[Study on protective effects and mechanisms of total glucosides of Paeony on Graves disease in BALB/c mice].

Objective: To investigate the protective effect and its immunoregulatory mechanism of Total Glucosides of Paeony (TGP) against Graves' Disease (GD) model on BALB/c mice. Methods: Fifty female (6 weeks old, weighing 16-18 g) BALB/c mice of specific pathogen free were divided into control group according to random number table method, model group, early low-dose TGP intervention group (250 mg·kg-1·d-1), early high-dose TGP intervention group (500 mg·kg-1·d-1), and late TGP intervention group, with 10 mice in each group. Except the control group, the other 4 groups were immunized 3 times (0, 3rd, and 6th week) with recombinant adenovirus expressing the thyroid stimulating hormone receptor (TSHR) A subunit to establish the GD model. The early low-dose and high-dose intervention group were given diets containing different doses of TGP throughout the whole process, and the late intervention group was given diets containing low doses of TGP from the 1st week after the 2nd immunization (week 4). The levels of thyrotropin receptor antibody (TRAb) and total thyroxine (TT4) were detected in the tail venous blood of mice at the 4th week. At the 10th week, the serum TRAb and TT4 levels and the ratio of regulatory T cells (Treg) in each group were detected, and the pathological changes of thyroid tissue were observed. Serum helper T cell 1(Th1) and Th2 cell-related factors interleukin-2 (IL-2), IL-4, IL-5, IL-10, IL-12p70, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ) and tumor necrosis factors-α (TNF-α) were detected to investigate the protective effect of TGP on GD model in BALB/c mice and its mechanism. Results: At the 4th week, The level of TT4 [(55.07±12.89) µg/L] in early high-dose intervention group was lower than that in model group [(74.33±8.63) µg/L] (all P<0.05). The level of TT4 in early low-dose intervention group and late intervention group and model group had no statistical significance (all P>0.05). TRAb level of mice between early low-dose, early high-dose, late intervention groups and model group was no significant difference (all P>0.05). At the 10th week, TRAb [(90.00±26.89) U/L] and TT4[(32.66±8.11) µg/L] levels in the early high-dose intervention group were lower than those in the model group [(396.97±95.35) U/L, (73.70±16.33) µg/L] (all P<0.05). The TRAb and TT4 levels in the early low-dose intervention group and late intervention group were not significantly different from those in the model group (all P>0.05). The thyroid tissue of hyperthyroidism mice in the early high dose intervention group showed focal hypertrophic changes, while the thyroid tissue of other hyperthyroidism mice showed diffuse hypertrophic changes. The CD4+CD25+/CD4+Treg ratio in early high-dose intervention group was higher than that in model group at the 10th week (4 weeks after three recombinant adenovirus immunization) (P<0.05). Compared with the model group at the 10th week, the levels of IL-2, IL-12p70 and IFN-γ in the early high-dose intervention group were all decreased (all P<0.05), and the levels of IL-10 were increased (P<0.05). Conclusion: Early high-dose (500 mg·kg-1·d-1) TGP intervention group displays a protective effect against GD mice, the mechanism of which may be related to regulatory T cell function changes and Th1/Th2 cytokine balance restoration.

[Clinical characteristics of 272 437 patients with different histopathological subtypes of primary esophageal malignant tumors].

Objective: To characterize the histopathological subtypes and their clinicopathological parameters of gender and onset age by common, rare and sparse primary esophageal malignant tumors (PEMT). Methods: A total of 272 437 patients with PEMT were enrolled in this study, and all of the patients were received radical surgery. The clinicopathological information of the patients was obtained from the database established by the State Key Laboratory of Esophageal Cancer Prevention & Treatment from September 1973 to December 2020, which included the clinical treatment, pathological diagnosis and follow-up information of esophagus and gastric cardia cancers. All patients were diagnosed and classified by the criteria of esophageal tumor histopathological diagnosis and classification (2019) of the World Health Organization (WHO). The esophageal tumors, which were not included in the WHO classification, were analyzed separately according to the postoperative pathological diagnosis. The χ2 test was performed by the SPSS 25.0 software on count data, and the test standard α=0.05. Results: A total of 32 histopathological types were identified in the enrolled PEMT patients, of which 10 subtypes were not included in the WHO classification. According to the frequency, PEMT were divided into common (esophageal squamous cell carcinoma, ESCC, accounting for 97.1%), rare (esophageal adenocarcinoma, EAC, accounting for 2.3%) and sparse (mainly esophageal small cell carcinoma, malignant melanoma, etc., accounting for 0.6%). All the common, rare, and sparse types occurred predominantly in male patients, and the gender difference of rare type was most significant (EAC, male∶ female, 2.67∶1), followed with common type (ESCC, male∶ female, 1.78∶1) and sparse type (male∶ female, 1.71∶1). The common type (ESCC) mainly occurred in the middle thoracic segment (65.2%), while the rare type (EAC) mainly occurred in the lower thoracic segment (56.8%). Among the sparse type, malignant melanoma and malignant fibrous histiocytoma were both predominantly located in the lower thoracic segment (51.7%, 66.7%), and the others were mainly in the middle thoracic segment. Conclusion: ESCC is the most common type among the 32 histopathological types of PEMT, followed by EAC as the rare type, and esophageal small cell carcinoma and malignant melanoma as the major sparse type, and all of which are mainly occur in male patients. The common type of ESCC mainly occur in the middle thoracic segment, while the rare type of EAC mainly in the lower thoracic segment. The mainly sparse type of malignant melanoma and malignant fibrous histiocytoma predominately occur in the lower thoracic segment, and the remaining sparse types mainly occur in the middle thoracic segment.

[Relationship of C1QA level and therapeutic effect and prognosis of DLBCL patients treated with R-CHOP].

Objective: To investigate the relationship between plasma levels of complements before treatment and the clinicopathological feathers and prognoses of diffuse large B-cell lymphoma (DLBCL) patients treated with Rituximab (R)-CHOP or R-CHOP-like therapy. Methods: The clinicopathological data of 105 DLBCL patients treated in cancer Hospital of Chinese Academy of Medical Sciences from 2010 to 2016 were collected. The plasma samples from 105 DLBCL patients treated with R-CHOP or R-CHOP-like therapy and 80 healthy controls were used to detect 34 complement levels before treatment by utilizing antibody microarray. The relationship between plasma levels of complements and the clinicopathological feathers and prognosis of DLBCL patients were analyzed. Results: The signal values of C1QA and CR1L in patients with international prognostic index (IPI) scores of 3-5 were 1 261.43±138.9 and 2 214.69±98.58, respectively, higher than 950.79±80.19 and 984.67±121.79 in patients with IPI scores of 0~2 (both P<0.05). The levels of C1QA and CR1L in the non-complete response (CR) group were 1 165.43±98.56 and 2 263.13±145.63, respectively, higher than 914.70±100.77 and 1 821.34±84.68 in the CR group (both P<0.05). Cox regression analysis showed that elevated C1QA signal value was associated with poor progression-free survival (PFS) and poor overall survival (OS) (PFS: HR=2.063, 95%CI: 1.220-3.489, P=0.007; OS: HR=2.23, 95%CI: 1.036~4.798, P=0.040). After IPI correction by Cox multivariate model, the elevated C1QA signal value was still correlated with poor PFS (HR=1.765, 95%CI 1.034~3.013, P=0.037). Conclusions: The baseline plasma levels of C1QA and CR1L are correlated with IPI scores and therapeutic effects of DLBCL patients treated with R-CHOP. The baseline plasma level of C1QA has a certain predictive value for the prognostic evaluation of DLBCL.

Amsp3 may act upstream of Amdnmt3 in female caste differentiation in the honeybee (Apis mellifera).

In honey bees, the process of producing two female castes, including queens and workers, is nutritionally controlled by differential feeding royal jelly to newly emerged larvae. Although they have almost identical genetic blueprints, these castes show striking differences in their morphologies, longevities and reproductive capabilities. DNA methyltransferase 3 (Amdnmt3) gene is involved in the regulatory network for honeybee caste differentiation. Due to the role of two zinc fingers containing transcription factors, SP1 and SP3 in controlling mammalian Dnmts, this study aimed to determine a similar interaction of SPs with Amdnmt3 in the honeybee. We confirmed that the promoter region of Amdnmt3 contained multiple predicted SP1/SP3 binding sites and then investigated the role of AmSP3 in queen-worker differentiation network. We observed that the expression level of Amsp3 was significantly higher in worker larvae than that in queen larvae at 48 h, 84 h and 120 h. Knockdown of Amsp3 expression by RNAi in worker larvae significantly reduced the expression level of Amdnmt3 and caused morphological changes in adult bees towards a queen-like phenotype. However, the expression levels of Amsp3 and Amdnmt3 were repressed by juvenile hormone (JH). Our results suggest that AmSP3 is an important part of the queen-worker differentiation network and supports the role of Amdnmt3 in determining the phenotypic outcome of developing larvae.

Nuclear export protein CSE1L interacts with P65 and promotes NSCLC growth via NF-κB/MAPK pathway.

Non-small cell lung cancer (NSCLC) is characterized with high morbidity and mortality, mainly due to frequent recurrence and metastasis. However, the underlying molecular mechanisms of NSCLC tumorigenesis are largely unclear. Through data mining in the ONCOMINE and Gene Expression Omnibus (GEO) databases, the expression of CSE1L (chromosome segregation like 1 protein/CAS), an exportin, was identified to be significantly upregulated in NSCLC and positively associated with poor prognosis of patients. By use of in vitro and in vivo gain- and loss-of-function experiments, we found that CSE1L can promote NSCLC cell proliferation while inhibiting cell apoptosis. Through immunoprecipitation and mass spectrometry experiments, we demonstrated that CSE1L interacted with RELA (named as P65) and affected its location in the nucleus. Moreover, we found that one of the mechanisms by which CSE1L promotes proliferation and inhibits apoptosis is through activating the nuclear factor-κB (NF-κB)/mitogen-activated protein kinase (MAPK) signaling pathway. In summary, our findings indicated an oncogenic role of CSE1L in NSCLC tumorigenesis.

[Programmed cell death-1 inhibitor combined with rituximab in refractory or relapsed diffuse large B-cell lymphoma: a preliminary efficacy and safety analysis].

Objective: To investigate the efficacy and safety of programmed cell death protein-1 (PD-1) inhibitor combined with rituximab in the treatment of refractory or relapsed diffuse large B-cell lymphoma (rrDLBCL) patients. Methods: The efficacy and safety of rrDLBCL patients treated with PD-1 inhibitor combined with rituximab as salvage therapeutic regimen after initially treated with rituximab, cyclophosphamide, anthracycline, vincristine and prednisone (R-CHOP) regimen in Cancer Hospital, Chinese Academy of Medical Science & Peking Union Medical College from October 2018 to Janurary 2020 were retrospectively analyzed.Patient who received at least one dose of PD-1 inhibitor combined with rituximab treatment and obtained the efficacy and safety evaluation were included. Results: A total of 22 patients were enrolled in this study. The median age was 51.5 years and the median number of prior treatment regimen was 2. The median time to progression (TTP) for the initial R-CHOP treatment was 9.3 months and the median interval time of rituximab administrations between the previous and the research regimen was 5.5 months. Patients were classified as germinal center B cell (GCB) origin (n=8), non-GCB origin (n=9) and primary mediastinal large B cell lymphoma (PMBCL, n=5). Four patients were double-expression lymphoma, one patient were triple-hit lymphoma. Nine patients had PD-L1 immunohistochemical staining and the proportion of PD-L1 positive tumor cells were 1%-90% for eight patients and negative for one patient.The objective response rate (ORR) and complete response rate (CR) were 72.7% (16/22) and 13.6% (3/22), respectively. The median progression free survival (PFS) was 8.0 (95%CI: 7.0-14.5) months, and overall survival (OS) was not reached. For the 17 patients of non-specific DLBCL, the ORR was 64.7% (11/17), the estimated median PFS was 4.0 (95%CI: 0-8.8) months, the 1-year PFS and OS rates were 39.2% (95%CI: 19.4%-43.4%)and 81.3% (95%CI: 71.4%-91.1%), respectively. All of 5 PMBCL cases achieved ORR, among them, one case was CR and 4 cases were partial responase (PR), and their PFS were 16.4, 9.3, 8.3, 7.9and 3.0 months, respectively. One patient had National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) version 5.0 grade 3 hypophysitis and one patient had NCI CTCAE grade 3 interstitial pneumonia. Conclusion: For rrDLBCL patients who have underwent rituximab treatment previously, PD-1 inhibitor combined with rituximab regimen shows a promising efficacy and tolerability, which can be a potential treatment option.

[Long-term outcome of childhood T-cell acute lymphoblastic leukemia treated with modified national protocol of childhood leukemia in China-acute lymphoblastic leukemia 2008].

Objective: To analyse the long-term efficacy in childhood T-cell acute lymphoblastic leukemia (T-ALL) cases enrolled in the national protocol of childhood leukemia in China-acute lymphoblastic leukemia (NPCLC-ALL) 2008. Methods: Clinical data of 96 patients diagnosed as T-ALL and treated with NPCLC-ALL2008 protocol between January 2009 and December 2017 in the Department of Hematology-Oncology, the Children's Hospital, Zhejiang University School of Medicine were analyzed retrospectively. Predictive value of minimal residual disease (MRD) monitored by flow cytometry was analyzed. Kaplan-Meier method was used for long-term survival analysis. Results: A total of 96 evaluable patients with newly diagnosed T-ALL were analysed, including 72 males and 24 females. The age was 9.5 (ranged from 1.0 to 16.0) years. The follow-up time was 5.7 (ranged from 1.0 to 9.7) years. Among 96 patients, 92 (96%) achieved complete remission. The 5-year event free survival (EFS) and overall survival (OS) rates were (61±6) % and (70±5) %, respectively. Relapse occurred in 18 cases and the 5-year cumulative incidence of relapse was (27±6) %. Twenty-four patients died. The 5-year OS rates of patients with MRD>5% on day 15 of induction therapy was significantly worse than those with MRD≤5% ((60±12) % vs. (72±6) %, χ(2)=3.904, P=0.048) . The 5-year EFS and OS rates were obviously lower in patients with MRD>10% before the consolidation therapy ((50±35) %). The 5-year OS rates of patients with relapsed disease was significantly worse than those without ((26±13) % vs. (81±5) %, χ(2)=18.411, P<0.01). The earlier the relapse, the worse the prognosis. The 5-year OS rates for patients relapsed within 6 months, within 3 years and more than 3 years, were (25±22) %, (30±14) % and (50±35) % respectively (χ(2)=13.207, P<0.01). Conclusions: NPCLC-ALL2008 protocol is effective for childhood T-ALL. The MRD guided accurate risk stratification and individualized treatment can reduce the relapse and improve the survival rate of pediatric T-ALL.

[Prenatal gene diagnosis of 200 fetuses at high risk of osteogenesis imperfect].

Objective: The authors aim to provide genetic counselling and prenatal gene diagnosis to the families with osteogenesis imperfecta(OI), based on the identification of pathogenetic mutations in large cohort genetic testing. Methods: DNA was extracted from the peripheral blood of parents of the fetuses, and from the villi tissue, amniotic fluid or cord blood of the fetuses using a standard sodium dodecyl sulfate-proteinase K-phenol/chloroform extraction method. PCR combined with Sanger DNA sequencing was performed to validate the pathogenic mutations of 200 fetuses at risk of OI and their parents from 158 families. Allelic analysis of microsatellite markers was applied to exclude the false positive caused by maternal DNA contamination, when both the fetus and the mother harbored the same pathogenic genotype. Results: A total of 83 affected fetuses (83/200, 41.5%) and 12 (12/200, 6.0%) recessive carriers were identified among the 200 fetuses. The 83 affected fetuses included 78 heterozygotes (45 of COL1A1, 32 of COL1A2, one of IFITM5), and 5 compound heterozygotes or homozygotes of recessive OI (two of FKBP10, one of SEC24D, one of WNT1 and one of CRTAP); The 12 recessive carriers included 7 of WNT1, 4 of SERPINF1 and one of SERPINH1. Maternal DNA contamination was excluded from the genomic DNA samples of OI fetuses when their mother with the same affected genotypes. Conclusion: In this study, the authors used an optimized gene diagnosis system of OI to perform prenatal genetic diagnosis to 200 fetuses at high risk of OI, and provided precisely genetic counselling to the OI families.

[The long coding RNA GSTM3TV2 acts an oncogene to promote chemoresistance in pancreatic cancer].

Objectives: To examine the expression of the long coding RNA GSTM3TV2 in pancreatic cancer tissues and to examine its role and mechanism in chemoresistance of pancreatic cancer cells. Methods: The expression of lncRNA GSTM3TV2 in 15 pancreatic cancer specimens and corresponding adjacent to cancer tissue samples diagnosed by Department of Pathology, Peking Union Medical College Hospital was detected by real-time PCR.And the expressions of GSTM3TV2 in pancreatic cancer cell AsPC-1, BxPC-3, MIAPaCa-2, PanC-1, SU86.86, T3M4, and chemoresistant cells AsPC-1/GR and MIAPaCa-2/GR, and human pancreatic nestin-expressing cells hTERT-HPNE were detected. Pancreatic cancer cell lines were transfected with GSTM3TV2-pcDNA3.1(+)in order to get cells with GSTM3TV2 overexpression.GSTM3TV2-siRNA was transfected into pancreatic cancer cells to knock down GSTM3TV2. The cell chemoresistance was measured by CCK-8 and flow cytometry assay when incubated with nab-paclitaxel. At the same time, subcutaneous xenograft tumor models were established in nude mice to observe the effect of GSTM3TV2 on chemoresistance of tumor growth in nude mice.Western blot assay was also performed to detect the molecular mechanism of chemoresistance of GSTM3TV2. Results: Comparing toadjacent tissues(0.084±0.019), GSTM3TV2 expression was significantly upregulated in the pancreatic cancer tissues(0.493±0.084) (t=5.146, P<0.05). GSTM3TV2 expression were higher in the chemotherapy resistance pancreatic cancer cells AsPC-1/GR(210.799±19.788) and MIAPaCa-2/GR(122.408±23.419) than that in the AsPC-1(3.793±0.615) and the MIAPaCa-2(5.179±1.095)(t=21.800,P<0.05;t=-18.490,P<0.05). The results of in vivo experiments showed that the volume of subcutaneously transplanted tumors in the overexpressing GSTM3TV2 group ((1 059.609±102.498)mm(3)) was significantly larger than that in the control group((566.414±81.087) mm(3)) by treated with nab-paclitaxel(t=4.230,P<0.05).Meanwhile, GSTM3TV2 could promote the expression of Cyclin D1, CDK6, Cyclin E1, Vimentin, N-cadherin, ZEB1, Snail and Slug; but decrease cleaved caspase-3, cleaved PARP in pancreatic cancer cells. Conclusions: The expression level of GSTM3TV2 in pancreatic canceris higher than that in paired adjacent tissues. GSTM3TV2 may act as an oncogene to promote chemoresistance in pancreatic cancer through regulation of cell proliferation, apoptosis, and epithelial-mesenchymal transition.

A novel silent RHCE allele in Chinese population.

OBJECTIVES: We aimed to analyse the molecular backgrounds of the family in which an eight-day-old baby was confirmed to have hemolytic disease of the newborn (HDN) and phenotype observed for the baby did not conform to the expected phenotype. BACKGROUND: The silent RHCE allele is rare in the Rh system. METHODS: To determine the antibody specificity, her family members' blood samples were collected and tested using routine serological methods. The Rh C + c-e + E- phenotype observed for the baby did not conform to the expected phenotype based on the maternal RhC-c + E + e- phenotype. The RH genes of the family members were further analysed by sequencing. RESULTS: The Rh phenotypes of the baby, her brother, her mother and father were CCDee, CcDEe, ccDEE and CCDee, respectively. IgG anti-e was confirmed to cause the HDN in the case. A heterozygous silent RHCE * 03(c.1059G > A) mutation in exon 7 was found in the baby and her mother, which is a novel nonsense allele caused by a premature termination codon (Trp353stop). CONCLUSION: The silent RHCE * 03(c.1059G > A) variant was observed in a heterozygous state in mother and baby. We predict that, had this occurred in the homozygous state, it would give rise to the rare D-- phenotype. To enhance the safety of transfusion, considerable attention should be paid to the RHCE gene in the Chinese population.

A novel double-variant RHAG allele leads to Rhmod phenotype.

AIMS/OBJECTIVES: We aimed to analyse the molecular backgrounds and red blood cell (RBC) antigen expression of a male blood donor with Rhmod phenotype and his family members. BACKGROUND: Rh deficiency phenotypes are rarely found worldwide and are characterised by the lack of Rh antigen expression on RBCs. During routine screening, we found a blood donor who seemingly lacked Rh antigens. Therefore, we recruited the donor and his family for further investigation. METHODS: RBC serotyping and antibody screening/identification were performed for each sample. A routine blood examination was also conducted. RHD, RHCE and RHAG were sequenced at the genomic DNA or RNA level. Eleven antigens or proteins associated with Rh complex were tested using flow cytometry analysis. RESULTS: The proband and one of his brothers showed extremely weak D antigen and Rh expression levels but did not manifest anaemia. Most of the expressed RBC antigens of the two Rh-deficient individuals were similar to the previously reported cases but with some exceptions. Molecular analyses demonstrated homozygous expression of a novel RHAG allele, namely, c.[572G>A;707A>C], both in the proband and one of his brothers. CONCLUSIONS: To our knowledge, we identified the second double-variant RHAG allele and the first one related to Rhmod phenotype. The novel allele was also confirmed to be heritable by family analyses.

The impact of ageing, fasting and high-fat diet on central and peripheral glucose tolerance and glucose-sensing neural networks in the arcuate nucleus.

Obesity and ageing are risk factors for diabetes. In the present study, we investigated the effects of ageing, obesity and fasting on central and peripheral glucose tolerance and on glucose-sensing neuronal function in the arcuate nucleus of rats, with a view to providing insight into the central mechanisms regulating glucose homeostasis and how they change or are subject to dysfunction with ageing and obesity. We show that, following a glucose load, central glucose tolerance at the level of the cerebrospinal fluid (CSF) and plasma is significantly reduced in rats maintained on a high-fat diet (HFD). With ageing, up to 2 years, central glucose tolerance was impaired in an age-dependent manner, whereas peripheral glucose tolerance remained unaffected. Ageing-induced peripheral glucose intolerance was improved by a 24-hour fast, whereas central glucose tolerance was not corrected. Pre-wean, immature animals have elevated basal plasma glucose levels and a delayed increase in central glucose levels following peripheral glucose injection compared to mature animals. Electrophysiological recording techniques revealed an energy-status-dependent role for glucose-excited, inhibited and adapting neurones, along with glucose-induced changes in synaptic transmission. We conclude that ageing affects central glucose tolerance, whereas HFD profoundly affects central and peripheral glucose tolerance and, in addition, glucose-sensing neurones adapt function in an energy-status-dependent manner.

Effectiveness of bacterial infection-related cytokine profile (BIRCP) determination for monitoring pathogen infections in children with hemopathy in the bone marrow inhibition phase.

Serum cytokine profiles were analyzed before and after infection in children with hemopathy in the bone marrow inhibition phase to explore the utility of cytokine variations for detecting infections. Serum Th1/Th2 cytokine levels, including tumor necrosis factor, interleukin (IL)-2, IL-4, IL-6, IL-10, and interferon, were quantitatively determined by cytometric bead array technology in 480 cases (230 children) of children with hemopathy in the bone marrow inhibition phase with signs of infection, such as fever, and without, to establish baseline and affected levels for comparison with healthy control children. We used the cytokine profile of infected, blood culture-positive children to establish a bacterial infection-related cytokine profile (BIRCP) for predicting infections by pathogens in blood culture-negative children. Overall, 82.9% of children with Gram-negative bacterial infections were accompanied by marked increases of IL-6 and IL-10 levels [>10 times (means ± SD)], whereas only a mild increase of IL-6 levels occurred in Gram-positive bacteria-infected children [>2 times (means ± SD)] and only a mild increase of IFN-γ levels occurred in fungal culture-positive children [>2 times (means ± SD)]. Gram-positive bacterial and fungal infections did not cause a marked increase in IL-6 or IL- 10 levels. The effective rate (86.05%, N = 43) of infectious cases predicted by BIRCP was significantly higher than that obtained using traditional methods for selecting antibiotics based on clinical indications (65.45%, N = 55, P < 0.05). In summary, BIRCP can be used to predict the infections by pathogens in children with hemopathy and to select appropriate antibiotics.

A method for simultaneously delineating multiple targets in 3D-FISH using limited channels, lasers, and fluorochromes.

Many studies have suggested a link between the spatial organization of genomes and fundamental biological processes such as genome reprogramming, gene expression, and differentiation. Multicolor fluorescence in situ hybridization on three-dimensionally preserved nuclei (3D-FISH), in combination with confocal microscopy, has become an effective technique for analyzing 3D genome structure and spatial patterns of defined nucleus targets including entire chromosome territories and single gene loci. This technique usually requires the simultaneous visualization of numerous targets labeled with different colored fluorochromes. Thus, the number of channels and lasers must be sufficient for the commonly used labeling scheme of 3D-FISH, "one probe-one target". However, these channels and lasers are usually restricted by a given microscope system. This paper presents a method for simultaneously delineating multiple targets in 3D-FISH using limited channels, lasers, and fluorochromes. In contrast to other labeling schemes, this method is convenient and simple for multicolor 3D-FISH studies, which may result in widespread adoption of the technique. Lastly, as an application of the method, the nucleus locations of chromosome territory 18/21 and centromere 18/21/13 in normal human lymphocytes were analyzed, which might present evidence of a radial higher order chromatin arrangement.

5-Azacytidine induces early stage apoptosis and promotes in vitro maturation by changing chromosomal construction in murine oocytes.

As an anticancer drug, 5-azacytidine (5-AzaC) has been widely used to treat various cancers. To investigate the effect of 5-AzaC on mouse oocytes cultured in vitro, we have performed morphological and molecular biology studies to examine the behavior of chromosomes and oocyte development. In 5-AzaC-treated oocytes, chromosomes were decondensed and unstable. The mRNA levels of Caspase3, Caspase8, and Caspase9 increased with the occurrence of early stage apoptosis in oocytes following 5-AzaC treatment. Furthermore, the mRNA levels of Gdf9 and Bmp15 also increased with the corresponding morphological changes in 5-AzaC-treated oocytes. In conclusion, 5-AzaC not only induced early apoptosis through both extrinsic and intrinsic pathways, but also had a positive effect on the developmental competence of mouse oocytes during in vitro maturation. These effects may be due to changes in chromosomal construction induced by DNA hypomethylation.

A clinicopathologic study on basaloid squamous cell carcinoma in the oral and maxillofacial region.

Basaloid squamous cell carcinoma (BSCC) is a rare distinct variant of squamous cell carcinoma (SCC). To investigate its clinical behavior and prognosis, 15 patients with BSCC in the oral and maxillofacial region were clinically analyzed and compared with 15 patients with conventional SCC matched for site, stage, gender and age. To understand its immunohistochemical features, sections for cytokeratin AE1/AE3, CK 13. CK 7, CK 8, proliferating cell nuclear antigen (PCNA) and p53 were reviewed from 12 patients with BSCC. The rate of cervical lymph node metastasis of BSCC was as high as 67% and that of distant metastasis 13%. The tumor recurrence rate was 33% and the 3-year and 5-year survival rates were 53% and 32%, respectively. For conventional SCC, the cervical lymph node metastasis rate was 27%, that of distant metastasis 7%, tumor recurrence rate was 33%, and 3-year and 5-year survival rates were 80% and 70%, respectively. In most BSCC patients (10/12) the PCNA index was over 50%. Twelve BSCC patients were diagnosed with grade II or III conventional SCC when the original records of the primary diagnosis for the 15 patients with BSCC were reviewed. The biological behavior and prognosis of BSCC are similar to those of poorly differentiated SCC.

Diffusion tensor feature in vasogenic brain edema in cats.

We investigated the correlation between the changes in diffusion tensor magnetic resonance imaging, regional water content, and tissue ultrastructure after vasogenic brain edema induced by cortical cold lesioning. In this cat model, E3 in the white matter was dominantly increased while fractional anisotropy (FA) was significantly decreased 8 hours after cortical cold lesioning. This finding indicates that water diffusion in the cortical white matter mainly increased perpendicularly rather than parallel to the direction of the nerve fibers. Additionally, in the area where edema is mild or moderate (tissues with water content of 65% to 75%), FA in the chronic phase was significantly lower than that in the acute phase. Histological examination demonstrated disordered arrangement of nerve fibers, highly dissociated neuronal fibers due to extracellular accumulation of protein rich-fluid, and enlarged interfiber spaces in the acute phase.

Functional projection distances of spinal interneurons mediating reciprocal inhibition during swimming in Xenopus tadpoles.

The basis for longitudinal coordination among spinal neurons during locomotion is still poorly understood. We have now examined the functional projection distances for the longitudinal axons of reciprocal inhibitory 'commissural interneurons' in the spinal cord of young Xenopus tadpoles. In quiescent animals, glycinergic inhibitory postsynaptic potentials (IPSPs) were evoked in ventral spinal neurons by stimulating small rostral and caudal groups of commissural interneuron somata at different distances on the opposite side of the hindbrain and spinal cord. Unitary IPSPs, produced by single synaptic contacts, could be distinguished from background noise. Local cord stimulation at different distances revealed maximum functional projection distances up to approximately 0.5 mm for both descending and ascending axons, but with the probability of recording connections falling steeply over this distance. These maximum longitudinal projection distances are smaller than predicted by axonal anatomy (approximately 1.2 mm). We then measured functional projection distances during swimming by examining the synaptic output of a surgically isolated group of rostral commissural interneurons, mapping the occurrence of the mid-cycle, reciprocal IPSPs they produced in more caudal neurons. IPSPs occurred with high probability up to 0.9 mm away, nearly twice the projection distance found in quiescent tadpoles. These results show that synaptic contacts from commissural interneurons could influence longitudinal coupling during swimming at distances of up to 0.9 mm (approximately 4-5 myotome segments or approximately 25% of the spinal cord). They provide direct evidence for functional projection distances of a characterized class of interneurons belonging to a spinal locomotor pattern generator.

Cerebral ischemic hypoxia: discrepancy between apparent diffusion coefficients and histologic changes in rats.

PURPOSE: To compare the apparent diffusion coefficients (ADCs) and histologic changes in young rats subjected to cerebral ischemic hypoxia (IH). MATERIALS AND METHODS: Fifteen 3-week-old rats were subjected to a 30-minute IH insult (unilateral common carotid arterial ligation and exposure to 8% oxygen) and were examined at diffusion-weighted magnetic resonance imaging and light and electron microscopy on cessation of the insult (n = 5), 60 minutes after resuscitation (n = 5), or 48 hours after resuscitation (n = 5). Twelve control rats either underwent unilateral common carotid arterial ligation or were subjected to hypoxia. RESULTS: The experimental rats showed primary ADC reduction during the insult, transient ADC recovery after resuscitation, and secondary ADC reduction 48 hours after the insult. Histologic examination revealed dendritic swelling and mild swelling of the perivascular astrocytic end-feet during the primary ADC reduction phase, dark neurons and pronounced swelling of the perivascular astrocytic end-feet during the transient ADC recovery phase, and severely retracted dark neurons and extensive swelling of the astrocytic end-feet during the secondary ADC reduction phase. CONCLUSION: Transient ADC normalization after cerebral IH does not necessarily mean that histologic normalization has occurred. The transient ADC recovery phase appeared to have limited potential for neuronal salvage.

Assessing the roles of glutamatergic and cholinergic synaptic drive in the control of fictive swimming frequency in young Xenopus tadpoles.

This paper investigates the proposal that the frequency of the swimming central pattern generator in young Xenopus tadpoles is partly determined by the population of glutamatergic premotor interneurons active on each cycle. During fictive swimming spinal neurons also receive cholinergic and electrotonic excitation from motoneurons. As frequency changes during swimming we make two predictions: first, since most motoneurons fire very reliably at all frequencies, the electrotonic and nicotinic drive from motoneurons should remain constant, and second, when swimming frequency decreases, the glutamatergic drive should decrease as the number of active premotor excitatory interneurons decreases. We have tested these predictions by measuring the excitatory synaptic drive to motoneurons as frequency changes during fictive swimming. The components of synaptic drive were revealed by the local microperfusion of strychnine together with different excitatory antagonists. After blocking the nicotinic acetylcholine receptor, the mainly glutamatergic excitatory synaptic drive still changed with frequency. However, when glutamate receptors or all chemical transmission was blocked, excitation did not change with frequency. Our predictions are confirmed, suggesting that premotor excitatory interneurons are a major factor in frequency control in the tadpole central pattern generator and that motoneurons provide a stable background excitation.