Interaction between mDia1 and ROCK in Rho-induced migration and adhesion of human dental pulp cells.

AIM: To investigate the effects of mammalian homologue of Drosophila diaphanous-1(mDia1) and Rho-associated coiled-coil-containing protein kinase (ROCK) on the migration and adhesion of dental pulp cells (DPCs). METHODOLOGY: Lysophosphatidic acid (LPA) was used to activate Rho signalling. mDia1 and ROCK were inhibited by short interfering RNA and the specific inhibitor, Y-27632, respectively. The migration of DPCs was assessed using the transwell migration assay and scratch test. Formation of cytoskeleton and focal adhesions(FAs) was observed by confocal laser scanning microscopy. Cell adhesion and spreading assays were performed. Phosphorylation of focal adhesion kinase (FAK) and paxillin was detected by Western blotting, and the bands were analysed using Adobe Photoshop CS5 software. All experiments were performed at least three times, and data were analysed with one-way anova and a post hoc test. RESULTS: LPA-triggered activation of Rho and inhibition of ROCK significantly increased the cell migration rate. Cell migration was inhibited by silencing mDia1. mDia1 silencing and ROCK inhibition suppressed the LPA-induced formation of the cytoskeleton, FA and phosphorylation of FAK and paxillin. Inhibition of ROCK or mDia1 facilitated early cell adhesion and spreading; by contrast, the combined inhibition of ROCK and mDia1 neutralized these effects. CONCLUSIONS: mDia1 promoted RhoA-induced migration of DPCs, but ROCK had an opposite effect. Both mDia1 and ROCK participated in cytoskeleton formation and adhesion of DPCs. The interactions between mDia1 and ROCK might influence dental pulp repair by determining the migration and adhesion of DPCs.

Effects of maternal folic acid supplementation on gene methylation and being small for gestational age.

BACKGROUND: Being small for gestational age (SGA), a foetal growth abnormality, has a long-lasting impact on childhood health. Its aetiology and underlying mechanisms are not well understood. Underlying epigenetic changes of imprinted genes have emerged as a potential pathological pathway because they may be associated with growth, including SGA. As a common methyl donor, folic acid (FA) is essential for DNA methylation, synthesis and repair, and FA supplementation is widely recommended for women planning pregnancy. The present study aimed to investigate the inter-relationships among methylation levels of two imprinted genes [H19 differentially methylated regions (DMRs) and MEST DMRs], maternal FA supplementation and SGA. METHODS: We conducted a case-control study. Umbilical cord blood was taken from 39 SGA infants and 49 controls whose birth weights are appropriate for gestational age (AGA). DNA methylation levels of H19 and MEST DMRs were determined by an analysis of mass array quantitative methylation. RESULTS: Statistically significantly higher methylation levels were observed at sites 7.8, 9 and 17.18 of H19 (P = 0.030, 0.016 and 0.050, respectively) in the SGA infants compared to the AGA group. In addition, the association was stronger in male births where the mothers took FA around conception at six H19 sites (P = 0.004, 0.005, 0.048, 0.002, 0.021 and 0.005, respectively). CONCLUSIONS: Methylation levels at H19 DMRs were higher in SGA infants compared to AGA controls. It appears that the association may be influenced by maternal peri-conception FA supplementation and also be sex-specific.

[Clinical analysis on effect of retroperitoneoscopic nephrectomy in elderly donors for renal transplantation].

OBJECTIVE: To evaluate the safety and effectiveness of retroperitoneoscopic donor nephrectomy in elderly donors for renal transplantation. METHODS: A retrospective analysis was conducted with 123 cases of retroperitoneoscopic living donor kidney transplantation in 309th Hospital of PLA from March 2011 to March 2014, including 44 elderly donors (age≥55 years) and 79 young to middle-aged donors (age <55 years). Comparisons were made in terms of postoperative complications in both donors and recipients, renal function recovery in the donors and function of graft in the recipients. RESULTS: The clinical baseline data of the two groups shows that glomerular filtration rate (GFR) of donors in the elderly donor group was lower than the young donor group (P=0.04). The 123 donors all underwent retroperitoneoscopic donor nephrectomy successfully. Postoperative complications in donors and recipients of both groups had no significant differences (P=0.60; P=1.00). In the elderly donor group, the mean serum creatinine level of donors was significantly higher than that in the young donors group [(115.8±22.3) vs (102.5±16.3) µmol/L, P<0.01] 3 days after operation; and estimated GFR (eGFR) was lower [(53.0±9.1)vs(59.6±8.3)ml·min(-1)·(1.73 m(2))(-1,) P<0.01]. Serum creatinine and eGFR of the two groups showed no significant differences one week and six months after surgery (all P>0.05). Four recipients in the elderly donor group had delayed graft function (DGF), 3 had acute rejection; 8 recipients in the young donor group had DGF, 5 had acute rejection; no statistically significant differences were observed between the 2 groups (both P=1.00). Recipients' eGFR were higher in the young donor group than in the elderly donor group at 1 week, 1 month, 3 months, 6 months and 12 months after surgery, but with no statistically significant differences(all P>0.05). After (27.8±12.6) months follow-up, 1 recipient in the elderly donor group died from pulmonary infection; two recipients in the young donor group had kidney dysfunction. Graft survival in the two groups showed no significant difference(P=0.95). CONCLUSIONS: Retroperitoneoscopic donor nephrectomy is safe and feasible for elderly donors. With careful preoperative evaluation, precise operation, and close postoperative monitoring and follow-up, it could provide satisfactory clinical outcome.

Casp11 Deficiency Alters Subgingival Microbiota and Attenuates Periodontitis.

Periodontitis (PD) is the primary cause of tooth loss in adults. Porphyromonas gingivalis (P.g), a keystone pathogen, has been identified as a crucial contributor to this process. Pyroptosis activation in PD is acknowledged, with accumulating evidence underscoring the crucial role of Caspase-11 (described as Caspase-4/5 in humans)-mediated noncanonical pyroptosis. However, the mechanism behind its impact on PD remains unclear. In this study, we delved into the interplay between the Caspase-11-mediated noncanonical pyroptosis, subgingival microbiota alteration, and macrophage polarization. Clinical samples from PD patients revealed heightened expression of Caspase-4, gasdermin-D, and their active fragments, pointing to the activation of the noncanonical pyroptosis. Single-cell sequencing analysis linked Caspase-4 with gingival macrophages, emphasizing their involvement in PD. In vitro cell experiments confirmed that P.g-induced pyroptosis was activated in macrophages, with Casp11 deficiency attenuating these effects. In an experimental PD mouse model, Casp11 deficiency led to an alteration in subgingival microbiota composition and reduced alveolar bone resorption. Casp11-/- mice cohousing with wild-type mice confirmed the alteration of the subgingival microbiota and aggravated the alveolar bone resorption. Notably, Casp11 deficiency led to decreased M1-polarized macrophages, corresponding with reduced alveolar bone resorption, uncovering a connection between subgingival microbiota alteration, macrophage M1 polarization, and alveolar bone resorption. Taken together, we showed that Caspase-11 fulfilled a crucial role in the noncanonical pyroptosis in PD, potentially influencing the subgingival microbiota and linking to M1 polarization, which was associated with alveolar bone resorption. These findings underscored the pivotal role of the Caspase-11-mediated noncanonical pyroptosis in PD pathogenesis and may provide critical insights into potential therapeutic avenues for mitigating PD.

[Analysis of genetic etiology of 234 deaths in the pediatric intensive care unit with suspected genetic diseases].

Objective: To explore the genetic etiology of pediatric intensive care unit (PICU) mortality cases and summarize their clinical characteristics. Methods: This was a retrospective cohort study. The study population consisted of 234 children who died within 7 d after admitted to the PICU of Children's Hospital of Fudan University from January 2017 to December 2021. The clinical diagnoses, laboratory test results, and genetic testing results were collected. These patients were divided into the pathogenic gene variation positive (PGVP) group and the pathogenic gene variation negative (PGVN) group according to the results of genetic testing. The Mann-Whitney U test and Pearson's chi-square test or Fisher's exact probability method were used to compare the clinical characteristics between the groups. Results: A total of 234 cases were enrolled, including 139 (59.4%) males and 95 (40.6%) females. The age at death was 1.0 (0.4, 3.7) years old and the length of PICU stay was 16 (6, 33) days. There were 62 cases (26.5%) PGVP, and the mutated pathogenic genes included immune genes (23 cases (37.1%)), metabolic genes (11 cases (17.7%)), neuromuscular genes (11 cases (17.7%)), cardiovascular genes (4 cases (6.5%)), and genes of other systems (13 cases (21.0%)). The age at death in PGVP cases was significantly lower than in PGVN cases (0.6 (0.3, 1.4) vs. 1.3(0.5, 4.3) years old, Z=3.85, P<0.001). Compared with the PGVN group, the PGVP group had a higher incidence of family history and chronic complex conditions (CCC) than the PGVN group (6.5% (4/62) vs. 0.6% (1/172) and 93.5% (58/62) vs. 76.2% (131/172), χ2=8.87, P=0.018 and 0.003, respectively). Children in the PGVP group were admitted with higher incidence of severe infection, decreased consciousness or coma, moderate-to-severe anemia, thrombocytopenia, protracted diarrhea, and abnormalities in muscle strength or tone than those in the PGVN group (74.2%(46/62) vs. 45.9%(79/172), 50.0%(31/62) vs. 35.5%(61/172), 32.3%(20/62) vs. 18.0%(31/172), 21.0%(13/62) vs. 10.5%(18/172), 25.8%(16/62) vs. 4.1%(7/172), 16.1%(10/62) vs. 5.2%(9/172), χ2=14.63, 4.04, 5.41, 4.37, 24.30, 7.25, all P<0.05). Pathogenic genes that occurred more than twice included IL2RG (5 cases), SMN1 (4 cases), and SH2D1A (3 cases, including 2 single gene varients and 1 copy number varient). Conclusions: Among the deceased cases in the PICU, the main genetic causes are immune-related, metabolic, and neuromuscular genetic disorders. Critically ill children with a family history, CCC, and early features such as severe infections, decreased consciousness or coma, moderate to severe anemia, thrombocytopenia, protracted diarrhea, or abnormalities in muscle strength or tone should be closely monitored and undergo early genetic testing.

The noggin2 gene of Gekko japonicus (Gekkonidae) is down-regulated in the spinal cord after tail amputation.

The cDNA encoding noggin2 protein was obtained from the brain and spinal cord cDNA library of Gekko japonicus. The size of the noggin2 transcript and its expression in different tissues were analyzed by Northern blot analysis. In situ hybridization revealed positive hybridization signals in both gray and white matter of the spinal cord. Changes in noggin2 expression in the spinal cord after tail amputation were examined by real-time PCR. The noggin2 was expressed in the normal spinal cord and down-regulated three days after tail amputation, reaching the lowest level at two weeks, during the time course when we followed the expression levels. We concluded that the expression of noggin2 is affected by the process of spinal cord injury and regeneration.

Short-term anti-CD25 monoclonal antibody administration down-regulated CD25 expression without eliminating the neogenetic functional regulatory T cells in kidney transplantation.

CD4(+)CD25(+) forkhead box P3 (FoxP3)(+)regulatory T (T(reg)) cells are generated and play a key role in the induction and maintenance of transplant tolerance in organ recipients. It has been proposed that interleukin (IL)-2/IL-2 receptor (IL-2R) signalling was essential for the development and proliferation of antigen-activated T cells that included both effector T cells and T(reg) cells. Basiliximab (Simulect), a chimeric monoclonal antibody directed against the alpha-chain of the IL-2R (CD25), can be expected to not only affect alloreactive effector T cells, but also reduce the number and function of T(reg) cells. We therefore examined the effect of basiliximab induction therapy on the number and function of the T(reg) cells in renal recipients. Basiliximab decreased the percentage of CD4(+)CD25(+)T cells, but failed to influence the percentage of CD4(+)FoxP3(+) T(reg) cells. The cellular CD25 expression was decreased significantly by basiliximab injection, but CD4(+)CD25(+) T cells was not depleted from the circulating pool through monoclonal antibody activation-associated apoptosis. Functional analysis revealed that inhibitory function of T(reg) cells from recipients with basiliximab injection was not significantly different from recipients without injection. These data indicate that the functional T(reg) population may not be influenced by short-term basiliximab treatment.

Does endothelial chimerism correlate with renal allograft rejection?

BACKGROUND: The blood vessels of a transplanted organ are an interface between the donor and the recipient. The endothelium is believed to be a major target for graft rejection. After transplantation endothelial cells of a transplanted organ may be of recipient origin. OBJECTIVES: In this study we sought to determine whether endothelial chimerism correlates with graft rejection. METHODS: Biopsy samples from 34 renal transplants of female recipients who received kidneys from male donors were studied for the presence of endothelial cells of recipient origin. Formalin-fixed, paraffin-embedded tissue sections of renal biopsy samples were examined by fluorescence in situ hybridization (FISH) for the presence of endothelial cells containing two X chromosomes, using a biotinylated Y-chromosome probe and digoxigenin-labeled X-chromosome probe. RESULTS: The FISH methods identified endothelial cells of recipient origin. Endothelial chimerism was common, irrespective of rejection. Its presence was focal with these elements, coexisting in the biopsy. CONCLUSIONS: We observed no correlation between the percentage of recipient endothelial cells among vascular elements and the type of graft rejection (P > .05).

Clinical significance of monitoring serum level of matrix metalloproteinase 9 in patients with acute kidney allograft rejection.

This study was designed to explore the clinical significance of dynamically monitoring the serum level of matrix metalloproteinase 9 (MMP-9) before and after renal transplantation. Before transplantation and 1, 3, 5, 7, 10, 15, and 20 days after transplantation, the peripheral blood was collected from 102 renal transplant recipients, including 8 with acute rejection (ARs) and 94 non-ARs. The serum MMP-9 level was detected by Luminex 200 analyzer (Luminex Corporation, Austin, TX, USA). By day 3 post-transplantation, the serum MMP-9 level in non-ARs had significantly reduced as compared to the pretransplantation level, and reached the lowest value on day 20 post-transplantation. In contrast, the serum MMP-9 level in ARs had significantly increased by day 3, reached the highest value on day 7, and remained significantly higher on day 20 as compared to the pretransplantation level. The receiver operating characteristic curve was plotted to evaluate the power of serum MMP-9 level on day 20 post-transplantation to differentiate the non-AR and AR groups. Our data revealed that with a threshold of 8473.26 pg/mL, the area under the curve was 0.758 (0.661, 0.856); the sensitivity and specificity of the diagnostic were 78.40% and 61.30%, respectively; the positive and the negative predictive values were 74.60% and 66.67%, respectively; and the accuracy rate was up to 71.57%. Taken together, the results indicated that dynamically monitoring serum MMP-9 levels in renal allograft recipients might be a convenient and safe method to diagnose ARs.

Thermomechanical analysis of shape memory devices.

Shape memory alloys (SMA) are being increasingly used in various industrial applications as actuators, connectors, or damping materials. In the medical field, superelastic devices such as eyeglass frames, stents or guide catheters have come to market in the recent years. The design of SMA devices has usually been based on trial and error, since until recently no general simulation model was available to assist application engineers. The purpose of this article is to describe the computational methodology developed, validated and used for several industrial projects at Ecole Polytechnique of Montréal to simulate the thermomechanical behavior of shape memory materials. This new approach includes three main stages: experimental characterization, construction of a nonlinear material law based on dual kriging interpolation and finally, calculation of the thermomechanical response of SMA devices. For complex geometry, finite element analysis is used, but for simple devices such as springs or electrically activated SMA wires, simplified calculation methods are satisfactory. Validation results recently obtained will also be presented, and examples of industrial applications briefly reviewed.

[Distribution and depth of internal mammary lymph nodes in breast cancer].

Lymphoscintigraphy was performed in 50 cases of breast cancer using 99mTc-colloid as a tracer. The results indicated that 277 lymph nodes, 128 on the right side and 149 on the left, were imaged. Most of the lymph nodes were located in the second and third intercostal spaces. The mean distance between these lymph nodes and mid line was 1.93 +/- 1.05 cm-2.70 +/- 0.93 cm. The mean depth was 2.42 +/- 0.52 cm-3.07 +/- 0.72 cm. It was over 4.0 cm in isolated cases. The lymph nodes which were not showed after repeated examination could have been involved by the cancer. Radionuclide lymphoscintigraphy, being safe, easily repeated and having low radiation dose and good image, is very useful for radiotherapy.

Regulation of autophagy by miR-30d impacts sensitivity of anaplastic thyroid carcinoma to cisplatin.

miR-30d has been observed to be significantly down-regulated in human anaplastic thyroid carcinoma (ATC), and is believed to be an important event in thyroid cell transformation. In this study, we found that miR-30d has a critical role in modulating sensitivity of ATC cells to cisplatin, a commonly used chemotherapeutic drug for treatment of this neoplasm. Using a mimic of miR-30d, we demonstrated that miR-30d could negatively regulate the expression of beclin 1, a key autophagy gene, leading to suppression of the cisplatin-activated autophagic response that protects ATC cells from apoptosis. A reporter gene assay demonstrated that the binding sequences of miR-30d in the beclin 1-3' UTR was the region required for the inhibition of beclin 1 expression by this miRNA. We further showed that inhibition of the beclin 1-mediated autophagy by the miR-30d mimic sensitized ATC cells to cisplatin both in vitro (cell culture) and in vivo (animal xenograft model). These results suggest that dysregulation of miR-30d in ATC cells is responsible for the insensitivity to cisplatin by promoting autophagic survival. Thus, miR-30d may be exploited as a potential target for therapeutic intervention in the treatment of ATC.

Antigen-specific suppression by induced CD4+CD25high regulatory T cells in kidney recipients.

The biology and function of induced CD4(+)CD25(high) regulatory T (Treg) cells have not been clarified for their specificity to a foreign antigen. To test whether the regulatory functions of the induced CD4(+)CD25(high) Treg cells after transplantation require antigen-specific triggering, we analyzed the capacity of induced CD4(+)CD25(high) Treg cells to inhibit the proliferation of conventional CD4(+)CD25(-) T cells in response to T-cell receptor stimulation using donor cells or HLA-mismatched third-party cells in vitro. CD4(+)CD25(high) Treg cells did not proliferate in response to allogeneic stimulation and suppressed proliferation of the co-cultured autologous CD4(+)CD25(-) populations in a dose-dependent manner. The proliferation of CD4(+)CD25(-)T cells from the same donor in mixed lymphocyte reactions was significantly inhibited at a 1:8 ratio of conventional T cells:Treg cells: 14,404 +/- 673 cpm without CD4(+)CD25(high) Treg cells versus 10,781 +/- 539 cpm with CD4(+)CD25(high) Treg cells P = .01). At the same 1:8 ratio, the proliferation of CD4(+)CD25(-) cells derived from major histocompatibility complex-mismatched patients was not significantly inhibited: 14,404 +/- 673 cpm without CD4(+)CD25(high) Treg cells versus 12,471 +/- 709 cpm with CD4(+)CD25(high) Treg cells (P = .06). Antigen specificity of the induced CD4(+)CD25(high) Treg cells was demonstrated, after transplantation, supporting the use of antigen-specific Treg cells as a therapeutic strategy.

Cyclosporin A is an adjuvant in murine IgE antibody responses.

Cyclosporin A (CsA) is an undecapeptide fungal metabolite and is generally regarded as a new generation of immunosuppressive drugs. We uncovered a novel immunomodulatory property of CsA as a potent immunologic stimulator in the murine IgE antibody system. The enhancement of IgE responses was observed in mice receiving as few as three daily i.m. injections before Ag priming. Our studies demonstrate the three points listed below. First, CsA potentiates murine IgE responses regardless of Ag specificities in inbred mice. A hierarchy of immunopotentiation by CsA follows the order of low, intermediate, and high IgE responder mice. Second, CsA, when administered along with Ag, exerts a thorough and long lasting impact on the Ag-specific IgE antibody response, and leads to an Ag-specific breakthrough of IgE antibody synthesis in mice rendered tolerant in the IgE antibody system by soluble Ag pretreatment or neonatal IgE treatment. Third, IgE enhancer cells become sensitive to a low dose of irradiation. Two enhancer cellular components are identified, those of the Th cells and B cells, which appear to favor the induction of IgE responses. Understanding the cellular basis of the immunopotentiating effect of CsA will provide further insight into the murine IgE antibody system.