Suppression of certain intestinal microbiota metabolites may lead to gestational diabetes in mice fed a high-fat diet.

Background: We aim to establish a gestational diabetes mellitus (GDM) mouse model with mice fed with a high-fat diet (HFD) in comparison with pregnant mice with normal blood glucose levels to investigate the role of intestinal microbiota in the development of HFD-induced GDM. Methods: We divided healthy 6-week-old female C57BL mice into an HFD-induced GDM group and a normal diet group. Their bacterial flora and metabolites in intestinal fecal exosomes were co-analyzed using 16 s multi-region sequencing and compared. Findings: Alpha (α) diversity was lower within the model group compared to the control group. Beta (ß) diversity was significantly different between the two groups. The relative abundances of Lactobacillus, Actinomyces, Rothia, and Bacteroidetes were significantly different between the two groups. Fermentation and nitrate consumption were significantly higher in the GDM group. Multiple bacteria were associated with glycerophosphocholine, S-methyl-5'-thioadenosine, quinolinate, galactinol, deoxyadenosine, DL-arginine, and 2-oxoadenic acid. Interpretation: Imbalances in the production of Lactobacillus, Bacteroidetes, Actinomyces, and Rothia and their related metabolites may lead to metabolic disturbances in GDM. These indicators may be used to assess changes affecting the intestinal microbiota during pregnancy and thus help modulate diet and alter blood glucose.

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.

Effects of resistance exercise on blood glucose level and pregnancy outcome in patients with gestational diabetes mellitus: a randomized controlled trial.

INTRODUCTION: To date, the effects of resistance exercise on diabetes-related parameters (blood glucose level and insulin use) and pregnancy outcome in participants with gestational diabetes mellitus (GDM) have not been compared with those of aerobic exercise. To investigate the effect of resistance exercise versus aerobic exercise on blood glucose level, insulin utilization rate, and pregnancy outcome in patients with GDM. RESEARCH DESIGN AND METHODS: From December 2019 to December 2020, 100 pregnant women with GDM were selected and divided into a resistance exercise group (49 patients) and an aerobic exercise group (51 patients) randomly. The aerobic exercise group received an aerobic exercise intervention, while the resistance exercise group received a resistance exercise intervention. Both groups received exercise intervention for 50-60 min, 3 times per week, lasting for 6 weeks. In addition, patients in both groups received the same routine care, including personalized dietary intervention, online education, and school courses for pregnant women. RESULTS: The blood glucose level in the resistance exercise group and the aerobic exercise group was lower after the intervention than before the intervention (p<0.05). After the intervention, no significant differences were observed in the fasting blood glucose level, insulin utilization rate, and incidence of adverse pregnancy outcomes between the two groups (p>0.05); however, significant differences were noted in 2-hour postprandial blood glucose level and exercise compliance between the two groups (p<0.05), with the resistance exercise group showing better outcomes than the aerobic exercise group. CONCLUSIONS: Resistance exercise is more compliant for pregnant women with GDM than aerobic exercise; hence, it is necessary to popularize resistance exercise in this specific population group. Long-term effects of resistance exercise should be evaluated in future studies. TRIAL REGISTRATION NUMBER: ChiCTR 1900027929.

Amelioration of murine beta-thalassemia through drug selection of hematopoietic stem cells transduced with a lentiviral vector encoding both gamma-globin and the MGMT drug-resistance gene.

Correction of murine models of beta-thalassemia has been achieved through high-level globin lentiviral vector gene transfer into mouse hematopoietic stem cells (HSCs). However, transduction of human HSCs is less robust and may be inadequate to achieve therapeutic levels of genetically modified erythroid cells. We therefore developed a double gene lentiviral vector encoding both human gamma-globin under the transcriptional control of erythroid regulatory elements and methylguanine methyltransferase (MGMT), driven by a constitutive cellular promoter. MGMT expression provides cellular resistance to alkylator drugs, which can be administered to kill residual untransduced, diseased HSCs, whereas transduced cells are protected. Mice transplanted with beta-thalassemic HSCs transduced with a gamma-globin/MGMT vector initially had subtherapeutic levels of red cells expressing gamma-globin. To enrich gamma-globin-expressing cells, transplanted mice were treated with the alkylator agent 1,3-bis-chloroethyl-1-nitrosourea. This resulted in significant increases in the number of gamma-globin-expressing red cells and the amount of fetal hemoglobin, leading to resolution of anemia. Selection of transduced HSCs was also obtained when cells were drug-treated before transplantation. Mice that received these cells demonstrated reconstitution with therapeutic levels of gamma-globin-expressing cells. These data suggest that MGMT-based drug selection holds promise as a modality to improve gene therapy for beta-thalassemia.

Public Knowledge, Attitudes, and Practices Behaviors Towards Coronavirus Disease 2019 (COVID-19) During a National Epidemic-China.

Background: The rapid outbreak of coronavirus disease 2019 (COVID-19) posed a serious threat to China, followed by compulsive measures taken against the national emergency to control its further spread. This study was designed to describe residents' knowledge, attitudes, and practice behaviors (KAP) during the outbreak of COVID-19. Methods: An anonymous online questionnaire was randomly administrated to residents in mainland China between Mar 7 and Mar 16, 2020. Residents' responses to KAP were quantified by descriptive and stratified analyses. A Multiple Logistic Regression model was employed to identify risk factors associated with KAP scores. Results: A total of 10,195 participants were enrolled from 32 provinces of China. Participants of the ≥61 years group had higher KAP scores [adjusted Odds Ratio (ORadj) = 4.8, 95% Confidence Interval (CI): 3.0-7.7, P < 0.0001], and the married participants and those in low-income families had higher scores of KAP (ORadj = 1.2, 95% CI: 1.1-1.3; ORadj = 1.8, 95% CI: 1.6-2.2, respectively, both P < 0.0001). The participants living with more than two family members had higher scores in an increasing ORs when the family members increased (ORadj = 1.3, 95% CI: 1.1-1.6, P = 0.013; ORadj = 1.3, 95% CI: 1.1-1.6, P = 0.003; ORadj = 1.3, 95% CI: 1.0-1.6, P = 0.02; for groups of 2, 3-4 and ≥5, respectively). Conclusions: Out of the enrolled participants who completed the survey, 85.5% responded positively toward the mandatory public health interventions implemented nationwide by the Chinese authorities. These effective practices seem to be related to a proper attitude generated by the increased knowledge and better awareness of the risks related to the COVID-19 pandemic and the consequent need for safe and responsible behavior.

Analysis of alpha hemoglobin stabilizing protein overexpression in murine ß-thalassemia.

Excess free alpha-globin is cytotoxic and contributes to the pathophysiology of b-thalassemia. Alpha hemoglobin stabilizing protein (AHSP) is a molecular chaperone that binds free alpha-globin to promote its folding and inhibit its ability to produce damaging reactive oxygen species. Reduced AHSP levels correlate with increased severity of b-thalassemia in some human cohorts, but causal mechanistic relationships are not established for these associations. We used transgenic and lentiviral gene transfer methods to investigate whether supraphysiologic AHSP levels could mitigate the severity of b-thalassemia intermedia by providing an increased sink for the excess pool of alpha-globin chains. We tested wild-type AHSP and two mutant versions with amino acid substitutions that confer 3- or 13-fold higher affinity for alpha-globin. Erythroid overexpression of these AHSP proteins up to 11-fold beyond endogenous levels had no major effects on hematologic parameters in b-thalassemic animals. Our results demonstrate that endogenous AHSP is not limiting for a-globin detoxification in a murine model of b-thalassemia.

Optimized lentiviral vector design improves titer and transgene expression of vectors containing the chicken beta-globin locus HS4 insulator element.

Hematopoietic cell gene therapy using retroviral vectors has achieved success in clinical trials. However, safety issues regarding vector insertional mutagenesis have emerged. In two different trials, vector insertion resulted in the transcriptional activation of proto-oncogenes. One strategy for potentially diminishing vector insertional mutagenesis is through the use of self-inactivating lentiviral vectors containing the 1.2-kb insulator element derived from the chicken beta-globin locus. However, use of this element can dramatically decrease both vector titer and transgene expression, thereby compromising its practical use. Here, we studied lentiviral vectors containing either the full-length 1.2-kb insulator or the smaller 0.25-kb core element in both orientations in the partially deleted long-terminal repeat. We show that use of the 0.25-kb core insulator rescued vector titer by alleviating a postentry block to reverse transcription associated with the 1.2-kb element. In addition, in an orientation-dependent manner, the 0.25-kb core element significantly increased transgene expression from an internal promoter due to improved transcriptional termination. This element also demonstrated barrier activity, reducing variability of expression due to position effects. As it is known that the 0.25-kb core insulator has enhancer-blocking activity, this particular insulated lentiviral vector design may be useful for clinical application.

[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.

Increased Engraftment of Human Short Term Repopulating Hematopoietic Cells in NOD/SCID/IL2rγnull Mice by Lentiviral Expression of NUP98-HOXA10HD.

Techniques to expand human hematopoietic stem cells ex-vivo could be beneficial to the fields of clinical hematopoietic stem cell transplantation and gene therapy targeted at hematopoietic stem cells. NUP98-HOXA10HD is a relatively newly discovered fusion gene that in mouse transplant experiments has been shown to increase numbers of hematopoietic stem cells. We evaluated whether this fusion gene could be used to expand engrafting human primitive CD34+ cells in an immunodeficient mouse model. Gene transfer was achieved using a lentiviral based vector. The engraftment of mobilized peripheral blood human CD34+ cells grown in culture for one week after gene transfer was evaluated 3-4 months after transplant and found to be 2-3 fold higher in the NUP98-HOXA10HD groups as compared to controls. These data suggest an expansive effect at least at the short term human repopulating cell level. Further evaluation in long term repopulating models and investment in a NUP98-HOXA10HD protein seems worthy of consideration. Additionally, the results here provide strong impetus to utilize NUP98-HOXA10HD as a tool to search for underlying genes and pathways involved in hematopoietic stem cell expansion that can be enhanced and have an even more potent expansive effect.

Amelioration of murine sickle cell disease by nonablative conditioning and γ-globin gene-corrected bone marrow cells.

Patients with severe sickle cell disease (SCD) are candidates for gene therapy using autologous hematopoietic stem cells (HSCs), but concomitant multi-organ disease may contraindicate pretransplant conditioning with full myeloablation. We tested whether nonmyeloablative conditioning, a regimen used successfully for allogeneic bone marrow transplantation of adult SCD patients, allows engraftment of γ-globin gene-corrected cells to a therapeutic level in the Berkeley mouse model of SCD. Animals transplanted according to this regimen averaged 35% engraftment of transduced hematopoietic stem cells with an average vector copy < 2.0. Fetal hemoglobin (HbF) levels ranged from 20 to 44% of total hemoglobin and approximately two-thirds of circulating red blood cells expressed HbF detected by immunofluorescence (F-cells). Gene therapy treatment of SCD mice ameliorated anemia, reduced hyperleukocytosis, improved renal function, and reduced iron accumulation in liver, spleen, and kidneys. Thus, modest levels of chimerism with donor cells expressing high levels of HbF from an insulated γ-globin lentiviral vector can improve the pathology of SCD in mice, thereby illustrating a potentially safe and effective strategy for gene therapy in humans.

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.

Gene therapy for canine leukocyte adhesion deficiency with lentiviral vectors using the murine stem cell virus and human phosphoglycerate kinase promoters.

Children with leukocyte adhesion deficiency type 1 (LAD-1) and dogs with canine LAD (CLAD) develop life-threatening bacterial infections due to mutations in the leukocyte integrin CD18. Here, we compared the human phosphoglycerate kinase (hPGK) promoter to the murine stem cell virus (MSCV) promoter/enhancer in a self-inactivating HIV-1-derived lentiviral vector to treat animals with CLAD. Four CLAD dogs were infused with CD34(+) cells transduced with the hPGK vector, and two CLAD dogs received MSCV vector-transduced CD34(+) cells. Infusions were preceded by a nonmyeloablative dose of 200 cGy total body irradiation. Comparable numbers of transduced cells were infused in each group of animals. Only one of four CLAD animals treated with the hPGK-cCD18 vector had reversal of CLAD, whereas both MSCV-cCD18 vector-treated dogs had reversal of the phenotype. Correction of CLAD depends both upon the percentage of CD18(+) myeloid cells and the level of expression of CD18 on individual myeloid cells. In this regard, the hPGK promoter directed low levels of expression of CD18 on neutrophils compared to the MSCV promoter, likely contributing to the suboptimal clinical outcome with the hPGK vector.

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.