Analysis of clinical and pathological characteristics of retroperitoneal paraganglioma and associated prognostic factors.

BACKGROUND AND OBJECTIVES: The aim of this study is to explore the long-term prognostic risk factors associated with patients diagnosed with retroperitoneal paraganglioma (RPGL) and examine their clinical and pathological characteristics. METHODS: Expressions of biomarkers were identified using immunohistochemistry (IHC) and case databases were retrospectively searched. Survival analysis was performed using Kaplan-Meier and Cox risk regression to identify the factors that influence the postoperative progression-free survival of patients with RPGL. RESULTS: A total of 105 patients, most of whom had tumors situated in the paraaortic region, and whose average tumor size was 8.6 cm, were enrolled in this study. The average follow-up duration was 51 months, with a mortality rate of 19% and a recurrence and metastasis rate of 41.9%. Tumors were assessed using the modified Grading system for Adrenal Pheochromocytoma and Paraganglioma (GAPP), and SDHB, S-100, and Ki-67 were stained using IHC in all cases. Out of the total cases examined, negative in SDHB expression were observed in 18.1% of cases, S-100 expression was negative in 36.2% of cases, and endovascular tumor enboluswas present in approximately 25.7% of cases. The results of the univariate analysis indicated that several factors significantly influenced the progression-free survival of patients with PGL as follow: maximum tumor diameter (>5.5 cm), tumor morphological features, tumor grading (modified GAPP score > 6), SDHB negative, S-100 negative, and expression of proliferation index Ki-67 (>3%) (X2 = 4.217-27.420, p < 0.05). The results of the multivariate analysis indicated that negative of S-100 (p = 0.021) and SDHB (p = 0.038), as well as intravascular tumor thrombus (p = 0.047) expression were independent risk factors for progression-free survival in patients. CONCLUSION: RPGL is characterized by diverse biological features and an elevated susceptibility to both recurrence and metastasis. Both SDHB and S-100 can be employed as traditional IHC indicators to predict the metastatic risk of PGL, whereas the tumor histomorphology-endovascular tumor enbolus assists in determining the metastasis risk of RPGL.

Phospholipase PLCE1 Promotes Transcription and Phosphorylation of MCM7 to Drive Tumor Progression in Esophageal Cancer.

Phospholipase C epsilon 1 (PLCE1) is a well-established susceptibility gene for esophageal squamous cell carcinoma (ESCC). Identification of the underlying mechanism(s) regulated by PLCE1 could lead to a better understanding of ESCC tumorigenesis. In this study, we found that PLCE1 enhances tumor progression by regulating the replicative helicase MCM7 via two pathways. PLCE1 activated PKCα-mediated phosphorylation of E2F1, which led to the transcriptional activation of MCM7 and miR-106b-5p. The increased expression of miR-106b-5p, located in intron 13 of MCM7, suppressed autophagy and apoptosis by targeting Beclin-1 and RBL2, respectively. Moreover, MCM7 cooperated with the miR-106b-25 cluster to promote PLCE1-dependent cell-cycle progression both in vivo and in vitro. In addition, PLCE1 potentiated the phosphorylation of MCM7 at six threonine residues by the atypical kinase RIOK2, which promoted MCM complex assembly, chromatin loading, and cell-cycle progression. Inhibition of PLCE1 or RIOK2 hampered MCM7-mediated DNA replication, resulting in G1-S arrest. Furthermore, MCM7 overexpression in ESCC correlated with poor patient survival. Overall, these findings provide insights into the role of PLCE1 as an oncogenic regulator, a promising prognostic biomarker, and a potential therapeutic target in ESCC. SIGNIFICANCE: PLCE1 promotes tumor progression in ESCC by activating PKCα-mediated phosphorylation of E2F1 to upregulate MCM7 and miR-106b-5p expression and by potentiating MCM7 phosphorylation by RIOK2.

Detection of various fusion genes by one-step RT-PCR and the association with clinicopathological features in 242 cases of soft tissue tumor.

Introduction: Over the past decades, an increasing number of chromosomal translocations have been found in different STSs, which not only has value for clinical diagnosis but also suggests the pathogenesis of STS. Fusion genes can be detected by FISH, RT-PCR, and next-generation sequencing. One-step RT-PCR is a convenient method to detect fusion genes with higher sensitivity and lower cost. Method: In this study, 242 cases of soft tissue tumors were included, which were detected by one-step RT-PCR in multicenter with seven types of tumors: rhabdomyosarcoma (RMS), peripheral primitive neuroectodermal tumor (pPNET), synovial sarcoma (SS), myxoid liposarcomas (MLPS), alveolar soft part sarcoma (ASPS), dermatofibrosarcoma protuberans (DFSP), and soft tissue angiofibroma (AFST). 18 cases detected by one-step RT-PCR were further tested by FISH. One case with novel fusion gene detected by RNA-sequencing was further validated by one-step RT-PCR. Results: The total positive rate of fusion genes was 60% (133/213) in the 242 samples detected by one-step RT-PCR, in which 29 samples could not be evaluated because of poor RNA quality. The positive rate of PAX3-FOXO1 was 88.6% (31/35) in alveolar rhabdomyosarcoma, EWSR1-FLI1 was 63% (17/27) in pPNET, SYT-SSX was 95.4% in SS (62/65), ASPSCR1-TFE3 was 100% in ASPS (10/10), FUS-DDIT3 was 80% in MLPS (4/5), and COL1A1-PDGFB was 66.7% in DFSP (8/12). For clinicopathological parameters, fusion gene status was correlated with age and location in 213 cases. The PAX3-FOXO1 fusion gene status was correlated with lymph node metastasis and distant metastasis in RMS. Furthermore, RMS patients with positive PAX3-FOXO1 fusion gene had a significantly shorter overall survival time than those patients with the negative fusion gene. Among them, the FISH result of 18 cases was concordant with one-step RT-PCR. As detected as the most common fusion types of AHRR-NCOA2 in one case of AFST were detected as negative by one-step RT-PCR. RNA-sequencing was used to determine the fusion genes, and a novel fusion gene PTCH1-PLAG1 was found. Moreover, the fusion gene was confirmed by one-step RT-PCR. Conclusion: Our study indicates that one-step RT-PCR displays a reliable tool to detect fusion genes with the advantage of high accuracy and low cost. Moreover, it is a great tool to identify novel fusion genes. Overall, it provides useful information for molecular pathological diagnosis and improves the diagnosis rate of STSs.

Hyperbaric Oxygen Therapy Promotes Hearing Gain with Increases in Serum IGF-1 and HSP70 in Patients with Idiopathic Sudden Sensorineural Hearing Loss.

Objective: Hyperbaric oxygen therapy (HBOT) has been recommended for the initial and salvage treatment of patients with idiopathic sudden sensorineural hearing loss (ISSHL), but its underlying mechanisms remain unclear. In this study, we investigated whether HBOT alters serum levels of insulin-like growth factor 1 (IGF-1) and heat shock protein 70 (HSP70) in patients with ISSHL. Then, we identified the relationship between hearing recovery and changes in serum IGF-1 and HSP70 levels. Methods: Moderately severe to profound unilateral ISSHL patients (n = 70) and healthy control participants (n = 30) were enrolled. The ISSHL patients were randomly assigned to receive medical therapy alone (MT group, n = 35) or both HBOT and medical therapy (HBOT + MT group, n = 35). Audiometric testing was performed before and after treatment. Serum IGF-1 and HSP70 levels were assessed by ELISA in ISSHL patients pre-and posttreatment and healthy controls. Results: Before treatment, compared with the healthy controls, serum IGF-1 and HSP70 were lower in ISSHL patients. After treatment, serum IGF-1 and HSP70 increased in both the HBOT + MT and MT groups, although they were significantly higher in the HBOT + MT group (p < 0.01). In the HBOT + MT group, these increases were associated with hearing gains. In addition, IGF-1 was strongly associated with HSP70 (r = 0.621, p = 0.001). No such association was found in the MT group (p = 0.757). Conclusion: Administering HBOT in addition to medical therapy can improve the hearing of patients with moderately severe to profound unilateral ISSHL. The improvement is related to the upregulation of IGF-1 and HSP70.

Food Allergy-Induced Autism-Like Behavior is Associated with Gut Microbiota and Brain mTOR Signaling.

Purpose: Food allergy-induced autism-like behavior has been increasing for decades, but the causal drivers of this association are unclear. We sought to test the association of gut microbiota and mammalian/mechanistic target of rapamycin (mTOR) signaling with cow's milk allergy (CMA)-induced autism pathogenesis. Methods: Mice were sensitized intragastrically with whey protein containing cholera toxin before sensitization on intraperitoneal injection with whey-containing alum, followed by intragastric allergen challenge to induce experimental CMA. The food allergic immune responses, ASD-like behavioral tests and changes in the mTOR signaling pathway and gut microbial community structure were performed. Results: CMA mice showed autism-like behavioral abnormalities and several distinct biomarkers. These include increased levels of 5-hydroxymethylcytosine (5-hmC) in the hypothalamus; c-Fos were predominantly located in the region of the lateral orbital prefrontal cortex (PFC), but not ventral; decreased serotonin 1A in amygdala and PFC. CMA mice exhibited a specific microbiota signature characterized by coordinate changes in the abundance of taxa of several bacterial genera, including the Lactobacillus. Interestingly, the changes were accompanied by promoted mTOR signaling in the brain of CMA mice. Conclusion: We found that disease-associated microbiota and mTOR activation may thus play a pathogenic role in the intestinal, immunological, and psychiatric Autism Spectrum Disorder (ASD)-like symptoms seen in CAM associated autism. However, this is only a preliminary study, and their mechanisms require further investigation.

Mechanistic Studies of Gypenosides in Microglial State Transition and its Implications in Depression-Like Behaviors: Role of TLR4/MyD88/NF-κB Signaling.

Depression is a prevalent psychiatric disorder. Microglial state transition has been found in many neurological disorders including depression. Gypenosides (Gypenosides I-LXXVIII, Gps) are saponin extracts isolated from the traditional Chinese herb Gynostemma pentaphyllum (Thunb.) Makino that exert anti-inflammatory and neuroprotective activities and regulate depression-like behaviors. However, its effect on microglial state transition in depression remains unknown. We aimed to evaluate the potential relationship between Gps and TLR4/MyD88/NF-κB signaling in microglial state transition in vitro and in vivo. First, BV-2 cells (microglial cell line) were exposed to lipopolysaccharides (LPS) and treated with 10 or 5 µg/ml Gps. Second, the chronic unpredictable mild stress (CUMS)-induced depression mouse model was used to investigate the antidepressant-like behaviors effects of Gps (100 or 50 mg/kg). We determined depression-like behaviors using the open-field test (OFT), forced swim test (FST), and sucrose preference test (SPT). Proteins and inflammatory factors in the TLR4/MyD88/NF-κB signaling pathway and the different microglial reaction states markers were subsequently conducted using enzyme-linked immunosorbent assay, immunocytochemistry, immunofluorescence, qPCR, or Western blotting analyses to evaluate the anti-inflammatory and antidepressant properties of Gps and the underlying molecular mechanisms. We found that Gps regulated the microglial cell line state transition in LPS-exposed BV-2 cells, as evidenced by the significantly decreased expression of inflammatory parameters iNOS, IL-1ß, IL-6, and TNF-α and significantly promoted anti-inflammatory microglial phenotypes markers CD206 (Mrc1) and IL-10. More importantly, Gps protected against the loss of monoamine neurotransmitters and depression-like behavior in a mouse model of depression, which was accompanied by a regulation of the microglial state transition. Mechanistically, Gps inhibited TLR4/MyD88/NF-κB signaling, which reduced the release of downstream inflammatory cytokines (IL-1ß, IL-6, and TNF-α) and promoted microglial phenotype transition, which all together contributed to the antidepressant effect. Our results suggest that Gps prevents depression-like behaviors by regulating the microglial state transition and inhibiting the TLR4/MyD88/NF-κB signaling pathway. Thus, Gps could be a promising therapeutic strategy to prevent and treat depression-like behaviors and other psychiatric disorders.

Using a System Pharmacology Method to Search for the Potential Targets and Pathways of Yinqiaosan against COVID-19.

The first reported case of coronavirus disease 2019 (COVID-19) occurred in Wuhan, Hubei, China. Thereafter, it spread through China and worldwide in only a few months, reaching a pandemic level. It can cause severe respiratory illnesses such as pneumonia and lung failure. Since the onset of the disease, the rapid response and intervention of traditional Chinese medicine (TCM) have played a significant role in the effective control of the epidemic. Yinqiaosan (YQS) was used to treat COVID-19 pneumonia, with good curative effects. However, a systematic overview of its active compounds and the therapeutic mechanisms underlying its action has yet to be performed. The purpose of the current study is to explore the compounds and mechanism of YQS in treating COVID-19 pneumonia using system pharmacology. A system pharmacology method involving drug-likeness assessment, oral bioavailability forecasting, virtual docking, and network analysis was applied to estimate the active compounds, hub targets, and key pathways of YQS in the treatment of COVID-19 pneumonia. With this method, 117 active compounds were successfully identified in YQS, and 77 potential targets were obtained from the targets of 95 compounds and COVID-19 pneumonia. The results show that YQS may act in treating COVID-19 pneumonia and its complications (atherosclerosis and nephropathy) through Kaposi sarcoma-related herpesvirus infection and the AGE-RAGE signaling pathway in diabetic complications and pathways in cancer. We distinguished the hub molecular targets within pathways such as TNF, GAPDH, MAPK3, MAPK1, EGFR, CASP3, MAPK8, mTOR, IL-2, and MAPK14. Five of the more highly active compounds (acacetin, kaempferol, luteolin, naringenin, and quercetin) have anti-inflammatory and antioxidative properties. In summary, by introducing a systematic network pharmacology method, our research perfectly forecasts the active compounds, potential targets, and key pathways of YQS applied to COVID-19 and helps to comprehensively clarify its mechanism of action.

Periodontal Status and Microbiologic Pathogens in Patients with Chronic Obstructive Pulmonary Disease and Periodontitis: A Case-Control Study.

Purpose: To evaluate clinical periodontal status and microbiologic pathogens in patients with chronic obstructive pulmonary disease (COPD) and periodontitis. Patients and Methods: We conducted a case-control study of 60 periodontitis patients with COPD (case group) and 60 periodontitis patients with normal pulmonary function (control group). Their periodontal status and respiratory function were clinically examined. Real-time polymerase chain reaction assays were used to measure five dental pathogens and four respiratory pathogens in subgingival dental plaque. Spearman's rank correlation coefficients (r2) were calculated to assess correlations of pathogens. Principal component analysis (PCA) was employed to assess the similarity of bacterial diversity between the two groups. Logistic regression was performed to examine the associations of periodontal variables and pathogens with COPD risk. Results: COPD patients had fewer remaining teeth, higher plaque index (PLI), and more severe site percentages of clinical attachment level (CAL) than the controls. Although COPD patients tended to have relatively higher ranked means of Porphyromonas gingivalis, Tannerella forsythensis, Treponema denticola, and Haemophilus influenza than control participants, the differences were not significant. Some periodontal pathogens and respiratory pathogens were positively correlated with each other (r2 =0.29 to 0.47, all P < 0.05). The PCA graph showed that the distributions of pathogens were more dispersed but less discriminated in the COPD group than those in the control group. PLI (P = 0.045) and CAL ≥ 5mm site percentages (P = 0.01) were significantly associated with an increased risk of COPD, while pathogens were not associated with COPD. Conclusion: Our results from this study do not indicate periodontal pathogens as potential predictors of COPD risk, despite significantly poor periodontal status associated with COPD.

Cosmc Disruption-Mediated Aberrant O-glycosylation Suppresses Breast Cancer Cell Growth via Impairment of CD44.

BACKGROUND: Breast cancer remains the most lethal malignancy in women worldwide. Aberrant O-glycosylation is closely related to many human diseases, including breast carcinoma; however, its precise role in cancer development is insufficiently understood. Cosmc is an endoplasmic reticulum-localized chaperone that regulates the O-glycosylation of proteins. Cosmc dysfunction results in inactive T-synthase and expression of truncated O-glycans such as Tn antigen. Here we investigated the impact of Cosmc disruption-mediated aberrant O-glycosylation on breast cancer cell development through in vitro and in vivo experiments. MATERIALS AND METHODS: We deleted the Cosmc gene in two breast cancer cell lines (MCF7, T47D) using the CRISPR/Cas-9 system and then measured the expression levels of Tn antigen. The proliferation of Tn-positive cells was examined by RTCA, colony formation and in vivo experiments. The effects of Cosmc deficiency on glycoprotein CD44 and MAPK pathway were also determined. RESULTS: Both in vitro and in vivo studies showed that Cosmc deficiency markedly suppressed breast cancer cell growth compared with the corresponding controls. Mechanistically, Cosmc disruption impaired the protein expression of CD44 and the associated MAPK signaling pathway; the latter plays a crucial role in cell proliferation. Reconstitution of CD44 substantially reversed the observed alterations, confirming that CD44 requires normal O-glycosylation for its proper expression and activation of the related signaling pathway. CONCLUSION: This study showed that Cosmc deficiency-mediated aberrant O-glycosylation suppressed breast cancer cell growth, which was likely mediated by the impairment of CD44 expression.

Physiologic Electrical Fields Direct Retinal Ganglion Cell Axon Growth In Vitro.

Purpose: The purpose of this study was to characterize the ability of applied electrical fields (EFs) to direct retinal ganglion cell (RGC) axon growth as well as to assess whether Rho GTPases play a role in translating electrical cues to directional cues. Methods: Full-thickness, early postnatal mouse retina was cultured in electrotaxis chambers and exposed to EFs of varying strengths (50-200 mV/mm). The direction of RGC axon growth was quantified from time-lapsed videos. The rate of axon growth and responsiveness to changes in EF polarity were also assessed. The effect of toxin B, a broad-spectrum inhibitor of Rho GTPase signaling, and Z62954982, a selective inhibitor of Rac1, on EF-directed growth was determined. Results: In the absence of an EF, RGC axons demonstrated indiscriminate directional growth from the explant edge. Retinal cultures exposed to an EF of 100 and 200 mV/mm showed markedly asymmetric growth, with 74.2% and 81.2% of axons oriented toward the cathode, respectively (P < 0.001). RGC axons responded to acute changes in EF polarity by redirecting their growth toward the "new" cathode. This galvanotropic effect was partially neutralized by toxin B and Rac1 inhibitor Z62954982. Conclusions: RGC axons exhibit cathode-directed growth in the presence of an EF. This effect is mediated in part by the Rho GTPase signaling cascade.

Detection of three common mutations causing ß-thalassemia by using a closed-tube multiplex PCR.

Small oligonucleotides mutations are the large majority causes of ß-thalassemia. Dual priming oligonucleotide PCR has been used to detect point mutations and thus could be applied to diagnose ß-thalassemia. The goal of this study was to establish a simple, quick and cost-effective screening assay by using modified dual priming oligonucleotide PCR for three most common mutations of ß-thalassemia [CD71-72 (+A), CD 41-42 (-CTTT), Pnt.-28 (A → G)] in Southeast Asia and southern China. Man-made 5 tandem mismatched bases instead of poly (I) were used as the linker in the specific PCR primers. Single closed-tube multiplex PCRs followed by dissociation curve (DC) analysis were included in the molecular screening assay. Denaturing high-performance liquid chromatography analysis was applied to distinguish compound heterozygotes from single mutations. A blinded study of 91 samples was performed using this new assay. There were 41 samples detected as the above three mutations and it was concordant with the original methods. In conclusion, the modified dual priming oligonucleotide multiplex PCR/DC can detect these three genotypes of common mutation of ß-thalassemia; this method is simple, rapid and cost-effective, which makes it suitable for large-scale screening and diagnosis.

Aberrant O-glycosylation contributes to tumorigenesis in human colorectal cancer.

Aberrant O-glycosylation is frequently observed in colorectal cancer (CRC) patients, but it is unclear if it contributes intrinsically to tumorigenesis. Here, we investigated the biological consequences of aberrant O-glycosylation in CRC. We first detected the expression profile of Tn antigen in a serial of human CRC tissues and then explored the genetic and biosynthetic mechanisms. Moreover, we used a human CRC cell line (LS174T), which express Tn antigen, to assess whether aberrant O-glycosylation can directly promote oncogenic properties. It showed that Tn antigen was detected in around 86% human primary and metastatic CRC tissues. Bio-functional investigations showed that T-synthase and Cosmc were both impaired in cancer tissues. A further analysis detected an occurrence of hypermethylation of Cosmc gene, which possibly caused its loss-of-function and a consequent inactive T-synthase. Transfection of LS174T cells with WT Cosmc restored mature O-glycosylation, which subsequently down-regulated cancer cell proliferation, migration and apoptotic-resistant ability. Significantly, the expression of MUC2, a heavily O-glycosylated glycoprotein that plays an essential role in intestinal function, was uniformly reduced in human CRC tissues as well as in LS174T cells. These data suggest that aberrant O-glycosylation contributes to the development of CRC through direct induction of oncogenic properties in cancer cells.

Effects of mechanical stretching on the morphology of extracellular polymers and the mRNA expression of collagens and small leucine-rich repeat proteoglycans in vaginal fibroblasts from women with pelvic organ prolapse.

To determine the effect of mechanical stretching load and the efficacy of postmenopausal estrogen therapy (ET) on pelvic organ prolapse (POP), vaginal fibroblasts isolated from postmenopausal women with or without POP were subjected to 0.1-Hz uniaxial cyclic mechanical stretching (CS) with 10% elongation and 10-8 M 17-ß-estradiol (E2) treatment. We investigated the morphological characteristics of extracellular polymers using scanning electron microscopy (SEM) and monitored the mRNA expression of type I collagen (COL I) and type III collagen (COL III) as well as the small leucine-rich proteoglycan (SLRP) family members decorin (DCN), biglycan (BGN), fibromodulin (FMO), and lumican (LUM), using real-time quantitative polymerase chain reaction (RT-PCR). Using SEM, certain viscoelastic polymers were found to be randomly distributed among fibroblasts, which for normal fibroblasts formed clusters of plum flower-like patterns under static-culture conditions and resembled stretched strips when stretched in culture, whereas polymers among POP fibroblasts resembled stretched strips under static-cultured conditions and presented broken networks when stretched in culture. RT-PCR revealed that COL I, DCN, BGN, FMO, and LUM mRNA expression was significantly higher in POP than in normal fibroblasts under static-culture condition. Following CS, COL I and BGN mRNA expression was significantly up-regulated in normal fibroblasts, and DCN and FMO mRNA expression was down-regulated in POP fibroblasts. Following concomitant CS and E2 treatment, significantly elevated COL I and DCN mRNA expression was observed in normal fibroblasts, and significantly elevated COL I and BGN mRNA expression was observed in POP fibroblasts. COL III mRNA expression was not significantly different between the POP and normal group, and CS did not significantly affect expression in either group, though COL III was down-regulated in normal fibroblasts concomitantly treated with E2 and CS. We conclude that the morphological distribution of extracellular polymers in POP fibroblasts exhibited higher sensitivity and lower tolerance to stretching loads than do normal fibroblasts. These mechanical properties were further reflected in the transcription of COL I. Defects in the compensatory function of BGN for DCN and LUM for FMO exist in POP fibroblasts, which further affect the structure and function of COL I in response to stretching load, ultimately resulting in abnormal reconstruction of pelvic supportive connective tissues and the occurrence of POP. ET can maintain stretching-induced elevations in COL I and DCN transcription in healthy women and improve stretching-induced COL I, DCN, BGN, and FMO transcriptional changes in POP women to prevent and improve POP. Only down-regulated COL III transcription was observed upon concomitant CS and E2 treatment in normal fibroblasts, which suggests that the tensile strength, not the elasticity, of the supportive connective tissues is damaged in POP and that the higher tensile strength induced by ET in healthy fibroblasts prevents POP. These findings confirm the role of higher sensitivity and lower tolerance to mechanical stretching in the pathogenesis of POP and further provide evidence supporting the use of ET to prevent and inhibit POP in postmenopausal women.

Kinetics of Label Retaining Cells in the Developing Rat Kidneys.

BACKGROUND: The kidney is a specialized low-regenerative organ with several different types of cellular lineages. The BrdU label-retaining cell (LRCs) approach has been used as part of a strategy to identify tissue-specific stem cells in the kidney; however, because the complementary base pairing in double-stranded DNA blocks the access of the anti-BrdU antibody to BrdU subunits, the stem cell marker expression in BrdU-labeled cells are often difficult to detect. In this study, we introduced a new cell labeling and detection method in which BrdU was replaced with 5-ethynyl-2-deoxyuridine (EdU) and examined the time-dependent dynamic changes of EdU-labeled cells and potential stem/progenitor markers in the development of kidney. METHODS: Newborn rats were intraperitoneally injected with EdU, and their kidneys were harvested respectively at different time points at 1 day, 3 days, 1 week, 2 weeks, and 6 weeks post-injection. The kidney tissues were processed for EdU and cellular markers by immunofluorescence staining. RESULTS: At the early stage, LRCs labeled by EdU were 2176.0 ± 355.6 cells at day one in each renal tissue section, but dropped to 168 ± 48.4 cells by week 6. As time increased, the numbers of LRCs were differentially expressed in the renal cortex and papilla. At the postnatal day one, nearly twice as many cells in the cortex were EdU-labeled as compared to the papilla (28.6 ± 3.6% vs. 15.6 ± 3.4%, P<0.05), while there were more LRCs within the renal papilla since the postnatal week one, and at the postnatal week 6, one third as many cells in the cortex were EdU-labeled as compared to the papilla (2.5 ± 0.1% vs. 7.7 ± 2.7%, P<0.05). The long-term LRCs at 6-week time point were associated exclusively with the glomeruli in the cortex and the renal tubules in the papilla. At 6 weeks, the EdU-labeled LRCs combined with expression of CD34, RECA-1, Nestin, and Synaptopodin were discretely but widely distributed within the glomeruli; Stro-1 around the glomeruli; and α-smooth muscle actin (SMA) in arteries. Conversely, co-expression of CD34, RECA-1, and Nestin with the long term EdU-labeled LRCs was significantly lower in renal tubules (P<0.01), while Stro-1 and Synaptopodin were not detected. CONCLUSION: Our data found that at 6-week time point, EdU-labeled LRCs existing in the glomeruli expressed undifferentiated podocyte and endothelial markers at high rates, while those in the renal tubules expressed Nestin and vascular markers at low rates. To understand the characterization and localization of these EdU-LRCs, further studies will be needed to test cell lineage tracing, clonogenicity and differentiation potency, and the contributions to the regeneration of the kidney in response to renal injury/repair.

Langerhans cell histiocytosis in Chinese adults: absence of BRAF mutations and increased FOXP3(+) regulatory T cells.

Langerhans cell histiocytosis (LCH) is a rare disorder characterized by the proliferation of abnormal Langerhans cells. Previous studies mainly focused on children with LCH. However, there is limited information on the clinical and pathological aspects of LCH in adults. Therefore, this study aimed to investigate the clinical and pathological aspects of LCH in Chinese adults. The results showed that the average age of 18 LCH patients was 35.22 ± 16.57 years old. The ratio of male to female was 3.5:1.14 patients (77.8%) had single-system involvement and 4 patients (22.2%) had multi-system diseases. The skin (38.9%) and lungs (44.4%) were the mainly affected organs. No BRAF mutations were detected in the lesions of 18 cases. The number of FOXP3(+) Tregs was significantly increased in LCH. In conclusion, clinical features of LCH in adults are distinct from those in children. Adult LCH has a relatively good prognosis and presents as a benign disease. Immune regulation plays an important role in the pathogenesis of adult LCH.

Detection of a novel large deletion causing α-thalassemia in South China.

α-Thalassemia is an inherited autosomal recessive disorder. It is one of the most common monogenic abnormalities known in the world and is prevalent in tropical and subtropical regions. α-Thalassemia is more frequently caused by deletional type than non-deletional type. Recently, we identified a novel large deletional type of α-thalassemia named --(FZ)/αα from a family in South China. Multiplex ligation-dependent probe amplification was used for diagnosing the carrier and prenatal diagnosing for a fetus. Real-time PCR was employed for characterizing the deletion breakpoints and the deletional segment was determined as 300 kb in length extending from the telomere to AXIN1 gene on the short arm of chromosome 16. The carriers in the family members were detected by real-time PCR using designed primers.

Hippocampal glutamate level and glutamate aspartate transporter (GLAST) are up-regulated in senior rat associated with isoflurane-induced spatial learning/memory impairment.

Postoperative cognitive decline is a clinical concern especially for senior patients. It is generally recognized that glutamatergic system plays a crucial role in the physiopathologic process of neurocognitive deterioration. However, alterations of glutamatergic system in prolonged isoflurane-induced learning/memory decline are still unclear. This study investigates the question whether glutamate concentration and corresponding transporters or receptors display any alternations in aged rat suffering from isoflurane-induced learning/memory impairment. 111 male Sprague-Dawley rats (>18 months) were randomly divided into two main groups: hippocampal microdialysis group (n = 38) and western blotting group (n = 73). Each group was subdivided into three subgroups including (1) control subgroup (n = 6 and 10, receiving no behavioral trial, anesthesia or air exposure); (2) air-exposed subgroup (n = 7 and 15, receiving behavioral trial and air exposure but not anesthesia); (3) isoflurane anesthesia subgroup (n = 25 and 48, receiving both behavioral trial and anesthesia). The isoflurane-exposed rats were further divided into a learning/memory-impaired subgroup and a non-learning/memory-impaired subgroup according to their behavioral performance, which was measured using Morris water maze. Hippocampal glutamate concentrations in microdialysates were determined by high-performance liquid chromatography. Expression levels of GLAST, GLT-1, NMDAR1, NMDAR2A/B, AMPAR and tau in hippocampus were assessed via quantitative Western blotting. The incidences of learning/memory impairment of isoflurane-exposed rats in hippocampal microdialysis group and western blotting group were 12.0 (3/25) and 10.4 % (5/48) respectively. The intra-anesthesia hippocampal glutamate levels were significantly lower than those of non-anesthesized rats. The learning/memory-impaired rats showed a long-lasting increased glutamate level from 24 h after isoflurane exposure to the end of the study, but the other 22 isoflurane-exposed rats did not. The learning/memory-impaired subgroup displayed a significantly higher GLAST level than the other three subgroups (p = 0.026, 0.02 and 0.032 respectively). The expression levels of GLT-1, NMDAR1, NMDAR2A/B and AMPAR of every subgroup were comparable. We found a continuous raised hippocampal glutamate and an up-regulation of GLAST rather than GLT-1, NMDAR1, NMDAR2A/B, AMPAR or tau in hippocampus of aged rats associated with isoflurane-induced learning/memory impairment.

Changes of learning and memory in aged rats after isoflurane inhalational anaesthesia correlated with hippocampal acetylcholine level.

BACKGROUND AND PURPOSE: It has not been clearly described for mechanisms of postoperative cognitive dysfunction (POCD), but not yet for narcotic in connection with POCD. Acetylcholine plays important roles in learning and memory especially in elderly people. It is not very clear that cholinergic changes in the hippocampus are in relation to spatial memory impairment. The effects of isoflurane, a commonly used inhalation anaesthetic, as well as acetylcholine on learning and memory in the brain tissue of aged rats were observed in present study. We investigated the inhalation anaesthesia drug's effect on cholinergic system to examine whether the regional and progressive cholinergic changes may lead to POCD. METHODS: Seventeen-month-old rats were randomly assigned to either an isoflurane anaesthesia group (n=17) or propofol anaesthesia group (n=17). In isoflurane anaesthesia experiment group, isoflurane (1.4 to 1.7% for 2 hours) was delivered via a ventilator to make freely moving microdialysis model. In propofol group, propofol was administered by continuous infusion via a tail vein catheter to make freely moving microdialysis model. They were all mechanically ventilated. Morris Water Maze test was used to assess the learning and memory abilities of all the two groups' rats twice a day for 5 days. Microdialysis was performed on the freely moving rats to determine the levels of acetylcholine in the brain tissues immediately after each Water Maze test. RESULTS: The isoflurane anaesthesia treatment increased the escape latency contrast to propofol anaesthesia group. The isoflurane anaesthesia's rats were then divided into isoflurane-induced severe learning/memory impairment group (n=6, mean escape latency is 1.96 times more than that of in propofol anaesthesia group) and the isoflurane-induced mild learning/memory impairment group (n=11, mean escape latency is equal or less than 1.96 times of that in propofol anaesthesia group). The results demonstrated that those rats that were categorized in the isoflurane-induced severe learning/memory impairment group had decreased levels of acetylcholine in the brain tissue as compared to those rats categorized in the mild learning/memory impairment group and in propofol anaesthesia group. CONCLUSION: The results indicated that isoflurane may impair learning and memory in aged rats.

[Detection of common deletions and mutations causing α-thalassemia in Southeast Asians and Southern Chinese with denaturing high performance liquid chromatography].

OBJECTIVE: To establish a comprehensive and simple assay using denaturing high performance liquid chromatography (DHPLC) for the diagnosis of most common mutations and deletions of α-thalassemia gene in Southeast Asians and Southern Chinese. METHODS: This assay has included a duplex polymerase chain reaction (PCR) followed by DHPLC analysis. An improved PCR was also performed followed by DHPLC analysis. With this assay, a blinded study of 160 samples was screened for three common mutations and three common deletions. RESULTS: The duplex PCR-DHPLC combined with the improved PCR-DHPLC analysis has detected all mutations and the wild-type allele. The results were consistent with those by the original methods. CONCLUSION: This molecular assay may be used for the diagnosis of α-thalassemia patients from this geographical region. The method is accurate, rapid, semi-automatic and cost-effective, which makes it suitable for large-scale screening.

Detection of α-globin gene deletions using denaturing high-performance liquid chromatography and multiplex ligation-dependent probe amplification.

BACKGROUND: Multiplex ligation-dependent probe amplification (MLPA) has been used to detect deletions and mutations of the α-globin gene for diagnosis of α-thalassemia. MLPA reaction products are usually separated and analyzed by high-voltage capillary gel electrophoresis (CGE). The goal of this study was to find and use a cost-effective method to separate and analyze MLPA products. METHODS: Blood samples were collected from China. DNA was extracted and amplified by PCR using fluorescently labeled primers. In this study, denaturing high-performance liquid chromatography (DHPLC) was used to separate and analyze the reaction products. And the optimal separation conditions were determined using nondenaturing columntemperature. RESULTS: The DHPLC conditions were optimized and have been applied to separate MLPA products and 27 of the MLPA products from 50 to 320 bp were well separated. DHPLC was able to separate up to 37 reaction products that differed by 4-12 base pairs and detected target gene deletions by differences in peak size. Compared with CGE, both the specificity and sensitivity of DHPLC for the 107 DNA samples were 100%. CONCLUSIONS: DHPLC could be used to test routinely for α-globin gene mutations and deletions. Combined with MLPA, DHPLC is a low-cost, simple to use, accurate technique with practical value.