Development of TaqMan-based real-time PCR based on ψ gene for quantitative detection of CAR-T cells.

Chimeric-antigen-receptor-T (CAR-T) have heralded a paradigm shift in the landscape of cancer immunotherapy. Retrovirus-mediated gene transfer serves to deliver the specific CAR expressing cassette into T cells across a spectrum of basic research and clinical contests in cancer therapy. However, it is necessary to devise a precise and validated quantitative methodology tailored to the diverse CAR constructs. In the investigation, a TaqMan real-time qPCR method was developed, utilizing primers targeting ψ gene sequence. This method offers a swift, sensitive, reproducible, and accurate tool for evaluating retroviral copy numbers at the integrated DNA level. Importantly, the established qPCR exhibits no cross-reactivity with non-transduced T cells or tissues. The regression equation characterizing TaqMan real-time PCR dynamics is y = -3.3841x + 41.402 (R2 = 0.999), showing an amplification efficiency of 97.47 %. Notably, the established qPCR method achieves a minimum detection of 43.1 copies/µL. Furthermore, both intra- and inter-group discrepancies remain below 4 %, underscoring the good repeatability of the established method. Our in vitro and in vivo results also support its sensitivity, specificity, and stability. Consequently, this method offers researchers with a cost-effective tool to quantify CAR copies both in vitro and in vivo.

Single-Cell Analyses Reveal the Metabolic Heterogeneity and Plasticity of the Tumor Microenvironment during Head and Neck Squamous Cell Carcinoma Progression.

Metabolic reprogramming is a hallmark of cancer. In addition to metabolic alterations in the tumor cells, multiple other metabolically active cell types in the tumor microenvironment (TME) contribute to the emergence of a tumor-specific metabolic milieu. Here, we defined the metabolic landscape of the TME during the progression of head and neck squamous cell carcinoma (HNSCC) by performing single-cell RNA sequencing on 26 human patient specimens, including normal tissue, precancerous lesions, early stage cancer, advanced-stage cancer, lymph node metastases, and recurrent tumors. The analysis revealed substantial heterogeneity at the transcriptional, developmental, metabolic, and functional levels in different cell types. SPP1+ macrophages were identified as a protumor and prometastatic macrophage subtype with high fructose and mannose metabolism, which was further substantiated by integrative analysis and validation experiments. An inhibitor of fructose metabolism reduced the proportion of SPP1+ macrophages, reshaped the immunosuppressive TME, and suppressed tumor growth. In conclusion, this work delineated the metabolic landscape of HNSCC at a single-cell resolution and identified fructose metabolism as a key metabolic feature of a protumor macrophage subpopulation. Significance: Fructose and mannose metabolism is a metabolic feature of a protumor and prometastasis macrophage subtype and can be targeted to reprogram macrophages and the microenvironment of head and neck squamous cell carcinoma.

Host Volatiles Potentially Drive Two Evolutionarily Related Weevils to Select Different Grains.

The Sitophilus zeamais (maize weevil) and Sitophilus oryzae (rice weevil) are two insect pests that have caused huge economic losses to stored grains worldwide. It is urgent to develop an environmentally friendly strategy for the control of these destructive pests. Here, the olfactory-mediated selection preference of the two weevil species to three stored grains was analyzed, which should help establish a pull-push system in managing them. Bioassays showed that maize weevil adults prefer to select maize, followed by paddy and wheat, while rice weevil adults mainly migrate towards wheat. Volatile analyses revealed that 2-ethylhexanol, piperitone, and (+)-Δ-cadiene are the major components in volatiles from both maize and wheat, but the abundance of these chemicals is much lower in maize than that in wheat. The volatile limonene was only detected in paddy. Y-tube bioassays suggest that 2-ethylhexanol, piperitone, and (+)-Δ-cadiene were all attractive to both weevils, whereas limonene was attractive only to rice weevils. Overall, maize weevil appeared more sensitive to the tested volatiles based on having much lower effective concentrations of these volatiles needed to attract them. The differences in volatile profiles among the grains and the sensitivity of the two species towards these volatiles may explain the behavioral differences between maize and rice weevils in selecting host grains. The differences in sensitivity of maize and rice weevils towards host volatile components with abundance differences are likely determinants driving the two insect species to migrate towards different host grains.

Potential of Lactobacillus plantarum A56 in relieving food allergy through immunoregulation, antioxidation, and reshaping intestinal microbiota.

Food allergy is an abnormal immune reaction triggered by food protein antigens. Relevant studies have suggested that probiotic supplementation was with the potential to alleviate food allergy. This study aimed to explore the effects of Lactobacillus plantarum A56 on the alleviation of ovalbumin (OVA)-induced food allergy via immunomodulatory function, antioxidation, and modification of intestinal microbiota. Balb/c mice were sensitized with OVA (20 µg/mouse) by intraperitoneal injection for 3 weeks and accompanied by oral administration of L. plantarum A56 (109 CFU/mL), subsequently with orally challenged twice by OVA at 50 mg/mL for 1 week. The results showed that oral supplementation of L. plantarum A56 could effectively relieve allergic symptoms of mice, and decreased OVA-specific IgE and IgG1 concentrations. It also declined interleukin (IL)-4 level, raised interferon-γ (IFN-γ) in serum, and splenocyte supernatant, and the qPCR results were consistent with above results. Moreover, L. plantarum A56 treatment also fortified superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels, and reduced malondialdehyde (MDA) level in serum. The increased nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and forkhead box O1 (Foxo1) expression indicated that L. plantarum A56 exerted antioxidation through Nrf2-Foxo1 pathway. In addition, L. plantarum A56 treatment elevated Bacteroidetes richness, ASV/OTU number, species diversity, etc. Notably, Spearman correlation analysis indicated that Bacteroidetes displayed obviously negative correlation with IgE and IgG1, but Actinobacteria and Acidobacteria exhibited significantly positive correlation with IgG1 and IgE. Collectively, these results suggested that L. plantarum A56 could alleviate OVA-induced food allergy by regulating Th1/Th2 imbalance, antioxidation, and modulating intestinal microbiota.

Syntheses of Thiophene-Thiophene-Linked Corrorin Dimers with Tunable Near-Infrared Absorption and Distinctive Reactivity.

Thiahexaphyrinone 1 and thia-dipyrrin-appended corrorin 2 have been synthesized. Surprisingly, further oxidation of compound 2 with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) in dichloromethane afforded dimer 3 with two molecules of compound 2 linked at the α-carbon atoms of the thienyl units. Treatment of compound 3 with DDQ in MeOH and SnCl2 in tetrahydrofuran/H2O afforded the dimethoxy-attached dimer 4 and hydrogenated dihydroxy-attached dimer 5, respectively. These results provide the first examples for synthesizing thiophene-linked porphyrinoid dimers with tunable near-infrared absorption and chirality.

The Correlation Between 18F-Fluorodeoxyglucose-Positron Emission Tomography/Computed Tomography Semiquantitative Parameters and the Clinical Features and Pathological Biological Indexes of Gastric Cancer.

Objective: This study explored the application value of the maximum standard uptake value (SUVmax) of 18F-fluorodeoxyglucose-positron emission tomography/computed tomography (18F-FDG PET/CT) in gastric cancer. Materials and Methods: Data of 164 patients with gastric cancer who had undergone18F-FDG PET/CT before a biopsy were collected, and the correlation of SUVmax with clinical stage, pathological differentiation degree, human epidermal growth factor receptor-2 (HER-2) status, and Ki-67 index of gastric cancer was analyzed. Results: The SUVmax of poorly differentiated adenocarcinoma was significantly higher than that of moderately differentiated adenocarcinoma and signet-ring cell carcinoma (p < 0.01), and SUVmax in the well-differentiated adenocarcinoma group was higher than that in the signet-ring cell carcinoma group (p < 0.01). The SUVmax in the HER-2 negative group was higher than that in the HER-2 positive group (p < 0.01). The SUVmax was higher in the Ki-67 high expression group than in the low expression group (p < 0.01), and there was a significant positive correlation between the two (p < 0.01). Conclusion: 18F-FDG PET/CT SUVmax can, to some extent, predict the degree of differentiation, HER-2 status, and Ki-67 index of gastric cancer patients.

Rectal gastrointestinal stromal tumor with metachronous liver metastasis demonstrated no relapse after multidisciplinary team discussion and comprehensive treatment: a case report.

Background: This study sought to explore the role and significance of multidisciplinary team (MDT) discussion and comprehensive treatment in the diagnosis and treatment of a gastrointestinal stromal tumor (GIST) with liver metastasis. For GIST patients with liver metastasis, MDT can evaluate whether the liver metastasis is resectable, so as to formulate accurate treatment goals and the best diagnosis and treatment plan. Case Description: A 53-year-old male patient with localized rectal GIST with metachronous liver metastasis (MLM) was admitted to Yunnan Cancer Hospital in October 2014. At the 1st visit, he was diagnosed with locally advanced rectal GIST, and a MDT discussion was held by departments of colorectal surgery, imaging, pathology and oncology. The tumor shrank after neoadjuvant targeted treatment with imatinib. A local resection of the rectal GIST was successfully performed via the anal approach. R0 resection was achieved and the function of the anal sphincter was preserved. Following the operation, oral imatinib treatment was discontinued after 2 years. The patient developed isolated liver metastasis 6 months later. After the MDT discussion by departments of colorectal surgery, hepatobiliary surgery, imaging, pathology, and oncology, R0 resection of the liver metastasis was achieved. After the operation, sunitinib was administered for 4.5 years. The patient's overall survival (OS) has reached 7.5 years. No tumor recurrence or metastasis was found in the re-examinations. The follow-up is ongoing. Conclusions: Targeted therapy combined with surgery is the most suitable way to cure GIST patients with liver metastasis. More importantly, the multi-disciplinary management and the standardized diagnosis and treatment of GIST patients with liver metastasis through MDT discussion can improve the quality of life and prolong the survival of patients.

Efficacy and safety of neoadjuvant imatinib therapy for patients with locally advanced rectal gastrointestinal stromal tumors: A multi-center cohort study.

Background: Several issues on neoadjuvant imatinib therapy remain controversial despite its widespread application for rectal gastrointestinal stromal tumors (GIST). We aimed to describe the clinicopathological characteristics of this specific population, and compare the surgical and oncologic outcomes between patients with or without neoadjuvant imatinib therapy. Patients and methods: A cohort of 58 consecutive locally advanced rectal GIST patients receiving surgical treatment between January 2007 and July 2019 at Sun Yat-sen University Cancer Center and Yunnan Cancer Hospital was retrospectively analyzed. Recurrence-free survival (RFS) and overall survival (OS) were estimated using Kaplan-Meier method. Results: There were 33 (56.9%) patients who received neoadjuvant imatinib therapy. Among them, 20 (60.6%) patients had partial response (PR) as their best response, 11 (33.3%) patients had stable disease (SD), and 2 (6.1%) patients had progressive disease (PD). The median tumor size reduced from 5.2 to 4.0 cm after treatment (p < 0.001), and an attained "maximal response" was primarily achieved (32/33) on the 12th month after treatment. The most common adverse event was anemia. There were 27 adverse events occurred, most of which were grade 1 (19/27). With respect to intraoperative and postoperative surgical outcomes, no significant difference was found between patients with or without neoadjuvant Imatinib therapy except that patients with neoadjuvant treatment had a significant higher rate of preventive ileostomy (p = 0.004). Patients received neoadjuvant treatment had a superior 2-years RFS outcome than those without, though the difference was no significant (91.7% vs. 78.9%, p = 0.203). There were no significant differences in the 2-years OS rates (95.2% vs. 91.3%, p = 0.441). Conclusion: Neoadjuvant imatinib therapy is an effective and safe treatment for locally advanced rectal GISTs. Further studies are warranted to validate the long-term prognostic benefit for patients with rectal GISTs receiving neoadjuvant imatinib therapy.

Plac1 Remodels the Tumor Immune Evasion Microenvironment and Predicts Therapeutic Response in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC or HNSC) is the sixth most common cancer worldwide. Placenta-specific 1 (Plac1) belongs to the cancer testis antigen family and is highly expressed in malignant cells in HNSC. However, the biological function and prognostic value of plac1 in HNSC are still unclear. In the current research, we performed a comprehensive analysis of plac1 using The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) bulk RNA sequencing databases as well as a single-cell sequencing dataset. We constructed a 15-gene prognostic signature through screening plac1-related immunomodulators and validated its efficiency and accuracy in immunotherapy cohorts and a pancancer database. We found that plac1 expression level is a prognostic predictor of poor overall survival in patients with HNSC. Plac1 is associated with epithelial-mesenchymal transition and tumor invasion. Plac1 has a "dual immunosuppressive function" on tumor microenvironment. On one hand, plac1-positive cells promote extracellular matrix formation and suppress immune cell infiltration. On the other hand, plac1-positive cells enhance the interaction between dendritic cells and macrophages, which further suppresses antitumor immunity. Finally, we constructed a 15-gene prognostic signature, the efficiency and accuracy of which were validated in immunotherapy cohorts and a pancancer database. In conclusion, plac1 is a promising candidate biomarker for prognosis, a potential target for immunotherapy, and a novel point for studying the immunosuppressive mechanisms of the tumor microenvironment in HNSC.

Long Non-Coding RNA (lncRNA) BMP/OP-Responsive Gene (BORG) Promotes Development of Chemoresistance of Colorectal Cancer Cells to Carboplatin.

BACKGROUND The function of long non-coding RNA (lncRNA) BMP/OP-responsive gene (BORG) has only been studied in breast cancer. We analyzed the role of BORG in colorectal cancer (CRC). MATERIAL AND METHODS BORG in CRC tissues and non-cancer tissues from 66 CRC patients was detected by performing quantitative reverse-transcription PCR (RT-qPCR). BORG in plasma of CRC patients was detected at 3 times-points: before treatment and at 3 and 6 months after treatment. p53 expression in tumor tissues was also detected by RT-qPCR. QPCR was performed to confirm the overexpression of p53 in cells of both CRC cell lines. RESULTS We found that BORG expression was upregulated in CRC tissues and was inversely correlated with p53. With application of carboplatin-based treatment, the expression level of BORG was further upregulated. In CRC cells, carboplatin upregulated the expression of BORG and BORG negatively regulated p53. Under carboplatin treatment, BORG positively regulated the viability of CRC cells. In addition, p53 overexpression attenuated the effects of BORG overexpression. CONCLUSIONS BORG promotes the development of chemoresistance of CRC cells to carboplatin.

Observation of therapeutic effect of 125I seed implantation combined with chemotherapy and antiviral therapy on HBV-related liver cancer.

PURPOSE: To investigate the therapeutic effect of 125I seed implantation combined with chemotherapy and antiviral therapy on hepatitis B virus (HBV)-related liver cancer. METHODS: A total of 126 patients with HBV-related liver cancer were selected and divided into observation group (n=63) and control group (n=63). The patients in the control group were treated with transcatheter arterial chemoembolization (TACE) and antiviral therapy, while those in the observation group were treated with 125I seed implantation combined with TACE and antiviral therapy. The therapeutic effect, liver function, serum HBV DNA and tumor marker levels, and changes in Child-Pugh score and Karnofsky performance status (KPS) score before and after treatment were compared between the two groups. RESULTS: After treatment in the observation group, the serum alanine aminotransferase (ALT), HBV DNA, alpha fetoprotein (AFP) levels and Child-Pugh score were lower than those in the control group, while the KPS score was significantly higher than in the control group (p<0.05). There was no statistically significant difference in the control rate of liver cancer after treatment between the two groups (p>0.05). The remission rate in the observation group was obviously higher than in the control group (p<0.05). CONCLUSION: 125I seed implantation combined with chemotherapy and antiviral therapy can effectively eliminate HBV DNA, improve liver function, increase quality of life and enhance the therapeutic effect in patients with HBV-related liver cancer, so it is worthy of clinical popularization.

Correlations between maximum standardized uptake value measured via 18F-fluorodeoxyglucose positron emission tomography/computed tomography and clinical variables and biochemical indicators in adult lymphoma.

OBJECTIVES: The aim of the current study was to investigate whether the maximum standardized uptake value (SUVmax) measured by 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET)/computed tomography (CT) could discriminate between aggressive and indolent non-Hodgkin lymphomas (NHLs) and correlations between the SUVmax and clinical variables and serum biochemical indicators in adult lymphoma. METHODS: A total of 103 patients with lymphoma confirmed by biopsy, pretreatment 18F-FDG PET/CT scans, and a complete medical record were retrospectively enrolled in the study. Clinical variables that were evaluated included stage, pathological subtype, International Prognostic Index (IPI) score, and Ki-67 index, as well as serum biochemical indicators (e.g., lactate dehydrogenase [LDH] and erythrocyte sedimentation rate [ESR]) and metabolic parameters (e.g., SUVmax of the biopsy site on PET/CT). Correlations between SUVmax and clinical variables and serum biochemical indicators were investigated. RESULTS: Of the 103 patients, 84 had NHL and 19 had Hodgkin lymphoma. The area under the receiver operating characteristic curve for examining the accuracy of SUVmax with regard to distinguishing between aggressive and indolent NHLs was 0.94 (95% confidence interval: 0.89-0.99), suggesting that SUVmax was a useful predictor of diagnosis. A cutoff value of 8.5 yielded a sensitivity of 76.3% and specificity of 92.0%. The SUVmax mean ± standard deviation of NHL (9.8 ± 6.0, range: 1.8-28.1) was higher than that of HL (7.5 ± 2.8, range: 3.5-13.9) (P = 0.016), but there was no statistically significant difference in SUVmax between NHL and HL (P > 0.05). SUVmax of the biopsy site was strongly positively correlated with Ki-67 index (r = 0.813, P < 0.001) and moderately positively correlated with IPI score (r = 0.332, P = 0.002), but it was not significantly correlated with clinical stage, LDH, or ESR (P > 0.05). CONCLUSIONS: 18F-FDG PET/CT may yield reliable measurements of tumor proliferation, and an SUVmax >8.5 may distinguish between aggressive and indolent NHLs. In adults with newly diagnosed lymphoma, SUVmax correlates with Ki-67 index and IPI score.

[Effects of insulin caliper for blood glucose control on glucose control in emergent and critical patients].

OBJECTIVE: To observe the effects of insulin caliper for blood glucose control on glycemic central tendency, fluctuation and incidence of hypoglycemia, etc., in emergent and critical patients to evaluate its application value. METHODS: A prospective single-blinded randomized parallel controlled intervention study was conducted. One hundred patients with severe hyperglycemia requiring treatment with insulin infusion admitted to emergency department and intensive care unit (ICU) of the First Hospital of Jiaxing from November 2015 to November 2017 were enrolled, and they were divided into the caliper group (used patented product insulin calipers for blood glucose control to adjust insulin dose for blood glucose control) and the conventional group (used paper-based insulin dose modification scheme to adjust insulin dose for blood glucose control) on average by random number table, 50 in each group. The gender, age, acute physiology and chronic health evaluation II (APACHE II), sequential organ failure assessment (SOFA), the principal diseases, main factors affecting blood glucose (hepatic and renal insufficiency, hypoglycemic drugs, glucocorticoids, mechanical ventilation, enteral nutrition, parenteral nutrition, intravenous glucose use, etc.), blood glucose levels at each time node (once every 2 hours after insulin use and once every 4 hours after 16-72 hours), glycemic coefficient of variance (CV), glycemic lability index (GLUGLI) and mean amplitude of glycemic excursion (GLUMAGE), insulin dose, incidence of hypoglycemia, proportion of achieving the glucose control target at each time point, the length of ICU stay and hospitalization cost per patient were recorded and compared between the two groups. RESULTS: After excluding those with incomplete data and withdraw in the midway, 92 patients were enrolled in the analysis finally, 47 in caliper group and 45 in conventional group. There were no significant differences in the incidence of the gender, age, APACHE II, SOFA, presence of infection at admission, previous diabetes history, glycosylated hemoglobin level, blood glucose at admission, proportion of patients after surgery, major diseases at admission and major factors affecting blood glucose between the two groups. A total of 1 379 blood glucose measurements were obtained in the caliper group and 1 332 blood glucose measurements were obtained in the conventional group. The glycemic measurements in caliper group were significantly lower than that in conventional group at each time point from 6-72 hours. Compared with conventional group, GLUGLI and GLUMAGE were significantly decreased in the caliper group [GLUGLI: 12.96 (8.73, 19.58) vs. 23.27 (13.07, 44.61), GLUMAGE (mmol/L): 0.66±0.22 vs. 0.87±0.28, both P < 0.01]; there was a tendency towards decreasing incidence of hypoglycemia in the caliper group [8.51% (4/47) vs. 15.56% (7/45)], but no statistical difference was found (P > 0.05); the proportion of achieving the glucose control target was significantly increased in the caliper group [41.99% (579/1 379) vs. 27.18% (362/1 332), P < 0.01]. There were no significant differences in glycemic CV, insulin dose, proportion of hypoglycemic measurements in total measurements, and the length of ICU stay, the length of hospital stay, incidence of nosocomial infection, patient prognosis and cost between the two groups. CONCLUSIONS: For emergent and critical patients, insulin caliper for blood glucose control presents favorable application value for achieving glucose control target, reducing glycemic fluctuation, and lowering the incidence of hypoglycemia. CLINICAL TRIAL REGISTRATION: China clinical trial registration center, ChiCTR1800015024.

[Effect of Bone Marrow Mesenchymal Stem Cells in Patients with Multiple Myelima on Migration of Myeloma Cells In Vitro].

OBJECTIVE: To explore the effect of bone marrow mesenchymal stem cells (MSC) in patients with multiple myeloma(MM) on chemotactic migration of myeloma cells in vitro. METHODS: By in vitro co-culture with diffferent MSC, the myeloma cell U266 was divided into 2 groups: group A in which the U266 cells were co-cultured with normal person MSC (N-MSC) and group B in which the U266 cells were co-cultured with MM-MSC. The expression level of CCR1 in U266 cells, migration rate of U266 cells in Transwell, and the effect of supernantant from co-culture of U266 cells with N-MSC and MM-MSC on the migration in Transwell were compared in condition with or without bortezomib. RESULTS: After co-culture of U266 cells with N-MSC or MM-MSC, the migration rate of U266 cells in Transwell in B group was higher than that in A group(P<0.05). The difference between 2 groups could not be eliminated after treatment of U266 cells with bortezomib. The CCR1 expression level of U266 cells in B group was higher than that in A group (P<0.05). The culture supernatant of bone marrow MSC showed that in condition without bortezomib the culture supernatant of MSC in MM patients and normal persons both possessed more strong chemotactic ability and enhanced the migration rate of cells in Transwell, compared with SDF-1, meanswhile the culture supernatant in 3 groups reduced the migration rate of cells in condition with bortezomib (P<0.05), but there were no statistical difference in migration rate of U266 cells in Transwell between supernatant of N-MSC and MM-MSC culture (P>0.05), no matter the bortezonib was used or not. CONCLUSION: The bone marrow MSC in MM patients have same intrinsic defects that affect the chemotaxis of cells in vitro by directly interacting with myeloma cells.

Treatment of thoracic hemorrhage due to rupture of traumatic mediastinal hematoma.

Patients in traffic accidents are usually presented with pain and bleeding due to fractures or soft tissue injury. On some occasions, more severe complications may be triggered by the trauma. A review of the published English language literature reveals no survival case once the traumatic mediastinal hematoma is ruptured. In our case, a 54-year-old man suffering motorcycle accident was admitted to emergency department. Computed tomography scan revealed subdural hematoma combined with posterior mediastinal hematoma. The patient was saved and discharged with a satisfactory outcome. Here we hope to share our treatment experience in dealing with the patient with severe multiple trauma.

Mimic of manganese superoxide dismutase induces apoptosis in human acute myeloid leukemia cells.

Abstract Increasing manganese superoxide dismutase (MnSOD) expression can suppress the malignant phenotype in various cancer cell lines and suppress tumor formation in xenograft and transgenic mouse models. A mimic of manganese superoxide dismutase (MnSODm), synthesized by a chemical method, has been shown to possess antitumor properties. However, the anticancer activity of MnSODm in acute myeloid leukemia (AML) is still obscure. In this study, we investigated the effects of MnSODm on the apoptosis of human leukemia HL-60 cells. Results showed that MnSODm significantly reduced the proliferation of HL-60 cells in a concentration- and time-dependent manner. By flow cytometric analysis, we found that MnSODm treatment resulted in increased apoptosis in HL-60 cells. Further analysis demonstrated involvement of activation of the caspase cascade, cleavage of poly(ADP-ribose) polymerase (PARP) and release of cytochrome c in MnSODm-induced apoptosis. The results also showed that the expression of anti-apoptotic Bcl-2 and Bid were dose-dependently decreased, whereas the expression of pro-apoptotic Bax protein was increased. Thus, MnSODm induced apoptosis in HL-60 cells via mitochondria-mediated, caspase-dependent pathways. MnSODm inhibition of Akt phosphorylation may contribute to MnSODm-mediated acute myeloid leukemia cell growth inhibition and apoptosis induction.

Mimic of manganese superoxide dismutase to induce apoptosis of human non-Hodgkin lymphoma Raji cells through mitochondrial pathways.

Increased reactive oxygen species (ROS) such as superoxide have been implicated as causal elements of oncogenesis. A variety of cancers have displayed changes in steady-state levels of key antioxidant enzymes, with the mitochondrial form of superoxide dismutase (MnSOD) being commonly implicated. Increasing MnSOD expression suppresses the malignant phenotype in various cancer cell lines and suppresses tumor formation in xenograft and transgenic mouse models. In this study, we examined the anti-proliferation effect of mimic of manganese superoxide dismutase (MnSODm) on human non-Hodgkin lymphoma Raji cells. The results showed that MnSODm significantly reduced the proliferation of Raji cells in a concentration and a time-dependent manner. By flow cytometric analysis, we found that MnSODm treatment resulted in an increased apoptosis in Raji cells. MnSODm also increased the production of ROS and the expression levels of cleaved caspase-9, caspase-3, poly (ADP-ribose) polymerase (PARP) and Bax in Raji cells. Moreover, the expression of Bcl-2 protein showed down-regulation in the MnSODm treatment group. In addition, MnSODm significantly elevated the level of cytochrome c in cytosol. These findings suggest that the activation of the mitochondrial pathway is involved in MnSODm-induced apoptosis in Raji cells.

Distinct contributions of replication and transcription to mutation rate variation of human genomes.

Here, we evaluate the contribution of two major biological processes--DNA replication and transcription--to mutation rate variation in human genomes. Based on analysis of the public human tissue transcriptomics data, high-resolution replicating map of Hela cells and dbSNP data, we present significant correlations between expression breadth, replication time in local regions and SNP density. SNP density of tissue-specific (TS) genes is significantly higher than that of housekeeping (HK) genes. TS genes tend to locate in late-replicating genomic regions and genes in such regions have a higher SNP density compared to those in early-replication regions. In addition, SNP density is found to be positively correlated with expression level among HK genes. We conclude that the process of DNA replication generates stronger mutational pressure than transcription-associated biological processes do, resulting in an increase of mutation rate in TS genes while having weaker effects on HK genes. In contrast, transcription-associated processes are mainly responsible for the accumulation of mutations in highly-expressed HK genes.

The disequilibrium of nucleosomes distribution along chromosomes plays a functional and evolutionarily role in regulating gene expression.

To further understand the relationship between nucleosome-space occupancy (NO) and global transcriptional activity in mammals, we acquired a set of genome-wide nucleosome distribution and transcriptome data from the mouse cerebrum and testis based on ChIP (H3)-seq and RNA-seq, respectively. We identified a nearly consistent NO patterns among three mouse tissues--cerebrum, testis, and ESCs--and found, through clustering analysis for transcriptional activation, that the NO variations among chromosomes are closely associated with distinct expression levels between house-keeping (HK) genes and tissue-specific (TS) genes. Both TS and HK genes form clusters albeit the obvious majority. This feature implies that NO patterns, i.e. nucleosome binding and clustering, are coupled with gene clustering that may be functionally and evolutionarily conserved in regulating gene expression among different cell types.

A comparison between ribo-minus RNA-sequencing and polyA-selected RNA-sequencing.

To compare the two RNA-sequencing protocols, ribo-minus RNA-sequencing (rmRNA-seq) and polyA-selected RNA-sequencing (mRNA-seq), we acquired transcriptomic data-52 and 32 million alignable reads of 35 bases in length-from the mouse cerebrum, respectively. We found that a higher proportion, 44% and 25%, of the uniquely alignable rmRNA-seq reads, is in intergenic and intronic regions, respectively, as compared to 23% and 15% from the mRNA-seq dataset. Further analysis made an additional discovery of transcripts of protein-coding genes (such as Histone, Heg1, and Dux), ncRNAs, snoRNAs, snRNAs, and novel ncRNAs as well as repeat elements in rmRNA-seq dataset. This result suggests that rmRNA-seq method should detect more polyA- or bimorphic transcripts. Finally, through comparative analyses of gene expression profiles among multiple datasets, we demonstrated that different RNA sample preparations may result in significant variations in gene expression profiles.