Feasibility analysis of rapid gene detection using intraoperative frozen tissues: comparison of intraoperative frozen tissues with paraffin-embedded tissues in epidermal growth factor receptor gene mutation detection of lung adenocarcinoma.

BACKGROUND: Epidermal growth factor receptor (EGFR) is the driver gene with the highest frequency of mutations in lung adenocarcinoma and can guide the development of targeted therapies. The detection of routine gene mutations must be performed after the preparation of paraffin samples in a standard polymerase chain reaction (PCR) laboratory, which is time-consuming. The Idylla™ EGFR fully automatic PCR system for rapid detection requires no special detection environment and completes the process in only 2.5 h. It has been applied to tissues embedded in paraffin. METHODS: The Idylla™ EGFR automated PCR system was used to detect EGFR gene mutations in intraoperative frozen fresh tissues and paraffin-embedded tissues from 47 enrolled patients with lung adenocarcinoma. The gold standard amplification refractory mutation system (ARMS) method for gene mutation detection was used for verification, and the concordance between the three detection results was compared, to investigate the feasibility of detecting rapid gene mutations in intraoperative frozen samples. RESULTS: The EGFR mutation rate in 47 fresh samples of lung adenocarcinoma was 61.7% (29/47), which is consistent with the mutation level of lung adenocarcinoma in the Asian population (38.8-64.0%). The concordance rate between the Idylla™ frozen tissues and paraffin-embedded tissues was 91.4% (43/47) when compared to the ARMS method, while the coincidence rate between the two methods was 93.6% (44/47). The three methods had a total consistency rate of 89.4% (42/47). CONCLUSIONS: The Idylla™ EGFR fully automatic PCR system directly detects EGFR mutations in fresh tissues. The operation is simple, the detection time is short, and the accuracy is high. The detection time is reduced to 1/4-1/3 of the original time while meeting clinical standards for detecting the gene status of patients, thus saving crucial time for individualized and accurate treatment of patients. The method has promising clinical application prospects.

A comparison of capillary electrophoresis and next-generation sequencing in the detection of immunoglobulin heavy chain H and light chain κ gene rearrangements in the diagnosis of classic hodgkin's lymphoma.

This study aimed to compare the application value of capillary electrophoresis and next-generation sequencing for immunoglobulin (IG) gene rearrangement in the diagnosis of classic Hodgkin's lymphoma. Twenty paraffin-embedded specimens from patients with classic Hodgkin's lymphoma were screened. For gene rearrangement detection, the ABI 3500 Genetic Analyzer and ABI Ion GeneStudio S5 Plus sequencing system were used, respectively, and the results were compared. Five cases with monoclonal rearrangements (25%, 5/20) were detected by Capillary Electrophoresis, and positivity for the FR1, FR2, FR3, and IGк loci was 5%, 10%, 10%, and 15%, respectively; 12 cases with monoclonal rearrangements (60%, 12/20) were detected by Next-generation Sequencing where the positivity of the above corresponding loci were 35%, 45%, 50%, and 30%, respectively. Among the 20 samples, 6 IGк clonal rearrangements were detected, and the usage frequency (66.7%) of IGкJ4 was the highest in the IGкJ subgroup. The usage frequency of IGкV1 and IGкV3 in the GкV sub-group was 33.3% and 33.3%, respectively. Twelve immunoglobulin heavy chain (IGH) clonal rearrangements were detected among the 20 samples, and the order of usage frequency in the IGH joining region J (IGHJ) subgroup was IGHJ4 > IGHJ5 > IGHJ6 > IGHJ3. The gene with the highest usage frequency in the IGH variable (IGHV) subgroup was IGHV3 (50%) and the percentage of IGHV mutations ranged from 0% ± 11.45% with an average frequency of 3.34%. Compared with Capillary Electrophoresis, Next-generation Sequencing showed a higher positivity in the detection of gene clonal rearrangements, was more accurate in the interpretation of results.

Effect of NTN and Lmx1α on the Notch Signaling Pathway during the Differentiation of Human Bone Marrow Mesenchymal Stem Cells into Dopaminergic Neuron-Like Cells.

Human bone marrow mesenchymal stem cells (h-BMSCs) have the potential to differentiate into dopaminergic neuron-like cells to treat Parkinson's disease. The Notch signaling pathway has been implicated in the regulation of cell fate decisions such as differentiation of BMSCs. This study investigated changes in the expression of Notch-related genes in the differentiation of BMSCs in vitro into dopaminergic (DA) neuron-like cells. BMSCs transfected with empty lentiviral vectors served as the control group and those transfected with NTN and Lmx1α recombinant lentiviral vectors served as the experimental group. After induction and culture of NTN and Lmx1α-transfected h-BMSCs for 21 days, the cells exhibited features of dopaminergic neuron-like cells, which were observed by transmission and scanning electron microscopy and verified by immunofluorescence of tyrosine hydroxylase (TH) and dopamine transporter (DAT). These induced cells could secrete dopamine and had basic action potentials. Expression of the neural stem cell (NSC) markers, including octamer-binding protein (Oct4), paired box gene 6 (Pax6), and sex determining region Y-box 1 (SOX1), increased on day 14 of induction and decreased on day 21 of induction during differentiation. The human Notch signaling pathway PCR array showed a differential expression of Notch-related genes during the differentiation of h-BMSCs into DA neuron-like cells in vitro relative to that in the control group. In conclusion, h-BMSCs overexpressing NTN and Lmx1α can successfully be induced to differentiate into dopaminergic neuron-like cells with a neuronal phenotype exhibiting fundamental biological functions in vitro, and NTN and Lmx1α may affect the expression of Notch-related genes during differentiation.

Screening and potential role of tRFs and tiRNAs derived from tRNAs in the carcinogenesis and development of lung adenocarcinoma.

Accumulating evidence has indicated that a group of novel molecules, known as transfer RNA (tRNA)-derived fragments (tRFs) and tRNA halves (tiRNAs), which are derived from tRNAs, serve an essential role in numerous types of human disease, in particular solid tumors. However, to the best of our knowledge, the underlying mechanisms of the effect of tRFs and tiRNAs in lung adenocarcinoma have not been reported. The present study aimed to determine the differential expression levels of tRFs and tiRNAs in lung adenocarcinoma and adjacent tissues using a NextSeq system, and further investigated their potential target genes via bioinformatics analysis. Kyoto Encyclopedia of Genes and Genomes signaling pathway and Gene Ontology functional term enrichment analyses were performed to investigate the function of these target genes in the occurrence and development of lung adenocarcinoma. In patients with lung adenocarcinoma, 338 types of tRFs and tiRNAs were detected via sequencing, 284 of which were not previously reported in the tRF database. Compared with the adjacent tissues, 17 types of tRFs and tiRNAs comprising 34 subtypes were found to be abnormally expressed in lung adenocarcinoma tissues, 20 of which were upregulated and 14 downregulated. Reverse transcription-quantitative PCR verification revealed that the expression levels of tiRNA-Lys-CTT-002, tRF-Val-CAC-010 and tRF-Val-CAC-011 were significantly upregulated, while those of tRF-Ser-TGA-005 were downregulated in lung adenocarcinoma tissues. Bioinformatics analysis identified that tRF-Ser-TGA-005 participated in the 'cellular response to transforming growth factor ß stimulus' and tRF-Val-CAC-010 and tRF-Val-CAC-011 participated in the 'Hedgehog signaling pathway'. In conclusion, the results of the present study suggested that tRFs and tiRNAs may be closely associated with the pathogenesis and development of lung adenocarcinoma, providing a novel insight for further studies into lung adenocarcinoma.

Efficient Definitive Endoderm Differentiation from Human Parthenogenetic Embryonic Stem Cells Induced by Activin A and Wnt3a.

OBJECTIVE: This study aimed to investigate the effects of combined activin A and Wnt3a treatment on definitive endoderm (DE) differentiation from human parthenogenetic embryonic stem cells (hPESCs). METHODS: hPESCs on human foreskin fibroblast feeder layers were induced to differentiate into DE using a combination of 50 ng/ml activin A and 25 ng/ml Wnt3a. Expression of the DE markers CXCR4, E-cadherin (ECD), Sox17, and Goosecoid (Gsc) were examined using flow cytometry and real-time quantitative PCR. RESULTS: The combination of activin A and Wnt3a significantly enhanced the percentages of CXCR4+, ECD+, Sox17+, and Gsc+ cells, culminating on day 2 of induction. This combined use promoted DE differentiation from hPESCs in vitro. CONCLUSIONS: Through the combination treatment using activin A and Wnt3a, DE differentiation from hPESCs culminated at 48 h, which can be regarded as the optimal time-point to induce differentiation of endodermal cells such as pancreatic, liver, and intestinal cells.

Lentivirus-Mediated hFGF21 Stable Expression in Liver of Diabetic Rats Model and Its Antidiabetic Effect Observation.

The incidence of type 2 diabetes mellitus (T2DM) has been increasing annually, which is a serious threat to human health. Fibroblast growth factor 21 (FGF21) is one of the most popular targets for the treatment of diabetes because it effectively improves glycolipid metabolism. In our experiment, human FGF21 (hFGF21) was injected and stably expressed in the liver tissues of a rat T2DM model with lentivirus system. Based on clinical and histopathological examinations, islet cells were protected and liver tissue lesions were repaired for >4 months. Glucose metabolism and histopathology were controlled perfectly when hFGF21 was stably expressed in partial liver of T2DM rats. The results showed that the liver tissue cell apoptosis was reduced, the lipid droplet content was decreased, the oxidative stress indexes were improved, the glycogen content was increased, and the islet cells were increased too. Besides, insulin sensitivity and glycogen synthesis-related genes expression were increased, but cell apoptosis-related genes caspase3 and NFκB expression were decreased. The effectiveness of results suggested that injecting hFGF21 to rats liver could effectively treat T2DM.

Dopaminergic neuron-like cells derived from bone marrow mesenchymal stem cells by Lmx1α and neurturin overexpression for autologous cytotherapy in hemiparkinsonian rhesus monkeys.

Bone marrow-derived mesenchymal stem cells hold great potential for cytotherapeutics of neurodegenerative disorders, including Parkinson's disease. The neurotrophic factor neurturin can rescue dopaminergic neurons damaged during the disease process. Lmx1α can promote mesencephalic dopaminergic differentiation during embryogenesis. In this study, we tested a cytotherapeutic strategy combining NTN/Lmx1α gene therapy and cell transplantation to ameliorate disease progression in hemiparkinsonian rhesus. Rhesus BMSCs were prepared for autologous grafting by transfection with recombinant adenoviral vectors expressing secreted NTN and Lmx1α,and cultured in the presence of induce factors, particularly the Lmx1α regulatory factor sonic hedgehog, to guide dopaminergic differentiation. These induced rh-BMSCs exhibited gene/protein expression phenotypes resembling nigral dopaminergic neurons. They survived and retained dopaminergic function following stereotaxic injection into the MPTP-lesioned right-side substantia nigra as indicated by SPECT measurement of DAT activity. Injected cells preserved and supplemented the remaining endogenous population of dopamine neurons (TH-positive cell ipsilateral/contralateral ratio was 56.81% ± 7.28% vs. 3.86%±1.22% in vehicle-injected controls; p<0.05). Cell injection also partially restored motor function and reduce apomorphine-evoked rotation (p<0.05). Moreover, function recovery occurred earlier than in previous studies on injected BMSCs. Our findings demonstrate a promising strategy for restoration of PD-associated motor dysfunction by transplantation of autologous BMSCs overexpressing NTN/Lmx1α.

A retrospective analysis of the clinicopathological characteristics of large cell carcinoma of the lung.

The aim of the present study was to analyze and summarize the clinicopathological characteristics of large-cell lung carcinoma (LCLC) of the lung, in order to improve the definite diagnosis rate of LCLC. Clinicopathological data of 174 patients with LCLC, confirmed pathologically, were retrospectively reviewed. The 174 cases of LCLC accounted for 5.7% of the total lung cancer cases during the corresponding time period at the Affiliated Cancer Hospital of Tianjin Medical University (Tianjin, China), among which there were 131 males and 43 females with an average age of 61.4 years. The postoperative pathological diagnosis of the 174 cases showed 80 cases of classic LCLC, 64 cases of large cell neuroendocrine carcinoma (LCNEC), six cases of combined LCNEC, 19 cases of basaloid carcinoma, three cases of clear cell carcinoma and two cases of lymphoepithelioma-like carcinoma. Of the total 174 LCLC cases, 96 patients exhibited lymph node metastasis. LCLC is a highly aggressive malignancy with a high tendency of invasion and metastasis, although the incidence rate is low. A definite diagnosis of LCLC primarily relies on the pathological diagnosis. Each subtype of LCLC has its own pathomorphological and immunohistochemical characteristics.

Conversion of human umbilical cord mesenchymal stem cells in Wharton's jelly to dopamine neurons mediated by the Lmx1a and neurturin in vitro: potential therapeutic application for Parkinson's disease in a rhesus monkey model.

hUC-MSCs hold great promise in vitro neuronal differentiation and therapy for neurodegenerative disorders including Parkinson's disease. Recent studies provided that Lmx1α play an important role in the midbrain dopamine cells differentiation. Neurturin is desired candidate gene for providing a neuroprotective to DA neurons. In this study, we investigated a novel neuronal differentiation strategy in vitro with Lmx1α and NTN. We transferred these two genes to hUC-MSCs by recombinant adenovirus combined with Lmx1α regulatory factor and other inductor to improve the efficiency of inducing. Then those induced cells were implanted into the striatum and substantia nigra of MPTP lesioned hemi-parkinsonian rhesus monkeys. Monkeys were monitored by using behavioral test for six months after implantation. The result showed that cells isolated from the umbilical cord were negative for CD45, CD34 and HLA-DR, but were positive for CD44, CD49d, CD29. After those cells were infected with recombinant adenovirus, RT-PCR result shows that both Lmx1α and NTN genes were transcribed in hUC-MSCs. We also observed that the exogenous were highly expressed in hUC-MSCs from immunofluorescence and western blot. Experiments in vitro have proved that secretion NTN could maintain the survival of rat fetal midbrain dopaminergic neurons. After hUC-MSCs were induced with endogenous and exogenous factors, the mature neurons specific gene TH, Pitx3 was transcripted and the neurons specific protein TH, ß-tubulinIII, NSE, Nestin, MAP-2 was expressed in those differentiated cells. In addition, the PD monkeys, transplanted with the induced cells demonstrated the animals' symptoms amelioration by the behavioral measures. Further more, pathological and immunohistochemistry data showed that there were neuronal-like cells survived in the right brain of those PD monkeys, which may play a role as dopaminergic neurons. The findings from this study may help us to better understand the inside mechanisms of PD pathogenesis and may also help developing effective therapy for Parkinson's disease.

Recovery of behavioral symptoms in hemi-parkinsonian rhesus monkeys through combined gene and stem cell therapy.

BACKGROUND AIMS: The use of adipose mesenchymal stromal cells (ASCs) in cellular and genic therapy has attracted considerable attention as a possible treatment for neurodegenerative disorders, including Parkinson disease. However, the effects of gene therapy combined with intracerebral cell transplantation have not been well defined. Recent studies have demonstrated the respective roles of LIM homeobox transcription factor 1, alpha (LMX1A) and Neurturin (NTN) in the commitment of embryonic stem cells (ESCs) to a midbrain dopaminergic neuronal fate and the commitment of mesenchymal stromal cells to cells supporting the nutrition and protection of neurons. METHODS: We investigated a novel in vitro neuronal differentiation strategy with the use of LMX1A and Neurturin. We were able to elicit a neural phenotype regarding cell morphology, specific gene/protein expression and physiological function. Neuronal-primed ASCs derived from rhesus monkey (rASCs) combined with adenovirus containing NTN and tyrosine hydroxylase (TH) (Ad-NTN-TH) were implanted into the striatum and substantia nigra of methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-lesioned hemi-parkinsonian rhesus monkeys. Monkeys were monitored with the use of behavioral tests and health measures until the fourth month after implantation. RESULTS: The differentiated cells transcribed and expressed a variety of dopaminergic neuron-specific genes involved in the SHH/LMX1A pathway. Single-photon emission computed tomography analysis and postmortem analysis revealed that the grafting of rASCs combined with Ad-NTN-TH had neuroprotective effects compared with Ad-NTN-TH or rASCs alone. Behavioral measures demonstrated autograft survival and symptom amelioration. CONCLUSIONS: These findings may lead to cellular sources for autologous transplantation of Parkinson disease. Combined transplantation of Ad-NTN-TH and induced rASCs expressing LMX1A and NTN may be a better therapy candidate for the treatment of Parkinson disease.

The delivery of tyrosine hydroxylase accelerates the neurorestoration of Macaca Rhesus model of Parkinson's disease provided by Neurturin.

Neurturin (NTN) is a desired candidate therapeutic gene for PD treatment, however, only neuroprotective effect may not work for PD clinical remission due to nearly 50% of the dopaminergic neurons have died way when symptoms appear. In this study, we constructed a bicistronic adenovirus expressing both Neurturin and tyrosine hydroxylase (TH). We hypothesized that the expression of NTN could provide neuroprotection to dopaminergic neurons and stop progressive neurodegeneration, while TH enhanced the synthesis of dopamine and accelerated the recovery of Parkinsonism. The chimeric adenovirus have been prepared and assayed in vitro and delivered into the target nucleuses of PD Macaca Rhesus models by MRI based stereotaxic injection. The observation assessments and physiological results indicated that compared to the group treated with NTN only, instant behavior recovery was seen after bicistronic adenovirus infusion. Although both groups displayed neuroprotection of dopaminergic neurons finally, the addition of TH genuinely accelerated animal behavior recovery, which showed great potential for clinical application.