Preimplantation genetic diagnosis and screening (PGD/S) using a semiconductor sequencing platform.

BACKGROUND: Recent advances in semiconductor sequencing platform (SSP) have provided new methods for preimplantation genetic diagnosis/screening (PGD/S). The present study aimed to evaluate the applicability and efficiency of SSP in PGD/S. METHODS: The artificial positive single-cell-like DNAs and normal single-cell samples were chosen to test our semiconductor sequencing platform for preimplantation genetic diagnosis/screening (SSP-PGD/S) method with two widely used whole-genome amplification (WGA) kits. A total of 557 single blastomeres were collected from in vitro fertilization (IVF) couples, and their WGA products were processed and analyzed by our SSP-PGD/S method in comparison with array comparative genomic hybridization (array-CGH). RESULTS: Our SSP-PGD/S method indicated high compatibilities with two commercial WGA kits. For 557 single blastomeres, our method with four million reads in average could detect 24-chromosome aneuploidies as well as microdeletion/microduplication of the size over 4 Mb, providing 100% consistent conclusion with array-CGH method in the classification of whether it was transplantable. CONCLUSIONS: Our studies suggested that SSP-PGD/S represents a valuable alternative to array-CGH and brought PGD/S into a new era of more rapid, accurate, and economic.

Mysm1 epigenetically regulates the immunomodulatory function of adipose-derived stem cells in part by targeting miR-150.

Adipose-derived stem cells (ASCs) are highly attractive for cell-based therapies in tissue repair and regeneration because they have multilineage differentiation capacity and are immunosuppressive. However, the detailed epigenetic mechanisms of their immunoregulatory capacity are not fully defined. In this study, we found that Mysm1 was induced in ASCs treated with inflammatory cytokines. Adipose-derived stem cells with Mysm1 knockdown exhibited attenuated immunosuppressive capacity, evidenced by less inhibition of T cell proliferation, more pro-inflammatory factor secretion and less nitric oxide (NO) production in vitro. Mysm1-deficient ASCs exacerbated inflammatory bowel diseases but inhibited tumour growth in vivo. Mysm1-deficient ASCs also showed depressed miR-150 expression. When transduced with Mysm1 overexpression lentivirus, ASCs exhibited enhanced miR-150 expression. Furthermore, Mysm1-deficient cells transduced with lentivirus containing miR-150 mimics produced less pro-inflammatory factors and more NO. Our study reveals a new role of Mysm1 in regulating the immunomodulatory activities of ASCs by targeting miR-150. These novel insights into the mechanisms through which ASCs regulate immune reactions may lead to better clinical utility of these cells.

CKIP-1 regulates the immunomodulatory function of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are self-renewing multipotent cells with immunoregulatory function, which makes them attractive candidates for regenerative medicine. However, the detailed mechanisms of their immunomodulatory capacity are not fully characterized. Here, we found that casein kinase 2 interacting protein-1 (CKIP-1) expression was induced in the murine MSC cell line C3H/10T1/2 by LPS. Knockdown of CKIP-1 did not cause significant differences on the cell cycle or immunophenotype of MSCs. However, MSCs with CKIP-1 knockdown showed enhanced immunosuppressive capacity. Real-time PCR and western blot analyses revealed that compared with the control group, MSCs with CKIP-1-knockdown exhibited higher IL-10 production and p38 MAPK phosphorylation following LPS treatment. Interestingly, the expression of CKIP-1 was decreased in MSCs following high glucose treatment. Furthermore, MSCs became more immunosuppressive after high glucose treatment, as shown by higher IL-10 production and enhanced inhibition of T cell proliferation. Collectively, our data reveal a novel role for CKIP-1 in regulating MSC-mediated immunomodulation, and indicate that MSCs become more immunosuppressive under high glucose conditions. These new insights may help in the development of future applications of MSCs.

Imaging the Transformation of Ipsilateral Internal Capsule Following Focal Cerebral Ischemia in Rat by Diffusion Kurtosis Imaging.

BACKGROUND: The purpose of our study is to explore the relationship between recovery of neural function and transformation of the internal capsule (IC) after transient middle cerebral artery occlusion (MCAO) by using diffusion kurtosis imaging (DKI). METHODS: Six male adult Sprague-Dawley rats implemented with transient MCAO were used in this study. Sensorimotor function was assessed according to repetitive behavioral testing on day 1, 3, 7, 14, and 28 after cerebral ischemia. Metrics of DKI were acquired, and the time course of the region-to-normal ratio was evaluated in IC. RESULTS: After cerebral ischemia, relative fractional anisotropy in IC decreased on day 3 (P < .01). Relative mean diffusivity (rMD) increased on day 28 (P < .05). Relative mean diffusional kurtosis (rMK) increased on day 3 (P < .01) and decreased on day 7 (P < .05). Relative axial diffusional kurtosis (rKa) increased on day 3 (P < .01) and declined on day 7 (P < .05). Relative radial diffusional kurtosis (rKr) was reduced on day 7 (P < .05). Changes in rMK were larger than changes in rMD on day 3 (P < .05). The factor of rKa and rKr revealed marked difference on day 7 (P < .05) and day 14 (P < .05). Neurological function score showed that rats exhibited functional recovery from day 7 (P < .01) post stroke. CONCLUSIONS: This longitudinal multiparametric magnetic resonance imaging study suggested that K metrics offers information complimentary to conventional diffusion metrics and revealed the procedure during the structural modification in the ipsilateral IC following focal cerebral ischemia. After transient MCAO, the neural transformation occurred in a time-dependent procedure.

A20 plays a critical role in the immunoregulatory function of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) possess an immunoregulatory capacity and are a therapeutic target for many inflammation-related diseases. However, the detailed mechanisms of MSC-mediated immunosuppression remain unclear. In this study, we provide new information to partly explain the molecular mechanisms of immunoregulation by MSCs. Specifically, we found that A20 expression was induced in MSCs by inflammatory cytokines. Knockdown of A20 in MSCs resulted in increased proliferation and reduced adipogenesis, and partly reversed the suppressive effect of MSCs on T cell proliferation in vitro and inhibited tumour growth in vivo. Mechanistic studies indicated that knockdown of A20 in MSCs inhibited activation of the p38 mitogen-activated protein kinase (MAPK) pathway, which potently promoted the production of tumour necrosis factor (TNF)-α and inhibited the production of interleukin (IL)-10. Collectively, these data reveal a crucial role of A20 in regulating the immunomodulatory activities of MSCs by controlling the expression of TNF-α and IL-10 in an inflammatory environment. These findings provide novel insights into the pathogenesis of various inflammatory-associated diseases, and are a new reference for the future development of treatments for such afflictions.

Combining systemic and stereotactic MEMRI to detect the correlation between gliosis and neuronal connective pathway at the chronic stage after stroke.

BACKGROUND: The early dysfunction and subsequent recovery after stroke, characterized by the destruction and remodeling of connective pathways between cortex and subcortical regions, is associated with neuroinflammation. As major components of the inflammatory process, reactive astrocytes have double-edged effects on pathological progression. The temporal patterns of astrocyte and neuronal pathway activity can be revealed by systemic and stereotactic manganese-enhanced magnetic resonance imaging (MEMRI), respectively. In the present study, we aimed to detect an association between astrocyte activity and recovery of neuronal connective pathways by combining systemic with stereotactic MEMRI. METHODS: Fifty adult rats, divided into two groups, underwent a 60-min occlusion of the middle cerebral artery. The groups were given either a systemic administration or stereotactic injection of MnCl2 at 1, 3, 7, and 14 days after stroke and underwent MRI 4 and 2 days later, respectively. Immunofluorescence (IF) of group 1 was conducted to corroborate the results. Repetitive behavioral testing was also performed with all rats at 1, 3, 7, and 14 days to obtain a functional score. RESULTS: Ring- or crescent-shaped enhancements formed in the striatal peri-infarct regions (STR) at 11 and 18 days. This was concurrent with the activity of glial fibrillary acidic protein (GFAP)-positive astrocytes, which mainly localized at the peri-infarct region and significantly increased in number at 11 and 18 days after stroke. Microglia/macrophages, detected by IF, mainly localized in the lesion core, rather than in the region of enhancement. The ipsilateral substantia nigra (SN) revealed Mn-related signal enhancement reduction and subsequent signs of the recovery process at 3 to 5 days and 9 to 16 days, respectively. Behavioral testing showed that sensorimotor functions were initially disturbed, but subsequently recovered at 7 and 14 days. CONCLUSIONS: We found a positive temporal correlation between astrogliosis and the recovery of neuronal connective pathways at the chronic stage by using the in vivo method of MEMRI. Our results highlighted the potential contribution of astrocytes to the neuronal recovery of these connective pathways.

Upregulation of CC Chemokine Receptor 7 (CCR7) Enables Migration of Xenogeneic Human Adipose-Derived Mesenchymal Stem Cells to Rat Secondary Lymphoid Organs.

BACKGROUND CC chemokine receptor 7 (CCR7) expression is vital for cell migration to secondary lymphoid organs (SLOs). Our previous work showed that inducing CCR7 expression enabled syngeneic mesenchymal stem cells (MSCs) to migrate into SLOs, resulting in enhanced immunosuppressive performance in mice. Given that human adipose-derived stem cells (hASCs) are widely used in clinical therapy, we further investigated whether upregulation of CCR7 enables xenogeneic hASCs to migrate to rat SLOs. MATERIAL AND METHODS hASCs rarely express CCR7; therefore, hASCs were transfected with lentivirus encoding rat CCR7 (rCCR7) plus green fluorescence protein (GFP) or GFP alone. CCR7 mRNA and cell surface expression of rCCR7-hASCs and GFP-hASCs were examined by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry (FCM), respectively. The phenotype, differentiation, and proliferation capacity of each cell type was also determined. To examine migration, rCCR7-hASCs and GFP-hASCs were injected intravenously into Lewis rats, and the proportion of GFP-positive cells in the spleen and lymph nodes was determined with FCM. RESULTS mRNA and cell surface protein expression of CCR7 was essentially undetectable in hASCs and GFP-ASCs; however, CCR7 was highly expressed in rCCR7-ASCs. rCCR7-hASCs, GFP-hASCs, and hASCs shared a similar immunophenotype, and maintained the ability of multilineage differentiation and proliferation. In addition, the average proportion of GFP-positive cells was significantly higher following transplantation of rCCR7-hASCs compared with GFP-hASCs (p<0.01). CONCLUSIONS These results suggest that upregulation of rat CCR7 expression does not change the phenotype, differentiation, or proliferation capacity of hASCs, but does enable efficient migration of hASCs to rat SLOs.

[Ecology suitability study of Chinese materia medica Gentianae Macrophyllae Radix].

This paper is aimed to predict ecology suitability distribution of Gentianae Macrophyllae Radix and search the main ecological factors affecting the suitability distribution. The 313 distribution information about G. macrophylla, 186 distribution information about G. straminea, 343 distribution information about G. dauricaand 131 distribution information about G. crasicaulis were collected though investigation and network sharing platform data . The ecology suitable distribution factors for production Gentianae Macrophyllae Radix was analyzed respectively by the software of ArcGIS and MaxEnt with 55 environmental factors. The result of MaxEnt prediction was very well (AUC was above 0.9). The results of predominant factors analysis showed that precipitation and altitude were all the major factors impacting the ecology suitable of Getiana Macrophylla Radix production. G. macrophylla ecology suitable region was mainly concentrated in south of Gansu, Shanxi, central of Shaanxi and east of Qinghai provinces. G. straminea ecology suitable region was mainly concentrated in southwest of Gansu, east of Qinghai, north and northwest of Sichuan, east of Xizang province. G. daurica ecology suitable region was mainly concentrated in south and southwest of Gansu, east of Qinghai, Shanxi and north of Shaanxi province. G. crasicaulis ecology suitable region was mainly concentrated in Sichuan and north of Yunnan, east of Xizang, south of Gansu and east of Qinghai province. The ecological suitability distribution result of Gentianae Macrophyllae Radix was consistent with each species actual distribution. The study could provide reference for the collection and protection of wild resources, meanwhile, provide the basis for the selection of cultivation area of Gentiana Macrophylla Radix.

[Analysis of commercial specifications and grades of wild and cultivated Gentianae Macrophyllae Radix based on multi-indicative constituents].

The study is aimed to analyze the commercial specifications and grades of wild and cultivated Gentianae Macrophllae Radix based on multi-indicative constituents. The seven kinds of main chemical components containing in Gentianae Macrophyllae Radix were determined by UPLC, and then the quality levels of chemical component of Gentianae Macrophyllae Radix were clustered and classified by modern statistical methods (canonical correspondence analysis, Fisher discriminant analysis and so on). The quality indices were selected and their correlations were analyzed. Lastly, comprehensively quantitative grade division for quality under different commodity-specifications and different grades of same commodity-specifications of wild and planting were divided. The results provide a basis for a reasonable division of specification and grade of the commodity of Gentianae Macrophyllae Radix. The range of quality evaluation of main index components (gentiopicrin, loganin acid and swertiamarin) was proposed, and the Herbal Quality Index (HQI) was introduced. The rank discriminant function was established based on the quality by Fisher discriminant analysis. According to the analysis, the quality of wild and cultivated Luobojiao, one of the commercial specification of Gentianae Macrophyllae Radix was the best, Mahuajiao, the other commercial specification, was average , Xiaoqinjiao was inferior. Among grades, the quality of first-class cultivated Luobojiao was the worst, of second class secondary, and the third class the best; The quality of the first-class of wild Luobojiao was secondary, and the second-class the best; The quality of the second-class of Mahuajiao was secondary, and the first-class was the best; the quality of first-class Xiaoqinjiao was secondary, and the second-class was the better one between the two grades, but not obvious significantly. The method provides a new idea and method for evaluation of comprehensively quantitative on the quality of Gentianae Macrophyllae Radix.

[Quality regionalization study on Gentianae Macrophyllae Radix].

The 79 samples of Gentianae Macrophyllae Radix were collected based on the distributed information by document literature. Based on sample information, and using the regression model of Gentianae Macrophyllae Radix index component and environmental factors, and combined with the prediction results of ecological suitability by MaxEnt and principal component analysis results of index component, the space distribution of Gentianae Macrophyllae Radix was estimated with the spatial analysis function of ArcGIS. The results showed that it had a higher comprehensive quality in south of Shaanxi, south of Gansu, middle of Sichuan and southeast of Xizang. The study results were coinciding with the producing regions of Gentianae Macrophyllae Radix. It can provide reference for Gentianae Macrophyllae Radix resource conservation, development and utilization.

[UPLC Fingerprint of Gentiana officinalis Root].

Objective: To establish UPLC fingerprints of Gentiana officinalis root and Gentiana macrophylla root,and to evaluate the similarity of the fingerprints,in order to provide theoretical basis for the quality control of Gentiana officinalis root. Methods: The separation was performed on an ACQUITY UPLC~BEH C18column( 50 mm × 2. 1 mm,1. 7 µm) through a gradient elution of methanol-0. 04% aqueous phosphoric at a flow rate of 0. 3 m L/min. The detection wavelength was 242 nm, and the column temperature was set at30 ℃. Similarity evaluation system for chromatographic fingerprint of TCM( Version 2004 A) was used to analyze the fingerprints. Results: 13 common peaks were selected as the fingerprint peaks from 13 batches of Gentiana officinalis root samples, with the similarities between 0. 976 to 0. 997, and Gentiana macrophylla root used for fingerprint applicability test has high similarity( 0. 998) with Gentiana officinalis root. Conclusion: This method with good precision and reliability should be used for the quality control of Gentiana officinalis root.

Normalization of T2 relaxation time and apparent diffusion coefficient in relation to the inflammatory changes in the substantia nigra of rats with focal cerebral ischemia.

BACKGROUND: Focal cerebral ischemia results in delayed neurodegeneration in remote brain regions, such as the substantia nigra. To date, a reasonable explanation is still lacking regarding the correlation of magnetic resonance (MR) signal pseudo-normalization following a transient abnormal change and subsequent progressive pathological damage. PURPOSE: To characterize the substantia nigra following middle cerebral artery occlusion and to evaluate the potential pathophysiological changes associated with the pseudo-normalization of MR signals in the substantia nigra at the subacute stage after stroke onset. MATERIAL AND METHODS: Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion. During the occlusion of single middle cerebral artery, computed tomography (CT) perfusion was acquired to observe the blood flow perfusion in the primary ischemic striatum and ipsilateral substantia nigra. Next, the MR T2 relaxation time and apparent diffusion coefficient changes within the substantia nigra were determined on days 1, 3, 7, and 14 after stroke onset, and compared with immunohistochemistry for microglia activation and astrogliosis. RESULTS: Twenty-four rats with strong hypoperfusion in the primary ischemic territory and no alterations of the perfusion in the ipsilateral substantia nigra detected both visually and measurably during the middle cerebral artery occlusion were further studied. All animals showed MR pseudo-normalization with T2 relaxation time and apparent diffusion coefficient recovered in the ipsilateral substantia nigra at the subacute phase following focal cerebral ischemia. Normalization of the MR signals corresponded well with the spatio-temporal occurrence of microglia activation and astrogliosis. CONCLUSION: The pseudo-normalization of T2 relaxation time and apparent diffusion coefficient reflects the neuroinflammatory changes that accompany activation of microglia and astrocytes in the ipsilateral substantia nigra following middle cerebral artery occlusion.

[Comparative Study on Morphological Characteristics of Root of Six Species in Sect. Cruciata].

OBJECTIVE: To provide evidences for the identification of Gentianae Macrophyllae Radix by comparing the morphological characteristics of six species of Sect. Cruciata (Gentiana macrophylla, G. crassicaulis, G. straminea, G. dahurica, G. officinalis and G. siphonantha). METHODS: By microscope, the tissue characteristics with freehand section of the upper, middle and lower of root and the powder characteristics with chloral hydrate were studied. RESULTS: The vascular cylinder of G. crassicaulis was not split. The vascular cylinder of G. macrophylla, G. dahurica and G. officinalis, were only split in the upper part. The root of G. straminea and G. siphonantha wre completely divided into several smaller roots twisting together. There were a lot of thick walled cells in the powder of G. dahurica, G. straminea and G. siphonantha, but their shapes were different. No thick walled cells were found in the other three species. CONCLUSION: There are obviously differences among the microscopic morphological characteristics of root of six species of Sect. Cruciata, which can provide the basis for identification of Gentianae macrophyllae Radix.

Expression and clinicopathological significance of Mel-18 mRNA in colorectal cancer.

Mel-18 is a member of the polycomb group (PcG) of proteins, which are chromatin regulatory factors that play an important role in oncogenesis. This study was designed to investigate the clinical and prognostic significance of Mel-18 in colorectal cancer (CRC) patients. For this purpose, expression of Mel-18 mRNA was evaluated in 82 primary CRC and paired noncancerous mucosa samples by qRT-PCR and Western blotting. We found that overall Mel-18 mRNA expression in the CRC tissue was significantly lower than in the noncancerous mucosal tissue (p = 0.007, Wilcoxon matched-pairs signed-ranks test). Mel-18 was conversely correlated with the pathological classifications (p = 0.003 for T, p < 0.001 for N, and p = 0.015 for M classifications, respectively) and clinical AJCC stage (p < 0.001). Furthermore, CRC patients with a higher level of Mel-18 showed prolonged disease-free survivals (DFS) (p < 0.001). In multivariate analysis, the diminished Mel-18 expression may be a risk factor for the patients' 3-year DFS (HR = 1.895; 95 % CI 1.032, 3.477; p = 0.039). It was therefore concluded that the lower Mel-18 expression might contribute to the CRC development/progression.

Prognostic significance of lymph node status in patients with metastatic colorectal carcinoma treated with lymphadenectomy.

BACKGROUND AND OBJECTIVES: To test prognostic significance of lymph node status in patients with metastatic colorectal carcinoma (mCRC). METHODS: Four hundred ninety six patients diagnosed with synchronous mCRC and treated with lymphadenectomy between 1995 and 2008 were identified and divided into groups pN0, pN1, and pN2 (140 (28.2%) in pN0, 223 (45.0%) in pN1, and 133 (26.8%) in pN2 group) according to their lymph node status. The Kaplan-Meier and Cox regression analyses were used to test associations and independent predictor status of lymph node involvement. RESULTS: The Cox proportional hazards regression showed pN as significantly associated with disease-specific survival (DSS) both in univariate (HR = 1.609, 95% CI 1.411 to 1.835, P < 0.001) and multivariate (HR = 1.630, 95% CI 1.422 to 1.868, P < 0.001) analyses. The Kaplan-Meier analysis demonstrated that patients with pN2 and pN1 had a significantly worse DSS compared with patients with pN0 tumors (respectively, 17.273 ± 1.020 and 27.145 ± 1.715 vs. 34.992 ± 2.143 months; P < 0.001). In accuracy analyses based on AUC values, nodal status demonstrated the highest accuracy (65.1%) out of all the variables. CONCLUSIONS: Our findings indicate that optimal TNM staging for mCRC should incorporate lymph node status to provide a more effective and predictive model.

Comparison of USPIO-enhanced MRI and Gd-DTPA enhancement during the subacute stage of focal cerebral ischemia in rats.

BACKGROUND: Being one class of magnetic resonance imaging (MRI) contrast agents, ultrasmall superparamagnetic particles of iron oxides (USPIO) bear the potential to study neuroinflammation following stroke, but there is still debate over whether the iron oxides particles may enter the brain tissue passively over a damaged blood-brain barrier (BBB). PURPOSE: To compare the enhancement patterns of USPIO and gadopentate dimeglumine (Gd-DTPA) during the subacute stage of focal cerebral ischemia, to examine the relationship between USPIO enhancement and BBB disturbance, as well as with neuroinflammatory cell response. MATERIAL AND METHODS: Multiple MR sequences were obtained on days 3 and 6 after transient middle cerebral artery occlusion induced in rats with and without the application of USPIO and Gd-DTPA. The enhancement patterns of the two contrast agents were compared and correlated to histology, including IgG for BBB permeability, Prussian Blue staining for iron particle detection, and CD68 immunohistochemistry staining to identify macrophage/microglia. RESULTS: Gd-DTPA enhancement depicted BBB breakdown being in line with IgG leakage. The USPIO enhanced images demonstrated both diffuse and focal signal alteration in ischemic lesions. The diffuse enhanced pattern had a similar spatial and temporal profile as with Gd-DTPA enhancement. In addition, focal enhanced signal loss was visible on T1-, T2-, and T2*-weighted images, with a peak tendency of MR signal loss, macrophage/microglia concentration and iron particle accumulation at a later phase of the study. CONCLUSION: After focal cerebral ischemia, Gd-DTPA-enhanced MRI showed a higher sensitivity in detecting BBB alterations than did USPIO enhancement. USPIO provided complementary information regarding inflammatory cell activity in neuroinflammatory to cerebral ischemia that had not been visualized using conventional Gd-DTPA. The assessment using multiple MR parameters may identify intracellular and extracellular USPIO in vivo.

An efficient molecular genetic testing strategy for incontinentia pigmenti based on single-tube long fragment read sequencing.

Incontinentia pigmenti (IP) is a rare X-linked dominant neuroectodermal dysplasia that primarily affects females. The only known causative gene is IKBKG, and the most common genetic cause is the recurrent IKBKG△4-10 deletion resulting from recombination between two MER67B repeats. Detection of variants in IKBKG is challenging due to the presence of a highly homologous non-pathogenic pseudogene IKBKGP1. In this study, we successfully identified four pathogenic variants in four IP patients using a strategy based on single-tube long fragment read (stLFR) sequencing with a specialized analysis pipeline. Three frameshift variants (c.519-3_519dupCAGG, c.1167dupC, and c.700dupT) were identified and subsequently validated by Sanger sequencing. Notably, c.519-3_519dupCAGG was found in both IKBKG and IKBKGP1, whereas the other two variants were only detected in the functional gene. The IKBKG△4-10 deletion was identified and confirmed in one patient. These results demonstrate that the proposed strategy can identify potential pathogenic variants and distinguish whether they are derived from IKBKG or its pseudogene. Thus, this strategy can be an efficient genetic testing method for IKBKG. By providing a comprehensive understanding of the whole genome, it may also enable the exploration of other genes potentially associated with IP. Furthermore, the strategy may also provide insights into other diseases with detection challenges due to pseudogenes.

Genistein induces G2/M arrest in gastric cancer cells by increasing the tumor suppressor PTEN expression.

Genistein, a major isoflavone found in soybeans, exhibits anticarcinogenic properties. The inhibitory effect of genistein on cell proliferation is associated with G2/M cell cycle arrest and inhibition of cdc2 activities. Here we assessed the role of PTEN in regulation of genistein-mediated G2/M cell cycle arrest in the gastric cancer cell lines (SGC-7901 and BGC-823). After 24 h following treatment, genistein induced a concentration-dependent accumulation of cells in the G2/M phase of the cell cycle. The sustained G2/M arrest by genistein in SGC-7901 and BGC-823 cells is associated with increased phospho-cdc2 (Tyr15) and decreased cdc2 protein. Genistein treatment increased Wee1 levels and decreased phospho-Wee1 (Ser 642). Moreover, genistein substantially decreased the Ser473 and Thr308 phosphorylation of Akt and upregulated PTEN expression. Downregulation of PTEN by siRNA in genistein-treated cells increased phospho-Wee1 (Ser642), whereas decreased phospho-Cdc2 (Tyr15), resulting in decreased the G2/M cell cycle arrest. Therefore, induction of G2/M cell cycle arrest by genistein involved upregulation of PTEN.

In vivo USPIO-enhanced MR signal characteristics of secondary degeneration in the ipsilateral substantia nigra after middle cerebral artery occlusion at 3T.

BACKGROUND AND PURPOSE: Ultrasmall superparamagnetic iron oxide (USPIO) particles to enhance MRI have been used to study neuroinflammation in vivo. Our purpose was to observe the USPIO-enhanced MR signal alterations in the primary ischemic lesion and ipsilateral substantia nigra after middle cerebral artery occlusion (MCAO) to verify the subsequent sequelae of neuroinflammation seen in the primary ischemic focus and secondary degeneration region. MATERIALS AND METHODS: Sprague-Dawley rats were subjected to transient MCAO. In addition to conventional T2-, T1-weighted imaging, USPIO-enhanced MRI was performed in USPIO-injected stroke rats, while Gd-enhanced imaging was acquired in control stroke rats, on days 3, 6 using a 3-T MR scanner. The MR signal characteristics in the primary ischemic striatum, ipsilateral substantia nigra were noted, compared on histopathological H&E, Prussian blue (PB) staining. RESULTS: After MCAO, USPIO-induced T2 hypointensity changes were observed in the primary ischemic region with BBB impairment at both time points. In the substantia nigra ipsilateral to the primary ischemic lesion, there was no evidence of USPIO accumulation detected by MRI and PB staining, and no BBB leakage reflected by Gd-enhanced imaging on days 3 and 6. CONCLUSION: USPIO-enhanced MR signals have variable characteristics in both primary and remote sites after focal cerebral ischemia. This suggests that the neuroinflammatory response to brain ischemia in the primary ischemic focus and secondary degeneration region have different temporal patterns and pathophysiological mechanisms.

Identification of potential biomarkers for idiopathic pulmonary fibrosis and validation of TDO2 as a potential therapeutic target.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease with a high mortality rate. On this basis, exploring potential therapeutic targets to meet the unmet needs of IPF patients is important. AIM: To explore novel hub genes for IPF therapy. METHODS: Here, we used public datasets to identify differentially expressed genes between IPF patients and healthy donors. Potential targets were considered based on multiple bioinformatics analyses, especially the correlation between hub genes and carbon monoxide diffusing capacity of carbon monoxide, forced vital capacity, and patient survival rate. The mRNA levels of the hub genes were determined through quantitative real-time polymerase chain reaction. RESULTS: We found that TDO2 was upregulated in IPF patients and predicted poor prognosis. Surprisingly, single-cell RNA sequencing data analysis revealed significant enrichment of TDO2 in alveolar fibroblasts, indicating that TDO2 may participate in the regulation of proliferation and survival. Therefore, we verified the upregulated expression of TDO2 in an experimental mouse model of transforming growth factor-ß (TGF-ß)-induced pulmonary fibrosis. Furthermore, the results showed that a TDO2 inhibitor effectively suppressed TGF-ß-induced fibroblast activation. These findings suggest that TDO2 may be a potential target for IPF treatment. Based on transcription factors-microRNA prediction and scRNA-seq analysis, elevated TDO2 promoted the IPF proliferation of fibroblasts and may be involved in the P53 pathway and aggravate ageing and persistent pulmonary fibrosis. CONCLUSION: We provided new target genes prediction and proposed blocking TGF-ß production as a potential treatment for IPF.