we reviewed the relationship between metabolic reprogramming and gestational diabetes mellitus to provide new therapeutic strategies for maternal health during pregnancy and reduce the risk of developing gestational diabetes mellitus.

Lentiviral Transfer of γ-Globin with Fusion Gene NUP98-HOXA10HD Expands Hematopoietic Stem Cells and Ameliorates Murine ß-Thalassemia.

Recently, an engineered Homeobox-nucleoporin fusion gene, NUP98-HOXA10HD or NA10HD, was reported to expand and maintain murine hematopoietic stem cells (HSCs). We postulated that NA10HD would increase the number of human γ-globin-expressing cells to therapeutic levels. We developed a double gene lentiviral vector encoding both human γ-globin and NA10HD, which was used to transduce human peripheral blood CD34+ cells and increased engraftment 2- to 2.5-fold at 15 weeks post-transplantation in immunodeficient mice. In ß-thalassemic mice transplanted with ß-thalassemic HSCs transduced with the γ-globin/NA10HD vector, the number of fetal hemoglobin (HbF)-expressing cells was significantly increased after 3 months, leading to resolution of the anemia. Furthermore, the increases in HbF were maintained at 6 months and persisted after secondary transplantation. In addition, NA10HD enrichment of transduced HSCs led to HbF increases without affecting homeostasis of the white blood cell lineages. Our results suggest that NA10HD increases the number of γ-globin-transduced HSCs that engraft, leading to an elevated number of fetal hemoglobin-containing red cells. These effects of NA10HD provide an improved platform for testing of the therapeutic efficacy of novel globin vectors and provide further impetus to develop safe and effective methods for selective expansion of genetically modified cells.

[Analysis of treatment and prognosis of orbital adenoid cystic carcinoma].

OBJECTIVE: To summarize the treatment outcomes of orbital adenoid cystic carcinoma and to evaluate prognostic factors. METHOD: A retrospective case series study was performed on 75 patients with orbital adenoid cystic carcinoma treated from 1991 to 2006. RESULTS: The 2- and 5-year local recurrence rate of solid type orbital adenoid cystic carcinoma was significantly higher than that of the adeno-tubiform type [2-year, 85% (17/20) vs 23.53% (8/34), chi(2) = 19.14, P = 0.000; 5-year, 100% (19/19) vs 64.52% (20/31), Fisher's exact test, P = 0.003]. The regional extension and distant metastasis of solid type were more than those of adeno-tubiform type. The 5-year local recurrence rate treated by postoperative radiation was lower than that treated with only surgical excision [70% (14/20) vs 92.86% (13/14); Fisher's exact test, P = 0.198]. The 5-year local recurrence rate in patients initially treated by orbital evisceration during the first time was lower than that of cases which evisceration procedure was used after the recurrence [25% (1/4) vs 75% (6/8), Fisher's exact test, P = 0.222]. Tumors may extend into intracalvarium, nasal cavity and temporal fossa. They may spread to the lung, bone, liver and lymph node. The 5-year metastasis rate was 25.71% (9/35). Both of the lung and bone metastasis rates were 33.33% (3/9). The overall 5-year accumulative survival was 74.29% (26/35), mortality was 25.71% (9/35), and rate of survival without tumor recurrence was 37.14% (13/35). The 10-year disease free survival rate was 17.14% (6/35). Patients were most likely to die with intracranial extension. Surgical excision with postoperative radiation improved the 5-year survival rate to 80% (16/20). CONCLUSIONS: Orbital adenoid cystic carcinoma is one of the most malignant tumors in the orbit. They have a high local recurrence rate and survival rate. Tumor histological types and the treatment procedure can influence the prognosis. Combined therapy may decrease the recurrence and increase the survival rate.

High-throughput screening of effective siRNAs from RNAi libraries delivered via bacterial invasion.

Use of RNA interference (RNAi) as a reverse genetics tool for silencing genes in mammalian cells is achieved by in vitro transfection of small interfering RNAs (siRNAs). For a target gene, several siRNAs must be designed according to the empirical rules. We demonstrated that functional short hairpin RNAs (shRNAs) could be synthesized in Escherichia coli and delivered directly via bacterial invasion to the near entirety of a mammalian cell population to trigger RNAi. Furthermore, using a luciferase-target gene transcript, we identified effective shRNAs and siRNAs from RNAi libraries delivered conveniently through bacterial invasion in 96-well plates without need for preparation, purification and transfection of shRNAs. Notably, several of the most highly effective shRNAs and siRNAs identified do not fit the empirical rules commonly used for siRNA design, suggesting that this approach is a powerful tool for RNAi research, and could be used complementarily to the empirical rules for RNAi applications.

A mammalian genetic system to screen for small molecules capable of disrupting protein-protein interactions.

A mammalian two-hybrid system was developed for high-throughput screening of compounds that disrupt specific protein-protein interactions. The existing mammalian systems are unsatisfactory for drug screening due to nonregulated expression of interacting proteins. To construct a tightly regulated system, the tetracycline repressor was fused with the inhibitory KRAB domain as a suppressor. The binding of the suppressor to the tet operator entirely blocked expression of two interacting proteins. When both the inducer doxycycline and drugs were added to the culture, the reporter gene was either activated by interaction of the paired proteins with ineffective drugs or remained silent due to disruption of the protein interactions by the effective drugs. We demonstrate that interactions of the type I receptor for TGFbeta with FKBP12 and the epidermal growth factor receptor (EGFR) with p85 are effectively disrupted by FK506 and EGFR kinase inhibitor AG1478, respectively. The power of this system for drug screening was further demonstrated by rapid identification of inhibitors from a druglike library for the receptor kinases.

A coumermycin/novobiocin-regulated gene expression system.

Regulated expression of transgenes in mammals is an important technique in both functional genomic studies and clinical applications. Here we describe a regulated gene expression system for mammals, based on coumarin-switched dimerization of the bacterial DNA gyrase B subunit (GyrB). The transactivator was constructed by fusing the GyrB activator to the bacterial lambda repressor-binding domain. The antibiotic coumermycin in nanomolar concentrations activated the transgene through binding of the homodimerized chimeric transactivator to the lambda operator located upstream of a minipromoter. More significantly, addition of novobiocin, an antagonist of coumermycin, promptly switched off expression of the gene by abolishing coumermycin-induced dimerization of the transactivator. Site-directed mutagenesis of the lambda repressor-binding domain resulted in significant reduction of basal expression levels and an induction reaching four orders of magnitude in stably transfected 293A cells in response to coumermycin. The capability of this inducible system for tightly regulated gene expression was demonstrated by the ready generation of stable cell lines inducibly expressing the proapoptotic bax gene in mammalian cells. Hence, this novel coumarin switch-on/switch-off system should broaden the utility of regulated gene expression, particularly when rapid on/off interchange is required.