A large deletion in TSC2 causes tuberous sclerosis complex by dysregulating PI3K/AKT/mTOR signaling pathway.

BACKGROUND/AIM: Tuberous sclerosis complex (TSC) is a multi-system syndrome caused by loss-of-function mutation in TSC1 or TSC2. Most TSC patients present with cardiac rhabdomyoma or cortical tubers during fetal life, and the symptoms are not uniform as their age. The gene products of TSC1/2 are components of the TSC protein complex and are important role in the PI3K/AKT/mTOR (PAM) signaling pathway. Based on three members of a family with variable expressivity, the purpose of this study was to clarify the clinical features of TSC in different age groups and to analyze the genetic characteristics of TSC2 gene. METHODS: Clinical exome sequencing and co-segregation were used to identify a three-generation family with four affected individuals. HEK-293T cell model was constructed for subsequent experiments. Quantitative RT-PCR, western blotting, and subcellular localization were used to analyze the expression effect of TSC2 mutation. CCK-8 assay, wound healing assay, and cell cycle analysis were used to analyze the function effect of TSC2 mutation. RESULT: We identified a TSC family with heterozygous deletion of exon 4 in TSC2 by clinical exon sequencing. Sanger sequencing indicated that the affected individuals have 2541-bp deletion that encompassed exon 4 and adjacent introns. Deletion of exon 4 decreased the TSC2 mRNA and protein levels in HEK-293T cells, and activated the PI3K/AKT/mTOR pathway, thereby altering the cell cycle and promoting cell proliferation and migration. CONCLUSION: We confirmed the pathogenicity of the large deletion in TSC2 in a three- generations family.. Deletion of exon 4 of TSC2 affected cell proliferation, migration, and cell cycle via abnormal activation of the PAM pathway. This study evaluated the pathogenic effect of deletion of exon 4 of TSC2 and investigated the underlying mechanism.

Plasma-derived exosomal miRNA profiles reveal potential epigenetic pathogenesis of premature ovarian failure.

The role of plasma-derived exosomal miRNA in premature ovarian failure (POF) remains unclear. This study aimed to investigate the epigenetic pathogenesis of POF through exosomal miRNA sequencing. Exosomes were isolated and characterized from six POF patients and four healthy individuals using nanoparticle tracking analysis, transmission electron microscopy and western blot analysis. Exosomal miRNA sequencing was performed to identify differentially expressed miRNAs with |fold change| greater than 1.5 and p value less than 0.05. Bioinformatics analysis in GSE39501 dataset and our sequencing data was conducted to investigate underlying mechanisms of POF. The functional role of hsa-miR-19b-3p was assessed using CCK8, western blot, flow cytometry and fluorescence staining. The regulatory effect of hsa-miR-19b-3p on BMPR2 was investigated through miRNA transfection, qPCR analysis, and luciferase reporter assay. Statistical significance was determined using t-tests and one-way ANOVA (p < 0.05). Exosomal miRNA sequencing revealed 18 dysregulated miRNAs in POF patients compared to healthy controls. Functional enrichment analysis demonstrated their involvement in cell growth, oocyte meiosis and PI3K-Akt signaling pathways. Moreover, the constructed miRNA-mRNA network unveiled potential regulatory mechanisms underlying POF, particularly implicating hsa-miR-19b-3p in the regulation of BMPR2. In vitro assays conducted on KGN cells confirmed that hsa-miR-19b-3p promoted apoptosis, as evidenced by reduced cell viability, decayed mitochondrial membrane potential and increased apoptotic rate, thereby supporting its role in POF. Notably, hsa-miR-19b-3p was found to significantly downregulate BMPR2 expression via targeting its 3'UTR, while co-expression analysis revealed strong associations between BMPR2 and POF-related processes. This study sheds light on the epigenetic pathogenesis of POF by investigating exosomal miRNA profiles. Particularly, hsa-miR-19b-3p emerged as a potential regulator of BMPR2 and demonstrated its functional significance in POF through modulation of apoptosis.

[Genetic analysis of two cases with MYC "negative" Burkitt lymphoma].

OBJECTIVE: To carry out combined genetic analysis on two patients suspected for Burkitt lymphoma to facilitate their diagnosis and treatment. METHODS: G banded karyotyping and interphase and metaphase fluorescence in situ hybridization (FISH) were used to detect the specific sites of chromosomes by using separate and fusion probes. RESULTS: The separate probe showed no presence of MYC gene abnormality, while fusion probe confirmed the IGH::MYC translocation in the samples. Combined with the clinical features and pathological characteristics, the two patients were finally diagnosed with Burkitt lymphoma, which was confirmed by targeted capture next generation sequencing. CONCLUSION: The separate probe for the MYC gene has some shortcomings and should be used together with dual fusion probe to improve the accuracy of diagnosis.

Intravenous Administration of an AAV9 Vector Ubiquitously Expressing C1orf194 Gene Improved CMT-Like Neuropathy in C1orf194-/- Mice.

Charcot-Marie-Tooth (CMT) disease, also known as hereditary motor sensory neuropathy, is a group of rare genetically heterogenous diseases characterized by progressive muscle weakness and atrophy, along with sensory deficits. Despite extensive pre-clinical and clinical research, no FDA-approved therapy is available for any CMT type. We previously identified C1ORF194, a novel causative gene for CMT, and found that both C1orf194 knock-in (I121N) and knockout mice developed clinical phenotypes similar to those in patients with CMT. Encouraging results of adeno-associated virus (AAV)-mediated gene therapy for spinal muscular atrophy have stimulated the use of AAVs as vehicles for CMT gene therapy. Here, we present a gene therapy approach to restore C1orf194 expression in a knockout background. We used C1orf194-/- mice treated with AAV serotype 9 (AAV9) vector carrying a codon-optimized WT human C1ORF194 cDNA whose expression was driven by a ubiquitously expressed chicken ß-actin promoter with a CMV enhancer. Our preclinical evaluation demonstrated the efficacy of AAV-mediated gene therapy in improving sensory and motor abilities, thus achieving largely normal gross motor performance and minimal signs of neuropathy, on the basis of neurophysiological and histopathological evaluation in C1orf194-/- mice administered AAV gene therapy. Our findings advance the techniques for delivering therapeutic interventions to individuals with CMT.

Double heterozygous pathogenic mutations in KIF3C and ZNF513 cause hereditary gingival fibromatosis.

Hereditary gingival fibromatosis (HGF) is a rare inherited condition with fibromatoid hyperplasia of the gingival tissue that exhibits great genetic heterogeneity. Five distinct loci related to non-syndromic HGF have been identified; however, only two disease-causing genes, SOS1 and REST, inducing HGF have been identified at two loci, GINGF1 and GINGF5, respectively. Here, based on a family pedigree with 26 members, including nine patients with HGF, we identified double heterozygous pathogenic mutations in the ZNF513 (c.C748T, p.R250W) and KIF3C (c.G1229A, p.R410H) genes within the GINGF3 locus related to HGF. Functional studies demonstrated that the ZNF513 p.R250W and KIF3C p.R410H variants significantly increased the expression of ZNF513 and KIF3C in vitro and in vivo. ZNF513, a transcription factor, binds to KIF3C exon 1 and participates in the positive regulation of KIF3C expression in gingival fibroblasts. Furthermore, a knock-in mouse model confirmed that heterozygous or homozygous mutations within Zfp513 (p.R250W) or Kif3c (p.R412H) alone do not led to clear phenotypes with gingival fibromatosis, whereas the double mutations led to gingival hyperplasia phenotypes. In addition, we found that ZNF513 binds to the SOS1 promoter and plays an important positive role in regulating the expression of SOS1. Moreover, the KIF3C p.R410H mutation could activate the PI3K and KCNQ1 potassium channels. ZNF513 combined with KIF3C regulates gingival fibroblast proliferation, migration, and fibrosis response via the PI3K/AKT/mTOR and Ras/Raf/MEK/ERK pathways. In summary, these results demonstrate ZNF513 + KIF3C as an important genetic combination in HGF manifestation and suggest that ZNF513 mutation may be a major risk factor for HGF.

The loss of function GBA1 c.231C > G mutation associated with Parkinson disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease characterized by bradykinesia, rigidity, and tremor. However, familial PD caused by single-gene mutations remain relatively rare. Herein, we described a Chinese family affected by PD, which associated with a missense heterozygous glucocerebrosidase 1 (GBA1) mutation (c.231C > G). Clinical data on the proband and her family members were collected. Brain MRI showed no difference between affected and unaffected family members. Whole-exome sequencing (WES) was performed to identify the pathogenic mutation. WES revealed that the proband carried a missense mutation (c.231C > G) in GBA1 gene, which was considered to be associated with PD in this family. Sanger sequencing and co-segregation analyses were used to validate the mutation. Bioinformatics analysis indicated that the mutation was predicted to be damaging. In vitro functional analyses were performed to investigated the mutant gene. A decrease in mRNA and protein expression was observed in HEK293T cells transfected with mutant plasmids. The GBA1 c.231C > G mutation caused a decreased GBA1 concentration and enzyme activity. In conclusion, a loss of function mutation (c.231C > G) in GBA1 was identified in a Chinese PD family and was confirmed to be pathogenic through functional studies. This study help the family members understand the disease progression and provide a new example for studying the pathogenesis of GBA1-associated Parkinson disease.

A Rare Heterozygous TINF2 Deletional Frameshift Mutation in a Chinese Pedigree With a Spectrum of TBDs Phenotypes.

Telomere biology disorders (TBDs) induced by TINF2 mutations manifest clinically with a spectrum of phenotypes, from silent carriers to a set of overlapping conditions. A rare TINF2 frameshift mutation (c.591delG) encoding a truncated mutant TIN2 protein (p.W198fs) was identified in a 6-years-and-3-month-old Chinese girl with neuroblastoma (NB) by next generation sequencing and confirmed by Sanger sequencing. To explore the possible implications of TINF2 mutations in TBDs development, the TINF2 mutant was transfected into the human embryonic kidney (HEK) 293T cells, and mRNA expression of the shelterin complex components as well as the cellular distribution of mutant TIN2 were examined. The TINF2 mutation was phenotypically associated with short stature in the proband, nail dystrophy and spotted hypopigmentation in her mother, and psoriasis in her older brother. I-TASSER modeling analysis revealed conformational changes of the mutant TIN2 protein and loss of pivotal domains downstream of the 198th amino acid. Additionally, mRNA expression of the shelterin components was downregulated, and TIN2 mutant protein expression was reduced in HEK293T cells transfected with mutant TINF2. Furthermore, instead of being restricted to the nucleus, the mutant TIN2 was identified in both the cytoplasm and the nucleus. The TINF2 gene mutation might impair the function of the shelterin complex and the telomere maintenance mechanisms, both of which are involved in the development of TBDs. TBDs have been associated with increased cancer risk. To the best of our knowledge, this is the first report of NB in patients with TBDs. The relationship between the TINF2 mutation and NB may need to further study.

A Heterozygous LMF1 Gene Mutation (c.1523C>T), Combined With an LPL Gene Mutation (c.590G>A), Aggravates the Clinical Symptoms in Hypertriglyceridemia.

Hypertriglyceridemia is an important contributor to atherosclerotic cardiovascular disease (ASCVD) and acute pancreatitis. Familial hypertriglyceridemia is often caused by mutations in genes involved in triglyceride metabolism. Here, we investigated the disease-causing gene mutations in a Chinese family with hypertriglyceridemia and assessed the functional significance in vitro. Whole-exome sequencing (WES) was performed revealing that the severe hypertriglyceridemic proband carried a missense mutation (c.590G > A) in exon 5 of the LPL gene, as well as a missense mutation (c.1523C > T) in exon 10 of the LMF1 gene. Conservation analysis by Polyphen-2 showed that the 508 locus in the LMF1 protein and 197 locus in the LPL protein were highly conserved between different species. I-TASSER analysis indicated that the LMF1 c.1523C > T mutation and the LPL c.590G > A mutation changed the tertiary structure of the protein. A decrease in mRNA and protein expression was observed in 293T cells transfected with plasmids carrying the LMF1 c.1523C > T mutation. Subcellular localization showed that both wild-type (WT) and mutant LMF1 protein were localized at the cell cytoplasm. In the cell medium and cell lysates, these LMF1 and LPL gene mutations both caused a decreased LPL mass. Moreover, the combination of LMF1 and LPL gene mutations significantly decreased LPL levels compared to their individual effects on the LPL concentration. Both the clinical and in vitro data suggest that severe hypertriglyceridemia was of digenic origin caused by LMF1 and LPL mutation double heterozygosity in this patient.

CircEXOC6B Suppresses the Proliferation and Motility and Sensitizes Ovarian Cancer Cells to Paclitaxel Through miR-376c-3p/FOXO3 Axis.

Background: Circular RNAs (circRNAs) are regarded as important regulators in the tumorigenesis of multiple cancers. However, the characterization of circRNA exocyst complex component 6B (circEXOC6B) in ovarian cancer is barely known. Materials and Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to detect the enrichment of circEXOC6B, microRNA-376c-3p (miR-376c-3p), and forkhead box O3 (FOXO3). Cell proliferation was examined by Cell Counting Kit-8 (CCK8) assay and colony formation assay. Cell metastasis was measured by transwell assays. Western blot assay was conducted to examine the expression of proliferation and metastasis-related proteins and FOXO3. The chemoresistance of ovarian cancer cells was analyzed by CCK8 assay. Flow cytometry was used to detect cell apoptosis. The activities of caspase3 and caspase9 were analyzed through using colorimetric assay kits. The direct interaction between miR-376c-3p and circEXOC6B or FOXO3 was predicted by StarBase software and confirmed by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Murine xenograft assay was conducted to verify the role of circEXOC6B on the paclitaxel (PTX) resistance of ovarian cancer cells in vivo. Results: The level of circEXOC6B was notably decreased in ovarian cancer tissues. Low level of circEXOC6B was associated with malignant pathological characteristics in ovarian cancer patients. CircEXOC6B suppressed the proliferation and motility and decreased the chemoresistance of ovarian cancer cells to PTX. CircEXOC6B functioned through directly targeting and downregulating miR-376c-3p. FOXO3 was a direct target of miR-376c-3p, and the abundance of FOXO3 was regulated by circEXOC6B/miR-376c-3p axis. CircEXOC6B accelerated the PTX sensitivity of ovarian cancer cells through acting as a decoy of miR-376c-3p to upregulate FOXO3 in vivo. Conclusion: CircEXOC6B suppressed the progression and PTX resistance of ovarian cancer cells through sequestering miR-376c-3p, thus enhancing FOXO3 level.

ACBD3 is up-regulated in gastric cancer and promotes cell cycle G1-to-S transition in an AKT-dependent manner.

It has been reported that ACBD3 is closely related to the malignant process of cells, but its role in gastric cancer has not been elucidated. This study aims to investigate the expression and function of ACBD3 in human gastric cancer. The Cancer Genome Atlas (TCGA) database were selected to analyze mRNA levels of ACBD3 in gastric cancer tissues and normal gastric epithelial tissues. qPCR and Western blot were conducted to detect the expression of ACBD3 in two normal gastric epithelial cell lines and five gastric cancer cell lines which were cultured in our laboratory. To exclude differences in individual background between different patients, we further detected the expression of ACBD3 in 8 pairs of malignant/non-malignant clinical gastric tissues. Through the establishment of stable cells, in vitro cell experiments and in vivo xenotransplantation models in mice, the role of ACBD3 in the proliferation of gastric cancer cells has been further explored. AKT inhibitors were used to deeply explore the molecular regulation mechanism of ACBD3. The results showed that the elevated ACBD3 in gastric cancer tissue were positively correlated with the clinical grade and prognosis of gastric cancer. In terms of molecular function, we found that ACBD3 can enhance the production and growth of gastric cancer cells. At the same time, the activation of AKT kinase played an important role in ACBD3's promotion of G1-to-S transition. The experiments generally indicate that ACBD3 is expected to become a potential diagnostic molecule or therapeutic target for gastric cancer.

A Novel Mutation in ATRX Causes Alpha-Thalassemia X-Linked Intellectual Disability Syndrome in a Han Chinese Family.

OBJECTIVE: To analyze genetic mutations in a Chinese pedigree affected with Alpha-thalassemia X-linked intellectual disability syndrome, providing a precise diagnosis and genetic counseling. METHODS: Clinical data was collected. A novel alternative splicing variant detected by whole-exome sequencing was validated by Sanger sequencing. The functional effect of the mutation was predicted with Mutation Tasting. The analysis of 5' splice site score was estimated with MaxEntScan. Changes in amino acid sequencing were predicted with Mutalyzer. The tertiary structures of the wild type and mutation-carrying protein were predicted by I-TASSER. RNA was extracted from peripheral blood lymphocytes from the proband, his mother and a healthy control. Quantitative Real-Time PCR was used to detect mRNA expression. RESULTS: The proband presented with severe intellectual disability, developmental delay, characteristic facies, seizures and cryptorchidism. A novel hemizygous duplication mutation in the ATRX gene in a splice site between exons 3 and 4, NM_000489: c.189+1dupG, was identified with WES in the proband. Sanger sequencing confirmed that the mutation was inherited from his mother, who carried a heterozygous mutation, while his father was not affected. Bioinformatics analysis indicated that the splicing region where the mutation was located is highly conserved and the variant was damaging, producing a truncated protein due to the premature translation of a stop codon. Sanger sequencing with the Quantitative Real-Time PCR product containing a G base inserted between bases 189 and 190. The level of mRNA expression showed that ATRX gene transcription decreased due to the mutation (P < 0.05). CONCLUSIONS: A novel mutation in ATRX was found in this pedigree and was confirmed to be pathogenic through functional studies. Our research expanded the spectrum of ATRX gene mutations, providing a precise diagnosis and a basis for genetic counseling.

The gain-of-function FAM83H mutation caused hypocalcification amelogenesis imperfecta in a Chinese family.

OBJECTIVES: Autosomal-dominant hypocalcification amelogenesis imperfecta (ADHCAI) is a hereditary disease characterized by enamel defects. ADHCAI is mainly caused by nonsense mutations in a gene called family with sequence similarity 83 member H (FAM83H). To study the pathogenesis of ADHCAI, a Chinese ADHCAI family was investigated. MATERIALS AND METHODS: The ultrastructure of enamel was analyzed by micro-CT and scanning electron microscopy. Whole-exome sequencing (WES) was performed to identify the pathogenic gene. The function of the mutant FAM83H was studied by real-time PCR, western blotting, subcellular localization, and protein degradation pathway analyses. RESULTS: WES identified a known nonsense mutation (c.1915A > T) in exon 5 of the FAM83H gene, causing a truncated protein (p.Lys639*). However, the cases reported herein exhibited significant differences in the clinical phenotype compared with that the previously reported case. An abnormal enamel rod head structure was observed in affected teeth. In vitro functional studies showed altered protein localization and a decreased protein degradation rate for mutant FAM83H. CONCLUSIONS: We verified the FAM83H p.Lys639* protein as a gain-of-function variant causing ADHCAI. Abnormal enamel rod head structure was observed in teeth with mutant FAM83H proteins. We also investigated the molecular pathogenesis and presented data on the abnormal degradation of mutant FAM83H proteins. CLINICAL RELEVANCE: This study helped the family members to understand the disease progression and provided new insights into the pathogenesis of ADHCAI. Due to the large heterogeneity of ADHCAI, this study also provided a genetic basis for individuals who exhibit similar clinical phenotypes.

Upregulated GRB7 promotes proliferation and tumorigenesis of Bladder Cancer via Phospho-AKT Pathway.

Growth factor receptor-bound protein 7 (GRB7) has been found closely related to the occurrence and development of various tumors, but its function in bladder cancer has not yet been elucidated. The study is aiming at investigating the expression and function of GRB7 in bladder cancer. The Cancer Genome Atlas (TCGA) database was selected to analyze mRNA levels of GRB7 in bladder cancer. RT-qPCR and Western blot were conducted to detect the expression of GRB7 in normal bladder epithelial cells, seven bladder cancer cell lines and eight pairs of malignant/nonmalignant bladder tissues. The role of GRB7 in tumor proliferation and tumorigenesis was explored by establishing stable cells, in vitro cell experiments and in vivo xenograft models. The molecular regulation mechanism of GRB7 in bladder cancer was investigated by treatment with AKT inhibitor. GRB7 mRNA was upregulated in bladder cancer samples compared with that in normal tissue samples. Overexpressing GRB7 significantly promoted the proliferation and tumorigenesis of bladder cancer. However, silencing GRB7 played the retarding part. GRB7 promoted G1/S transition by activating the AKT pathway. Our results indicate that GRB7 plays an important role in promoting proliferation and tumorigenesis of bladder cancer.

A Marine Alkaloid, Ascomylactam A, Suppresses Lung Tumorigenesis via Inducing Cell Cycle G1/S Arrest through ROS/Akt/Rb Pathway.

Ascomylactam A was reported for the first time as a new 13-membered-ring macrocyclic alkaloid in 2019 from the mangrove endophytic fungus Didymella sp. CYSK-4 from the South China Sea. The aim of our study was to delineate the effects of ascomylactam A (AsA) on lung cancer cells and explore the antitumor molecular mechanisms underlying of AsA. In vitro, AsA markedly inhibited the cell proliferation with half-maximal inhibitory concentration (IC50) values from 4 to 8 µM on six lung cancer cell lines, respectively. In vivo, AsA suppressed the tumor growth of A549, NCI-H460 and NCI-H1975 xenografts significantly in mice. Furthermore, by analyses of the soft agar colony formation, 5-ethynyl-20-deoxyuridine (EdU) assay, reactive oxygen species (ROS) imaging, flow cytometry and Western blotting, AsA demonstrated the ability to induce cell cycle arrest in G1 and G1/S phases by increasing ROS generation and decreasing of Akt activity. Conversely, ROS inhibitors and overexpression of Akt could decrease cell growth inhibition and cell cycle arrest induced by AsA. Therefore, we believe that AsA blocks the cell cycle via an ROS-dependent Akt/Cyclin D1/Rb signaling pathway, which consequently leads to the observed antitumor effect both in vitro and in vivo. Our results suggest a novel leading compound for antitumor drug development.

Antibiotic fidaxomicin is an RdRp inhibitor as a potential new therapeutic agent against Zika virus.

BACKGROUND: Zika virus (ZIKV) infection is a global health problem, and its complications, including congenital Zika syndrome and Guillain-Barré syndrome, constitute a continued threat to humans. Unfortunately, effective therapeutics against ZIKV infection are not available thus far. METHODS: We screened the compounds collection consisting of 1789 FDA-approved drugs by a computational docking method to obtain anti-ZIKV candidate compounds targeting ZIKV RNA-dependent RNA polymerase (RdRp). SPR (BIAcore) assay was employed to demonstrate the candidate compounds' direct binding to ZIKV RdRp, and polymerase activity assay was used to determine the inhibitory effect on ZIKV RdRp-catalyzed RNA synthesis. The antiviral effects on ZIKV in vitro and in vivo were detected in infected cultured cells and in Ifnar1-/- mice infected by ZIKV virus using plaque assay, western blotting, tissue immunofluorescence, and immunohistochemistry. RESULTS: Here, we report that a first-in-class macrocyclic antibiotic, which has been clinically used to treat Clostridium difficile infection, fidaxomicin, potently inhibits ZIKV replication in vitro and in vivo. Our data showed that fidaxomicin was effective against African and Asian lineage ZIKV in a wide variety of cell lines of various tissue origins, and prominently suppressed ZIKV infection and significantly improved survival of infected mice. In addition, fidaxomicin treatment reduced the virus load in the brains and testes, and alleviated ZIKV-associated pathological damages, such as paralysis, hunching, and neuronal necrosis in the cerebra. Furthermore, our mechanistic study showed that fidaxomicin directly bound ZIKV NS5 protein and inhibited the RNA synthesis-catalyzing activity of ZIKV RdRp. CONCLUSIONS: Our data suggest that fidaxomicin may represent an effective anti-ZIKV agent. In the light that fidaxomicin is already a clinically used drug, there might be a promising prospect in the development of fidaxomicin to be an antiviral therapeutic.

[Structural characteristics of the deciduous teeth of Tibetan miniature pigs].

OBJECTIVE: To explore the histological structure of the deciduous teeth and the tooth germs of Tibetan miniature pigs for studies of dental tissue diseases and tooth regeneration. METHODS: The structure of the deciduous teeth of Tibetan miniature pigs was observed by X-ray. The ultrastructure of the enamel and dentin of deciduous teeth was characterized by scanning electron microscopy. The jaws and teeth were three-dimensionally reconstructed using Mimics software based on Micro-CT scanning of the deciduous teeth. Image J software was used to calculate the gray value and the mineralization density of the deciduous teeth. Hisotological structure of the tooth germ and the pulp tissue of Tibetan miniature pigs was observed using HE staining. RESULTS: The deciduous teeth of Tibetan miniature pigs were composed of enamel, dentin and medullary pulp tissue. The permanent tooth germ were formed during the deciduous dentition. The enamel and dentin ultrastructure of deciduous teeth were consistent with that of human deciduous teeth. The enamel and dentin mineralization densities were 2.47±0.09 g/cm3 and 1.72±0.07 g/cm3, respectively. The pathological structures of tooth germ and pulp tissue were similar to those of human teeth, and the pulp tissue of the deciduous teeth was in an undifferentiated state. CONCLUSIONS: The deciduous teeth of Tibetan miniature pig have similar anatomy, ultrastructure and histopathological structure to human teeth and can serve as a good animal model for studying human dental tissue diseases and the mechanisms of tooth regeneration.

Mesenchymal stem cells derived from multiple myeloma patients protect against chemotherapy through autophagy-dependent activation of NF-κB signaling.

Chemotherapy resistance has been considered as a major problem for multiple myeloma (MM) treatment and bone marrow microenvironment plays a crucial role in the MM progression and chemoresistance. Recent studies reported that bone marrow mesenchymal stem cells derived from MM patients (MM-MSCs) revealed various characteristics compared with these from healthy subjects (NM-MSCs). However, the functions and mechanisms by which MM-MSCs mediate the chemotherapy resistance of MM remain unclear. In this study, we show that MM-MSCs decreased melphalan or doxorubicin-induced cell cycle arrest and apoptosis in two MM cell lines (U266 and RPMI-8226). Mechanistically, we demonstrate that MM-MSCs promote the expressions of autophagy related genes to activate autophagy, followed by ultimately NF-κB signaling activation in MM cells. Inhibition of NF-κB signaling reversed the protective effects of MM-MSC on MM cells. Moreover, autophagy inhibitor chloroquine (CQ) or 3-Methyladenine (3MA) treatment significantly suppressed phosphorylation and consequently degradation of NF-κB inhibitor I-κBα, reduced MM-MSCs-mediate activation of NF-κB and prevented MM-MSCs-induced resistance. Taken together, our findings indicate MM-MSCs are involved in the mechanism of the chemotherapy resistance of MM. Therefore, the inhibition of MM-MSCs-induced autophagy may combat chemotherapy resistance and provide a promising therapeutic strategy for MM treatment.

[Clinical and Cytogenetic Characteristics of Two AML Patients with High-level MLL Expression].

OBJECTIVE: To investigate the clinical and cytogenetic characteristics of high-level mixed-lineage leukaemia (MLL) gene amplification in patients with acute myeloid leukemia (AML). METHODS: The clinical and cytogenetic data of 2 AML patients with high-level MLL amplification from January 2010 to August 2016 were analyzed retrospectively. RESULTS: The two AML cases were in middle-aged population. They were diagnosed as FAB subtype M5b and M2a respectively. Both of them had complex karyotypes with the aberrations of chromosome 11. One case was confirmed as MLL-PTD involving exons 2-9 by RT-PCR and sequencing. The other case without MLL-PTD was further analyzed by CytoScan HD analysis. The CMA results showed partial gain of 11q accompanied with partial loss in 11q, deletion of regions in 3p, 3q, 4q, 5q, 7q, 8q, 10p, 10q, 12p and 18q, as well as gain of 4p. CONCLUSION: The co-existence of -5/5q-, -7/7q- and highly complex karyotype may accelerate the poor prognosis. Thus how those cytogenetic abnormalities influencing the disease prognosis need to be further explored.

[Application of Combined Detection of Fusion Gene and BIOMED-2 Standardized Ig Gene Rearrangement System in Childhood B-cell Acute Lymphoblastic Leukemia].

OBJECTIVE: To explore the application of combined detection of fusion gene and BIOMED-2 standardized immunoglobulin (Ig) gene rearrangement system in diagnosis and treatment of children with acute lymphoblastic leukemia (ALL). METHODS: Multiplex-PCR amplifications and RQ-PCR of RNA/DNA were performed using ALL fusion gene detection kit and BIOMED-2 primer. The Ig gene rearrangements were analyzed by using PCR fragment analysis system. RESULTS: Out of 251 children with B-ALL, 77 cases were TEL-AML1(+) , 28 cases were E2A-PBX1(+) , 10 cases were MLL-AF4(+) , 11 cases were BCR-ABL(+) , the total positive rate was 50.2%, 82.5% showed IgH VH-JH rearrangement, 53.4% showed IgK rearrangement. The positive rate of combined detection of fusion gene and gene rearrangement was 99%. E2A-PBX1(+) and MLL-AF4(+) with IgK(+) gene rearrangement group was compared with negative control group, the difference was statistically significant (P < 0.001 or P = 0.005); 105 ALL fusion gene positive cases had been detected by fluorescence in situ hybridization (FISH) simultaneously, the accordance rate of fusion gene and FISH was more than 94%. CONCLUSION: The combined detection of ALL fusion gene and BIOMED-2 standardized clonality analysis system can improve the positive detected rate of B-ALL dramatically, and make the grouping of disease prognosis more accurately; this combined detection is a more faster and sensitive method than FISH.