Cell Line T84 Research Articles

Targeted Editing of Myostatin Gene in Sheep by Transcription Activator-like Effector Nucleases.

Myostatin (MSTN) is a secreted growth factor expressed in skeletal muscle and adipose tissue that negatively regulates skeletal muscle mass. Gene knockout of MSTN can result in increasing muscle mass in sheep. The objectives were to investigate whether myostatin gene can be edited in sheep by transcription activator-like effector nucleases (TALENs) in tandem with single-stranded DNA oligonucleotides (ssODNs). We designed a pair of TALENs to target a highly conserved sequence in the coding region of the sheep MSTN gene. The activity of the TALENs was verified by using luciferase single-strand annealing reporter assay in HEK 293T cell line. Co-transfection of TALENs and ssODNs oligonucleotides induced precise gene editing of myostatin gene in sheep primary fibroblasts. MSTN gene-edited cells were successfully used as nuclear donors for generating cloned embryos. TALENs combined with ssDNA oligonucleotides provide a useful approach for precise gene modification in livestock animals.

Identification of long-non coding RNA UCA1 as an oncogene in renal cell carcinoma

Renal cell carcinoma (RCC) is the most common type of kidney cancer in adults, which is associated with poor prognosis and high recurrence. Long non‑coding RNAs (lncRNAs) have been reported to be dysregulated in cancer and to be important in the regulation of carcinogenesis, thus suggesting that this class of molecules may be used as biomarkers in cancer. The lncRNA urothelial carcinoma associated 1 (UCA1) has been observed to be upregulated and to function as an oncogene in certain types of cancer; however, the role of UCA1 in RCC remains to be elucidated. The present study aimed to determine the expression and function of UCA1 in RCC. Quantitative polymerase chain reaction (qPCR) was used to determine the expression levels of UCA1 in 46 paired RCC and adjacent normal tissue samples. Furthermore, qPCR was used to determine the expression levels of UCA1 in four RCC cell lines compared with the human embryonic kidney 293T cell line. The impact of UCA1 on cell migration, proliferation and apoptosis was investigated by wound scratch assay, MTT and flow cytometry, respectively. The results of the present study demonstrated that UCA1 expression levels were significantly increased in RCC tissues and cells, as compared with the controls. Ectopic expression and gene silencing of UCA1 in RCC cell lines exerted opposite effects on cellular proliferation, migration and apoptosis, and the results suggested that UCA1 may function as an oncogene in RCC. These results indicated that UCA1 may be considered as a promising biomarker for diagnosis, and a therapeutic target in RCC. Further research is required to elucidate the role and target genes of UCA1 in RCC.

Total Synthesis and Biological Evaluation of Natural and Designed Tubulysins.

A streamlined total synthesis of N(14)-desacetoxytubulysin H (Tb1) based on a C-H activation strategy and a short total synthesis of pretubulysin D (PTb-D43) are described. Applications of the developed synthetic strategies and technologies to the synthesis of a series of tubulysin analogues (Tb2-Tb41 and PTb-D42) are also reported. Biological evaluation of the synthesized compounds against an array of cancer cells revealed a number of novel analogues (e.g., Tb14), some with exceptional potencies against certain cell lines [e.g., Tb32 with IC50 = 12 pM against MES SA (uterine sarcoma) cell line and 2 pM against HEK 293T (human embryonic kidney) cell line], and a set of valuable structure-activity relationships. The highly potent cytotoxic compounds discovered in this study are highly desirable as payloads for antibody-drug conjugates and other drug delivery systems for personalized targeted cancer chemotherapies.

Is Hydrogen Peroxide a Suitable Apoptosis Inducer for All Cell Types?

Hydrogen peroxide is currently the most widely used apoptosis inducer due to its broad cytotoxic efficacy against nearly all cell types. However, equivalent cytotoxicity is achieved over a wide range of doses, although the reasons for this differential sensitivity are not always clear. In this study, three kinds of cells, the 293T cell line, primary fibroblasts, and terminally differentiated myocardial cells, were treated with a wide range of H2O2 doses. Times to apoptosis initiation and end were measured cytochemically and the changes in expression of caspase-9, P53, NF-κB, and RIP were determined by RT-PCR. The 293T cell line was the most sensitive to H2O2, undergoing necroptosis and/or apoptosis at all concentrations from 0.1 to 1.6 mM. At > 0.4 mM, H2O2 also caused necroptosis in primary cells. At < 0.4 mM, however, primary cells exhibited classic signs of apoptosis, although they tended to survive for 36 hours in < 0.2 mM H2O2. Thus, H2O2 is a broadly effective apoptosis inducer, but the dose range differs by cell type. For cell lines, a low dose is required and the exposure time must be reduced compared to primary cells to avoid cell death primarily by necroptosis or necrosis.

Absorption process of salazosulfapyridine in human intestinal epithelial cells and rat intestine

Introduction: Salazosulfapyridine (SASP) is an oral medication used to treat rheumatoid arthritis and inflammatory bowel disease, particularly ulcerative colitis. It has been reported that various transporters such as P-glycoprotein (Pgp) and multidrug resistance-associated protein 2 (MRP2), are involved in the transport of SASP. P-gp and MRP2 are expressed in the brain, intestine, and various tissues in both humans and rats. In the intestine, P-gp limits the absorption of certain drugs, however, its mode of absorptive action with regard to SASP has not, as of yet been studied. The aim of this study was to investigate the intestinal transport of SASP and examine whether transporters such as P-gp and MRP2 are involved in this process. Method: Bidirectional permeability and inhibition transport studies were performed using Caco-2 and T84 cell lines. Transcellular transport studies were conducted using isolated rat intestinal tissue mounted in an Ussing-type chamber. The intestinal absorption was examined using an in-situ closed-loop experiment in rats. Results: SASP showed secretory-directed transport across Caco-2 and T84 cells, as well as rat intestinal tissue. Cyclosporine A (CsA), a P-gp inhibitor, was found to decrease this secretory-directed transport. In the intestinal closedloop experiment, addition of CsA resulted in increased accumulation of SASP; however, this was too miniscule to be considered. The inhibition study showed that both secretory and absorptive transporters sensitive to probenecid are involved in the process of SASP absorption in the small intestine. Conclusion: In the process of SASP absorption in the intestine, CsA-sensitive secretory transporters including P-gp as well as probenecid-sensitive absorptive and secretory-transporters are involved and the total of the absorption of SASP is regulated by these transporters. It is likely that these transporters are coordinated in a complex manner to effectively regulate absorption of SASP and other biochemically similar drugs.

Δ42PD1稳定表达细胞系的建立及其功能的初步研究

目的：建立稳定表达Δ42PD1的293T细胞系，并探索其对AKT，NF-κB和Erk1/2磷酸化的影响。方法：分别将含有人PD1和Δ42PD1基因的真核表达质粒稳定转染293T细胞，用流式细胞术筛选稳定细胞系，用RT-PCR和Western blot进一步鉴定目基因的表达。将稳定细胞系分别于PBMCs进行孵育，以激活Δ42PD1和PD1，用流式细胞术检测细胞内AKT，NF-κB和Erk1/2的磷酸化水平。结果：通过流式细胞术筛选出稳定表达PD1和Δ42PD1的293T细胞系，RT-PCR和Western blot确认了目的基因的表达。与PBMC混合孵育后，293T细胞内AKT的磷酸化水平被Δ42PD1或PD1显著抑制，而NF-κB和Erk1/2的磷酸化水平没有显著变化。结论：本研究发现Δ42PD1与PD1的功能类似，可能是一种重要的抑制性免疫调节受体。 Objective: To establish Δ42PD1-expressing stable 293T cell line and explore the effect of Δ42PD1 on phosphorylation of AKT, NF-κB and Erk1/2. Methods: The human PD1- and Δ42PD1-carrying eukaryotic expression plasmids were stably transfected into 293T cells respectively. Stable cell lines were screened using flow cytometry, and confirmed by RT-PCR and Western blot. The cell lines were co-cultured with PBMC to activate PD1 and Δ42PD1, and then analyzed the phosphorylation of AKT, NF-κB and Erk1/2 via flow cytometry. Results: We obtained PD1- and Δ42PD1-stably expressing 293T cell lines by flow cytometry. The expression of genes of interest was confirmed by RT-PCR and Western blot. After co-culture with PBMC, phosphorylation of AKT but not NF-κB or Erk1/2 in 293T cells was significantly inhibited by Δ42PD1 or PD1. Conclusion: The present study found that functionally similar to PD1, Δ42PD1 could be an important inhibitory immune-regula- tory receptor.

Transient Expression of Green Fluorescent Protein in Integrase-Defective Lentiviral Vector-Transduced 293T Cell Line.

Non-integrating lentiviral vectors or also known as integrase-defective lentiviral (IDLV) hold a great promise for gene therapy application. They retain high transduction efficiency for efficient gene transfer in various cell types both in vitro and in vivo. IDLV is produced via a combined mutations introduced on the HIV-based lentiviral to disable their integration potency. Therefore, IDLV is considered safer than the wild-type integrase-proficient lentiviral vector as they could avoid the potential insertional mutagenesis associated with the nonspecific integration of transgene into target cell genome afforded by the wild-type vectors.Here we describe the system of IDLV which is produced through mutation in the integrase enzymes at the position of D64 located within the catalytic core domain. The efficiency of the IDLV in expressing the enhanced green fluorescent protein (GFP) reporter gene in transduced human monocyte (U937) cell lines was investigated. Expression of the transgene was driven by the spleen focus-forming virus (SFFV) LTRs. Transduction efficiency was studied using both the IDLV (ID-SFFV-GFP) and their wild-type counterparts (integrase-proficient SFFV-GFP). GFP expression was analyzed by fluorescence microscope and FACS analysis.Based on the results, the number of the GFP-positive cells in ID-SFFV-GFP-transduced U937 cells decreased rapidly over time. The percentage of GFP-positive cells decreased from ~50 % to almost 0, up to 10 days post-transduction. In wild-type SFFV-GFP-transduced cells, GFP expression is remained consistently at about 100 %. These data confirmed that the transgene expression in the ID-SFFV-GFP-transduced cells is transient in dividing cells. The lack of an origin of replication due to mutation of integrase enzymes in the ID-SFFV-GFP virus vector has caused the progressive loss of the GFP expression in dividing cells.Integrase-defective lentivirus will be a suitable choice for safer clinical applications. It preserves the advantages of the wild-type lentiviral vectors but with the benefit of transgene expression without stable integration into host genome, therefore reducing the potential risk of insertional mutagenesis.

Escherichia coli Heat-Stable Enterotoxin Mediates Na+/H+ Exchanger 4 Inhibition Involving cAMP in T84 Human Intestinal Epithelial Cells.

The enterotoxigenic Escherichia coli strains lead to diarrhoea in humans due to heat-labile and heat-stable (STa) enterotoxins. STa increases Cl-release in intestinal cells, including the human colonic carcinoma T84 cell line, involving increased cGMP and membrane alkalization due to reduced Na+/H+ exchangers (NHEs) activity. Since NHEs modulate intracellular pH (pHi), and NHE1, NHE2, and NHE4 are expressed in T84 cells, we characterized the STa role as modulator of these exchangers. pHi was assayed by the NH4Cl pulse technique and measured by fluorescence microscopy in BCECF–preloaded cells. pHi recovery rate (dpHi/dt) was determined in the absence or presence of 0.25 μmol/L STa (30 minutes), 25 μmol/L HOE-694 (concentration inhibiting NHE1 and NHE2), 500 μmol/L sodium nitroprusside (SNP, spontaneous nitric oxide donor), 100 μmol/L dibutyryl cyclic GMP (db-cGMP), 100 nmol/L H89 (protein kinase A inhibitor), or 10 μmol/L forskolin (adenylyl cyclase activator). cGMP and cAMP were measured in cell extracts by radioimmunoassay, and buffering capacity (ßi) and H+ efflux (J H +) was determined. NHE4 protein abundance was determined by western blotting. STa and HOE-694 caused comparable reduction in dpHi/dt and J H + (~63%), without altering basal pHi (range 7.144–7.172). STa did not alter ßi value in a range of 1.6 pHi units. The dpHi/dt and J H + was almost abolished (~94% inhibition) by STa + HOE-694. STa effect was unaltered by db-cGMP or SNP. However, STa and forskolin increased cAMP level. STa–decreased dpHi/dt and J H + was mimicked by forskolin, and STa + HOE-694 effect was abolished by H89. Thus, incubation of T84 cells with STa results in reduced NHE4 activity leading to a lower capacity of pHi recovery requiring cAMP, but not cGMP. STa effect results in a causal phenomenon (STa/increased cAMP/increased PKA activity/reduced NHE4 activity) ending with intracellular acidification that could have consequences in the gastrointestinal cells function promoting human diarrhoea.

Development of a Stable Cell Line, Overexpressing Human T-cell Immunoglobulin Mucin 1.

Recent researches have demonstrated that human T-cell immunoglobulin mucin 1 (TIM-1) glycoprotein plays important roles in regulation of autoimmune and allergic diseases, as well as in tumor immunity and response to viral infections. Therefore, targeting TIM-1 could be a potential therapeutic approach against such diseases. In this study, we aimed to express TIM-1 protein on Human Embryonic kidney (HEK) 293T cell line in order to have an available source of the TIM-1 antigen. The cDNA was synthesized after RNA extraction from peripheral blood mononuclear cells (PBMC) and TIM-1 cDNA was amplified by PCR with specific primers. The PCR product was cloned in pcDNA™3.1/Hygro (+) and transformed in Escherichia coli TOP 10 F'. After cloning, authenticity of DNA sequence was checked and expressed in HEK 293T cells. Finally, expression of TIM-1 was analyzed by flow cytometry and real-time PCR. The result of DNA sequencing demonstrated correctness of TIM-1 DNA sequence. The flow cytometry results indicated that TIM-1 was expressed in about 90% of transfected HEK 293T cells. The real-time PCR analysis showed TIM-1 mRNA expression increased 195-fold in transfected cells compared with un-transfected cells. Findings of present study demonstrated the successful cloning and expression of TIM-1 on HEK 293T cells. These cells could be used as an immunogenic source for production of specific monoclonal antibodies, nanobodies and aptamers against human TIM-1.

MicroRNA-20b-5p functions as a tumor suppressor in renal cell carcinoma by regulating cellular proliferation, migration and apoptosis.

Renal cell carcinoma (RCC) is the most common type of kidney cancer in adults and is associated with a poor prognosis due to a lack of early‑warning signs, protean clinical manifestations, and resistance to radiotherapy and chemotherapy. Recently, increasing evidence has suggested that microRNAs (miRNAs) are involved in the proliferation, invasion and apoptosis of various types of human cancer cells. In a previous study, miRNA expression profiles from renal cell carcinoma (RCC) revealed that expression of miR‑20b‑5p was significantly downregulated in RCC tissues. The aim of this study was to investigate the expression and functional significance of miR‑20b‑5p in RCC. The expression of miR‑20b‑5p was quantified in 48 paired RCC tissues and cell lines, and compared with adjacent normal tissues and the 293T cell line by reverse transcription‑quantitative polymerase chain reaction. The functional impact of miR‑20b‑5p on cell proliferation, cell migration and apoptosis in the 786‑O and ACHN RCC cell lines, was determined by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, a scratch assay and flow cytometry. To the best of our knowledge, the present study was the first to reveal that miR‑20b‑5p was downregulated in RCC tissues and cell lines. It also demonstrated that upregulation of miR‑20b‑5p inhibited cellular migration and proliferation, and promoted cellular apoptosis, suggesting that miR‑20b‑5p functioned as a potential tumor suppressor. However, further studies are required to fully determine the effects of miR‑20b‑5p and the miR‑20b‑5p‑mediated molecular pathway in RCC and other types of cancer. In conclusion, these results imply that miR‑20b‑5p may be a biomarker for early detection and prognosis prediction, as well as a therapeutic target for RCC.

Effects of shear stress on the cellular distribution of polystyrene nanoparticles in a biomimetic microfluidic system

Effects of shear stress on the intracellular uptake of nanoparticles were originally investigated using a calibrated biomimetic microfluidic system (BMS) that mimics the dynamic environment of cells. Positively or negatively charged polystyrene nanoparticles (PSNs) were chosen as a model. PSNs were delivered to HEK 293T and MS1 cell lines using a BMS. To evaluate intracellular uptake of PSNs under static and dynamic conditions (0.5, 1.0, 3.0 dyne/cm2), the fluorescence intensity of intracellular PSNs was measured by flow cytometric analysis and confocal laser scanning microscopy. When delivering cationic PSNs to cells, the intracellular uptake increased as the exposure time and PSN concentration increased under both static and dynamic conditions. Under dynamic conditions, the intracellular uptake of cationic PSN was highly increased in both HEK 293T and MS1 cell lines compared to static conditions. However, intracellular uptake of cationic PSNs was maximized when shear stress was at 0.5 dyne/cm2 and then gradually decreased as the magnitude of fluidic shear stress increased to 3.0 dyne/cm2. Contrarily, the anionic PSNs showed no significant difference of cellular uptake in presence of shear stress. Thus, shear stress should be considered to investigate the cellular distribution of various nanoparticles and drug delivery systems.

Analysis of 54 Follicular Lymphomas By Whole Exome Sequencing Identifies Multiple Novel Recurrently Mutated Pathways

Introduction: Follicular lymphoma (FL) constitutes the second most common non-Hodgkin's lymphoma in the Western world. FL carries multiple recurrently mutated genes that are under active investigation. However, due to the relatively small number of published sequenced cases, knowledge regarding the coding genome in FL is still evolving.Methods: To further our understanding of the genetic basis of FL, we used solution exon capture of sheared and processed genomic DNA isolated from highly purified light chain restricted B-cells and paired CD3+ T-cells from 54 FL cases for paired-end massively parallel sequencing (WES). Data were subsequently analyzed using bioinformatics pipelines including the variant callers MuTect v.1.1.4, Strelka v.1.0.13, and VarScan2 v.2.3.7. Candidate somatically acquired gene mutations with variant allele frequencies (VAFs) >0.15 were confirmed using Sanger sequencing. Selected mutations were validated in an expansion cohort of 120 FL.Results: We identified heterozygous missense mutations in the mTOR regulator RRAGC in 10% of FL. The RRAGC mutations targeted multiple hotspot residues (amino acid 115, 118 and 119). RRAGC forms heterodimers with either RRAGA or RRAGB that under conditions of amino acid sufficiency facilitate recruitment of mTOR through the raptor subunit to lysosomal membranes. At the lysosomal surface, multiple protein complexes, each containing various proteins regulate mTOR activation through RHEB.To gain insights into the functional consequences of RRAGC mutations, we performed 3-dimensional modeling of FL-associated RRAGC mutations and located the mutations into relatively close proximity to the RRAGC GTP/GDP binding site. Energy calculations did not identify strong effects of mutated amino acid residues on the binding of GTP/GDP to RRAGC.We performed studies of the effects of RRAGC mutants on mTOR activity as measured through S6-kinase phosphorylation. In transient transfection systems (293T and HELA) achieving expression slightly above endogenous RRAGC levels, performed under conditions of leucine starvation or sufficiency, we did not identify differences in baseline mTOR activation. In stably transfected 293T cell lines (expressing RRAGB and RRAGC proteins above endogenous levels), that were starved for leucine for 1 hour, we detected modestly elevated p-S6K levels in RRAGC mutant versus wild type transfectants, suggesting a mild intrinsic activation phenotype of RRAGC mutations. Experiments in lentivirally-transfected lymphoma cell lines, including RRAGC binding studies to raptor and folliculin (a RRAGC regulator) are in progress and will be updated at the meeting. Curiously, we did not identify mutations in the other three small GTP binding proteins that are part of the same amino acid sensing pathway (RRAGA, RRAGB or RRAGD), potentially pointing to a unique advantage conferred by RRAGC mutants on FL B cells.We identified additional mutations (combined ~15%) in other mTOR components linked to lysosomal amino acid sensing, including recurrent mutations in the v-ATPase subunit ATP6V1B2 and the accessory subunit ATP6VAP1. The mutations in RRAGC and v-ATPase together highlight a previously unidentified role of the amino acid sensing pathway that regulates mTOR in FL pathogenesis.We have discovered a high frequency of mutations (40%) in the surrogate light chain gene IGLL5 in FL, a critical component of the pre-B-cell receptor. Mutations sharply cluster in the N-terminal 70 amino acid of IGLL5, a region known as the non-Ig domain of IGLL5. The non-Ig domain of IGLL5 has been implicated in influencing pre-B-cell receptor signaling and receptor surface expression as well as interaction with extracellular ligands. The mutational data suggest an unexpected role of IGLL5 in the pathogenesis of FL and work is in progress studying IGLL5 expression in primary FL samples.Conclusion: This large WES study of 54 FL identifies novel recurrently mutated genes and pathways in FL, including frequent mutations in genes involved in amino acid signaling to mTOR (RRAGC and v-ATPase) as well as pre-B-cell receptor signaling (the surrogate light chain gene IGLL5) and multiple other novel recurrently mutated genes that will be updated at the meeting. These data substantially broaden our understanding of the genetic basis of FL and provide clues to therapeutically targeting specific pathways in FL. DisclosuresMalek:Abbvie: Equity Ownership; Gilead Sciences: Equity Ownership; Janssen Pharmaceuticals: Research Funding.

Chemotherapeutic agents attenuate CXCL12-mediated migration of colon cancer cells by selecting for CXCR4-negative cells and increasing peptidase CD26.

BackgroundRecurrence of colorectal cancer (CRC) may arise due to the persistence of drug-resistant and cancer-initiating cells that survive exposure to chemotherapy. Proteins responsible for this recurrence include the chemokine receptor CXCR4, which is known to enable CRC metastasis, as well as the cancer-initiating cell marker and peptidase CD26, which terminates activity of its chemokine CXCL12.MethodsWe evaluated the expression and function of CXCR4 and CD26 in colon cancer cell lines and xenografts following treatment with common chemotherapies using radioligand binding, flow cytometry, immunofluorescence, and enzymatic assays.Results5-Fluorouracil, oxaliplatin and SN-38 (the active metabolite of irinotecan), as well as cisplatin, methotrexate and vinblastine, each caused decreases in cell-surface CXCR4 and concomitant increases in CD26 on HT-29, T84, HRT-18, SW480 and SW620 CRC cell lines. Flow cytometry indicated that the decline in CXCR4 was associated with a significant loss of CXCR4+/CD26- cells. Elevations in CD26 were paralleled by increases in both the intrinsic dipeptidyl peptidase activity of CD26 as well as its capacity to bind extracellular adenosine deaminase. Orthotopic HT-29 xenografts treated with standard CRC chemotherapeutics 5-fluorouracil, irinotecan, or oxaliplatin showed dramatic increases in CD26 compared to untreated tumors. Consistent with the loss of CXCR4 and gain in CD26, migratory responses to exogenous CXCL12 were eliminated in cells pretreated with cytotoxic agents, although cells retained basal motility. Analysis of cancer-initiating cell CD44 and CD133 subsets revealed drug-dependent responses of CD26/CD44/CD133 populations, suggesting that the benefits of combining standard chemotherapies 5-fluoruracil and oxaliplatin may be derived from their complementary elimination of cell populations.ConclusionOur results indicate that conventional anticancer agents may act to inhibit chemokine-mediated migration through eradication of CXCR4+ cells and attenuation of chemokine gradients through elevation of CD26 activity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1702-2) contains supplementary material, which is available to authorized users.

Trolox induces inhibition of cell proliferation and apoptosis in human colon cancer cells

&lt;p class="Abstract"&gt;In the present study, the effect of trolox on human colon cancer cell lines was investigated. The results revealed that trolox treatment caused inhibition of cell growth in T84 and HCT-15 colon cancer cell lines in a dose-dependent manner. The inhibition was significant at 50 µM of trolox after 48 hours in both cell lines. Trolox treatment promoted expression of p38 and inhibited expression of survivin and Akt. It also induced cleavage of PARP and caspase-3 and ultimately induced apoptosis in T84 and HCT-15 cells. The tumor growth was inhibited significantly in the xenotransplanted mice on treatment with trolox compared to the control group. Since trolox treatment exhibits inhibitory effect on the proliferation of colon cancer cells and inhibits tumor growth in vivo therefore, can be of therapeutic importance for the treatment of colon cancer.&lt;/p&gt;&lt;p&gt; &lt;/p&gt;

Water-soluble octahedral molybdenum cluster compounds Na2[Mo6I8(N3)6] and Na2[Mo6I8(NCS)6]: Syntheses, luminescence, and in vitro studies

The octahedral molybdenum cluster compounds Na2[Mo6I8(N3)6] and Na2[Mo6I8(NCS)6] were synthesized and their luminescent properties and biological activity were investigated. Most molybdenum cluster compounds reported so far have had low solubility in water and/or underwent hydrolysis connected with aggregation, sedimentation, and diminishing luminescence and singlet oxygen-sensitizing properties. This behavior prevented the investigation of their photophysical properties and in vitro biological activities in aqueous media. Title compounds behave differently. They are well soluble in water and have red luminescence, which is quenched by oxygen to produce singlet oxygen, O2(1Δg), in high quantum yields. Even though [Mo6I8(N3)6]2− and [Mo6I8(NCS)6]2− gradually hydrolyze to mono-hydroxy derivatives, they do not form sedimenting aggregates, luminescence properties are well-defined, and the quantum yields of O2(1Δg) remain unchanged. These attractive features, suitable for luminescent probes or O2(1Δg) sensitizers in living cells, are overshadowed by the toxicity, especially of [Mo6I8(NCS)6]2−, and by the limited uptake by the HeLa and HEK 293T cell lines.

HuR antagonizes the effect of an intronic pyrimidine-rich sequence in regulating WT1 +/−KTS isoforms

WT1 + KTS and −KTS isoforms only differ in 3 amino acids in protein sequence but show significant functional difference. The +/−KTS isoforms were generated by alternative usage of 2 adjacent 5’ splice sites at RNA level, however, how these 2 isoforms are regulated is still elusive. Here we report the identification of an intronic pyrimidine-rich sequence that is critical for the ratio of +/−KTS isoforms, deletion or partial replacement of the sequence led to full/significant shift to -KTS isoform. To identify trans-factors that can regulate +/−KTS isoforms via the binding to the element, we performed RNP assembly using in vitro transcribed RNA with or without the pyrimidine-rich sequence. Mass spectrometry analysis of purified RNPs showed that the element associated with many splicing factors. Co-transfection of these factors with WT1 reporter revealed that HuR promoted the production of −KTS isoform at the reporter level. RNA immuno-precipitation experiment indicated that HuR interacted with the pyrimidine-rich element in WT1 intron 9. We further presented evidence that transient or stable over-expression of HuR led to enhanced expression of endogenous −KTS isoform. Moreover, knockdown of HuR resulted in decreased expression of endogenous −KTS isoform in 293T, SW620, SNU-387 and AGS cell lines. Together, these data indicate that HuR binds to the pyrimidine-rich sequence and antagonize its effect in regulating WT1 +/−KTS isoforms.

MicroRNA-33a disturbs influenza A virus replication by targeting ARCN1 and inhibiting viral ribonucleoprotein activity

In order to explore the roles of microRNA(s) [miRNA(s)] in the influenza A virus life cycle, we compared the miRNA profiles of 293T and HeLa cell lines, as influenza A virus can replicate efficiently in 293T cells but only poorly in HeLa cells. We analysed differentially expressed miRNAs and identified five, including miR-33a, that could disturb influenza A virus replication significantly. Using TargetScan analysis, we found that ARCN1 could be a potential target of miR-33a. To confirm whether miR-33a could truly target ARCN1, we generated a luciferase reporter for the ARCN1 3' untranslated region (UTR) and performed a luciferase assay. The data indicated that miR-33a could suppress the luciferase activity of the reporter for the ARCN1 3' UTR but not a reporter in which the predicted miR-33a targeting sites on ARCN1 3' UTR were mutated. We performed immunoblotting to confirm that miR-33a could downregulate the protein level of ARCN1. Consistently, the level of ARCN1 protein in HeLa cells was significantly lower than that in 293T cells. We also demonstrated that ectopic expression of ARCN1 could partially rescue the inhibitory effect of miR-33a on virus replication. Furthermore, we demonstrated that miR-33a could impede virus replication at the stage of virus internalization, which was similar to the pattern for knockdown of ARCN1, indicating that miR-33a inhibits influenza virus infection by suppressing ARCN1 expression. In addition, we found that miR-33a could also weaken the viral ribonucleoprotein activity in an ARCN1-independent manner. In conclusion, we found that miR-33a is a novel inhibitory factor for influenza A virus replication.

Polyamide Nanogels from Generally Recognized as Safe Components and Their Toxicity in Mouse Preimplantation Embryos.

Safe delivery systems that can not only encapsulate hydrophobic drug molecules, but also release them in response to specific triggers are important in several therapeutic and biomedical applications. In this paper, we have designed a nanogel based on molecules that are generally recognized as safe (GRAS). We have shown that the resultant polymeric nanogels exhibit responsive molecular release and also show high in vitro cellular viability on HEK 293T, HeLa, MCF 7, and A549 cell lines. The toxicity of these nanogels was further evaluated with a highly sensitive assay using mouse preimplantation embryo development, where blastocysts were formed after 4 days of in vitro culture, and live pups were born when morulae/early blastocysts were transferred to the uteri of surrogate recipients. Our results indicate that these nanogels are nontoxic during mammalian development and do not alter normal growth or early embryo success rate.

Research of methods to detect genomic mutations induced by CRISPR/Cas systems

The indel-forming non-homologous end joining (NHEJ) pathway repairs double strand breaks in mammalian genomes, resulting in mutation formation following genome editing. Common techniques employed to identify these mutations include the amplified fragment length polymorphism (AFLP) and SURVEYOR assays, which are time consuming, laborious, and only offer a low level of sensitivity. An alternative to these approaches, which is examined in this study, is based on the quantitative PCR high-resolution melting (qPCR-HRM) curve analysis technique and offers simple implementation, is capable of handling large sample sizes, takes no more than 90min, and produces sensitive results. Using the newly discovered RNA-guided CRISPR/Cas systems, the IL2RG and EMX1 genes were edited in the human 293T cell line in order to compare the mutation detection accuracies of the aforementioned methods. Genomic mutations were simulated by mixing mutated DNA fragments with normal fragments along a concentration gradient. The results of this comparative study showed that the HRM approach was both reproducible and accurate.

Increased miR-196a expression represses PGR in infertile women with minimal or mild endometriosis by increased activationof the MEK/ERK signal

Endometriosis-related infertility is common in women of reproductive age. Progesterone resistance, especially aberrantly expressed progesterone receptor in the eutopic endometrium, is considered as a key causal factor. Our objective is to explore microRNA-mediated mechanism controlling aberrant progesterone receptor expression in infertile women with minimal or mild endometriosis. microRNA Array, combined with Human Gene Expression microarrays, was used to screen eutopic endometrium of infertile women with minimal or mild endometriosis. Bioinformatics analysis predicted that microRNA-196a target the PGR 3′UTR. We studied the relationship between microRNA-196a level and PGR expression in endometrial stromal cells (ESCs). According to mRNAs microarray result, MAPK pathway is activated in eutopic mid-secretory endometrium. The role of MAPK pathway involved regulation of miR-196a on PGR was also investigated. The study was conducted at West China Second University Hospital of Sichuan University. 22 infertile women with r-AFSI-IIendometriosis and 20 disease-free control subjects were enrolled. MiRCURY LNATM microRNA Array(v.18.0)(Exiqon), Human 4×44K Gene Expression Microarrays v2(Agilent), qRT-PCR, cell culture, transfections, luciferase reporter assays, and western bolt were used in this study. 66 differently expressed microRNAs (fold change≥2.00 and p<0.05) were found by microRNA array in eutopic endometrium of infertile women with minimal or mild endometriosis. Among them, the expression of 54 microRNAs were increased, while 12 microRNAs were decreased. Meanwhile, mRNA array screened 357 differently expressed mRNAs (fold change≥2.00 and p<0.05).Among them, 224 mRNAs over-expressed and 133 mRNAs down-expressed. miR-196a was the highest over-expression microRNA, which was confirmed by qRT-PCR. The transfection of ESCs with miR-196a mimics significantly decreased expression of total PGR and PGR-B mRNA levels, while total PGR and PGR-B mRNA expression were up-regulated by miR-196a inhibitor. Luciferase reporter failed to confirm the target regulation of miR-196a on PGR in 293T cell line. Based on mRNA microarray, Western blot confirmed that P-MEK1/2 and P-ERK1/2, the key molecular of MAPK pathway, were upregulated in eutopic endometrium in infertile women with minimal or mild endometriosis. P-MEK1/2 and P-ERK1/2 expression were up-regulated, PGR-A and PGR-B expression were inhibited, and the ratio of PGR-A/PGR-B was up-regulated by miR-196a overexpression in eutopic ESCs. Meanwhile, a specific P-MEK/P-ERK inhibitor, U0126 resulted in upregulation of PGR-A and PGR-B protein, and the downregulation of the protein ratio of PGR-A/PGR-B in ESCs. Aberrantly expressed miR-196a represses PGR expression and upregulate the protein ratio of PGR-A/PGR-B via activation of MAPK pathway in eutopic endometrium of infertile women with minimal or mild endometriosis.