Inhibitory effect of a short Z-DNA forming sequence on transcription elongation by T7 RNA polymerase

DNA sequences capable of forming unusual secondary structures can be a source of genomic instability. In some cases that instability might be affected by transcription, as recently shown for the Z-DNA forming sequence (CG)14, which causes genomic instability both in mammalian cells and in bacteria, and this effect increases with its transcription. We have investigated the effect of this (CG)14 sequence on transcription with T7 RNA polymerase in vitro. We detected partial transcription blockage within the sequence; the blockage increased with negative supercoiling of the template DNA. This effect was not observed in a control self-complementary sequence of identical length and base composition as the (CG)14 sequence, when the purine–pyrimidine alternation required for Z-DNA formation was disrupted. These findings suggest that the inhibitory effect on T7 transcription results from Z-DNA formation in the (CG)14 sequence rather than from an effect of the sequence composition or from hairpin formation in either the DNA or the RNA product

Evaluation of Lu-177-Dotatate treatment in patients with metastatic neuroendocrine tumors and prognostic factors

BACKGROUND: (177)Lu peptide receptor radionuclide therapy (PRRT) is a recently approved therapy in Spain that has been demonstrated to be a well-tolerated therapy for positive somatostatin receptor advanced gastroenteropancreatic neuroendocrine tumors. AIM: To determine the impact of PRRT on quality of life, radiologic and metabolic response, overall survival, prognostic factors and toxicity. METHODS: Thirty-six patients treated with (177)Lu-PRRT from 2016 to 2019 were included. The most frequent location of the primary tumor was the gastrointestinal tract (52.8%), pancreas (27.8%), and nongastropancreatic neuroendocrine tumor (11.1%). The liver was the most common site of metastasis (91.7%), followed by distant nodes (50.0%), bone (27.8%), peritoneum (25.0%) and lung (11.1%). Toxicity was evaluated after the administration of each dose. Treatment efficacy was evaluated by two parameters: stable disease and disease progression in response evaluation criteria in solid tumors 1.1 criterion and prognostic factors were tested. RESULTS: From 36 patients, 55.6% were men, with a median age of 61.1 +/- 11.8 years. Regarding previous treatments, 55.6% of patients underwent surgery of the primary tumor, 100% of patients were treated with long-acting somatostatin analogues, 66.7% of patients were treated with everolimus, 27.8% of patients were treated with tyrosine kinase inhibitor, and 27.8% of patients were treated with interferon. One patient received radioembolization, three patients received chemoembolization, six patients received chemotherapy. Hematological toxicity was registered in 14 patients (G1-G2: 55.5% and G3: 3.1%). Other events presented were intestinal suboclusion in 4 cases, cholestasis in 2 cases and carcinoid crisis in 1 case. The median follow-up time was 3 years. Currently, 24 patients completed treatment. Nineteen are alive with stable disease, two have disease progression, eight have died, and nine are still receiving treatment. The median overall survival was 12.5 mo (95% confidence interval range: 9.8-15.2), being inversely proportional to toxicity in previous treatments (P < 0.02), tumor grade (P < 0.01) and the presence of bone lesions (P = 0.009) and directly proportional with matching lesion findings between Octreoscan and computed tomography pre-PRRT (P < 0.01), , primary tumor surgery (P = 0.03) and metastasis surgery (P = 0.045). In a multivariate Cox regression analysis, a high Ki67 index (P = 0.003), a mismatch in the lesion findings between Octreoscan and computed tomography pre-PRRT (P < 0.01) and a preceding toxicity in previous treatments (P < 0.05) were risk factors to overall survival. CONCLUSION: Overall survival was inversely proportional to previous toxicity, tumor grade and the presence of bone metastasis and directly proportional to matching lesion findings between Octreoscan and computed tomography pre-PRRT and primary tumor and metastasis surgery

Dissecting Microrna Requirements In Early Human Embryonic Development

DICER1 is the enzyme responsible for cleaving double-stranded RNAs (dsRNAs) into functionally mature ~20-24 nucleotide (nt) long microRNAs (miRNAs). miRNAs are the effectors of an RNA-induced gene silencing system that functions at the post-transcriptional and post-translational levels, and are essential for mouse development. Due to maternal Dicer1 contribution in the early stages of embryonic development, the exact timing for miRNA requirement has not yet been established. Furthermore, precise miRNA functional studies in early embryonic development have been lacking since miRNA knockout studies are hindered due to redundancy in the miRNA network. Thus, miRNA function in early embryonic development remains elusive. To address this knowledge gap, we set out to knockout DICER1 in human ESCs (hESCs) and assess human-specific miRNA requirements in the primed state. We report that DICER1 is essential in hESCs unlike in mouse embryonic stem cells (mESCs), and that this likely reflects a unique requirement for DICER1 in primed versus naïve pluripotency. Additionally, we designed an inducible DICER1 system to bypass the lethality of DICER1 loss in hESCs and enable the generation of homozygous mutants. A targeted mature miRNA rescue screen identified members of the miRNA-302-367 and miRNA-371-373 clusters, but surprisingly not miRNA-17-92, as having pro-survival functions in hESCs. Since it bypasses the common issue of redundancy in the miRNA network, our screening platform is particularly suited to dissect the roles of individual miRNAs and miRNA clusters in early human development and hESC differentiation

T7 RNA polymerase

sequence on transcription elongation b

DICER1 Is Essential for Self-Renewal of Human Embryonic Stem Cells

Summary: MicroRNAs (miRNAs) are the effectors of a conserved gene-silencing system with broad roles in post-transcriptional regulation. Due to functional overlaps, assigning specific functions to individual miRNAs has been challenging. DICER1 cleaves pre-miRNA hairpins into mature miRNAs, and previously Dicer1 knockout mouse embryonic stem cells have been generated to study miRNA function in early mouse development. Here we report an essential requirement of DICER1 for the self-renewal of human embryonic stem cells (hESCs). Utilizing a conditional knockout approach, we found that DICER1 deletion led to increased death receptor-mediated apoptosis and failure of hESC self-renewal. We further devised a targeted miRNA screening strategy and uncovered essential pro-survival roles of members of the mir-302-367 and mir-371-373 clusters that bear the seed sequence AAGUGC. This platform is uniquely suitable for dissecting the roles of individual miRNAs in hESC self-renewal and differentiation, which may help us better understand the early development of human embryos. : Huangfu and colleagues report an unexpected requirement for DICER1 in preventing death-receptor-mediated apoptosis in hESCs. This essential pro-survival role is largely mediated by mir-302-367 and mir-371-373 cluster members that bear the seed sequence “AAGUGC” shared by the ESC-specific cell-cycle-regulating family of miRNAs. This work provides a robust platform for interrogating miRNA function in hESC pluripotency and differentiation. Keywords: human embryonic stem cells (hESCs), human pluripotent stem cells (hPSCs), DICER1, microRNAs (miRNAs), self-renewal, apoptosis, ESCC miRNA

Hyperacetylation of α-tubulin correlates with starvation induced autophagy in tobacco BY-2 cells

Autophagy is known as an important adaptive stress response in eukaryotic cells. Also it is well known that autophagosome maturation and its intracellular transport to merge with lysosomes or lytic vacuoles are mediated by the cytoskeleton. However, the mechanism of participation of microtubules in the autophagosome formation discovered insufficiently as yet. It is assumed that regulation of cytoskeleton functional state in autophagy can be realized through posttranslational modifications of á-tubulin acetylation in particular. The objective was to study the autophagy dynamics in plant cells under metabolic stress conditions induced by long-term cultivation and functional relationship of this process with acetylation of á-tubulin. Experiments were performed using suspension culture cells of tobacco (Nicotiana tabacum L.) BY-2. Autophagy development was induced by increase in the duration of cell culture between passages (from 7 to 11 days). The first reliable signs of autophagy, namely the appearance of membrane structures stained with monodansylcadaverine were observed on 8th cultivation day and this hallmark correlated with decreasing of glucose concentration in the medium to a critical level - 1mg/ml. Following cultivation was resulted in complete glucose depletion in the medium which in turn correlated with increasing of cell number containing autophagosomes (up to 29.4% on 11th day). It should be noted that BY-2 cells demonstrate high survival rate in these stress conditions. Thus on 11th day the number of dead cells did not exceed 11% indicating the reliability of used experimental approach for simulation of autophagic processes in plant cell. Cell lysates of various cultivation stages were analyzed by Western blot using monoclonal antibodies against á-tubulin and acetylated á-tubulin. Blotted proteins were re-probed by both antibodies on the same membrane using stripping technique to quantify the acetylation level of tubulin comparing with whole tubulin amount. Following densitometric analysis revealed an essential increase of á-tubulin acetylation at the later stages of cultivation. Notably acetylation level of á-tubulin was increased in 3,5 and 11,5 folds on the 8th and 11th as compared to 7th day, respectively. Thus, glucose depletion in culture medium and the appearance of autophagosomes in BY-2 cells were correlated with á-tubulin hyperacetylation indicating the role of cytoskeleton in autophagy mediation in plant cell