Protocol for rapidly inducing genome-wide RNA Pol II hyperphosphorylation by selectively disrupting INTAC phosphatase activity.

While several inhibitors targeting RNA polymerase II (Pol II) kinases have been applied for inhibiting RNA Pol II phosphorylation, there are few approaches for inducing RNA Pol II hyperphosphorylation. Here, we present a protocol for constructing the INTS8 degradation tag (dTAG) system combined with ectopic expression of N-terminally truncated INTS8 (INTS8-ΔN) in DLD-1 cells. We describe steps for INTS8-dTAG cell line construction, validation of knockin and degradation, and INTS8-ΔN rescue. We then detail validation of RNA Pol II phosphorylation upregulation. For complete details on the use and execution of this protocol, please refer to Hu et al. (2023).1.

INTAC endonuclease and phosphatase modules differentially regulate transcription by RNA polymerase II.

Gene expression in metazoans is controlled by promoter-proximal pausing of RNA polymerase II, which can undergo productive elongation or promoter-proximal termination. Integrator-PP2A (INTAC) plays a crucial role in determining the fate of paused polymerases, but the underlying mechanisms remain unclear. Here, we establish a rapid degradation system to dissect the functions of INTAC RNA endonuclease and phosphatase modules. We find that both catalytic modules function at most if not all active promoters and enhancers, yet differentially affect polymerase fate. The endonuclease module induces promoter-proximal termination, with its disruption leading to accumulation of elongation-incompetent polymerases and downregulation of highly expressed genes, while elongation-competent polymerases accumulate at lowly expressed genes and non-coding elements, leading to their upregulation. The phosphatase module primarily prevents the release of paused polymerases and limits transcriptional activation, especially for highly paused genes. Thus, both INTAC catalytic modules have unexpectedly general yet distinct roles in dynamic transcriptional control.

Increased salinity improves the thermotolerance of mesophilic anammox consortia.

While the application of anammox-based process for mesophilic sidestream treatment is at present the state of the art and mainstream treatment at ambient temperature is also in development, the feasibility of thermophilic anammox process is still unclear. This study investigated the effects of salinity on the thermotolerance of mesophilic anammox sludge. In batch activity tests, 45 °C seems to be the critical temperature for the tolerance of mesophilic anammox consortia without acclimatization or amendments. The optimal anammox activity at 40, 42.5, and 45 °C can be achieved with the amendment of salt at 5-8, 8-10, and ~12 g NaCl L-1, respectively. However, this improvement effect was limited at 50 °C or when the shock duration was longer than 24 h even at 45 °C. In continuous-flow bioreactors, mesophilic anammox consortia could gradually adapt to 40-50 °C under a transition of 2.5 °C, and the performance was enhanced by an increase in salinity, which may be associated with the increase in extracellular polymeric substances. A nitrogen removal rate of 0.53 kgN m-3 d-1 was finally obtained at 50 °C. Overall, these interesting results facilitate further opportunities for thermophilic anammox process.

Transient and long-term effects of bicarbonate on the ANAMMOX process.

In this study, the effects of both transient and long-term inorganic carbon (IC) addition on the anaerobic ammonium oxidation (ANAMMOX) process under pseudo-steady-state and substrate inhibitions were analyzed using reactor performance and measures of sludge activity. Compared with the nitrogen removal rate (NRR) of 3.42 kg N m(-3) day(-1) in the control bioreactor (ICDR) without IC, the peak NRR reached 21.0 kg N m(-3) day(-1) in the reactor (ICAR) with sufficient IC added. It was revealed that the long-term addition of bicarbonate significantly enhanced the performance of the ANAMMOX reactor. The optimum HCO3 (-)/TN ratio was considered to be 1.20, which is lower than that in normal conditions. The IC concentration affected biomass activity, and the transient addition or removal of IC to differing sludge media caused a significant loss of activity. Sufficient addition of IC alleviated the inhibition of excess substrates, while the inhibition was aggravated by the IC limitation. The half-maximal (50 %) inhibitory concentrations of substrate for the sludge were 295 mg L(-1) NO2 (-)-N and 361 mg L(-1) NH4 (+)-N with 120 mg L(-1) of fixed HCO3 (-) and 346 mg L(-1) NO2 (-)-N and 456 mg L(-1) NH4 (+)-N with unlimited IC, respectively. Changing the HCO3 (-)/TN (in milligrams per milligram) ratio resulted in the variation of ANAMMOX stoichiometric ratios. Sludge characterization parameters in the ICDR, including biomass, extracellular polymeric substances, heme C, and so on, were lower than those in ICAR. Filamentous bacteria and spherical bacteria were also observed in the reactor with limited IC.

Enhancement of ANAMMOX activity by low-intensity ultrasound irradiation at ambient temperature.

This paper aims to investigate the enhancement effect of low intensity intermittent ultrasound irradiation on the efficiency of anaerobic ammonium oxidation (ANAMMOX) process at ambient temperature. With intermittently irradiated (ultrasound intensity of 0.19 w/cm(2), exposure time of 0.2 min), the reactor (RU) had a nitrogen removal rate (NRR) of 5.49 kgTN/m(3)/d at 14.8°C, while the NRR was 1.53 kgTN/m(3)/d in the control reactor (RC). At the end of operation, the contents of polysaccharide, protein, TTC-dehydrogenase and VSS were 6.82 mg/mgVSS, 26.79 mg/mgVSS, 0.58 mgTF/L/H and 10.11 gVSS/L in RU, higher than the levels in the RC. These results demonstrated that it is possible to achieve stable and highly efficient operation in an ANAMMOX reactor at low ambient temperature by implementation of ultrasonication.

Evaluating the recovery performance of the ANAMMOX process following inhibition by phenol and sulfide.

In this study, the recovery performance of two anaerobic ammonium oxidation (ANAMMOX) reactors (R1, R2) that were previously subjected to phenol and sulfide for nearly 200 days with respective levels of 12.5-50 and 8-40 mg L(-1) and then operated in the absence of these suppressors was investigated. High nitrogen removal rates of greater than 36 kg-Nm(-3)d(-1) were achieved through the 81 and 75 days restoration of R1 and R2, respectively. The recovery performance was determined by specific sludge removal rate, heme c contents, specific ANAMMOX activity, settling properties and morphology of ANAMMOX granules. In addition, the modified Boltzmann model, the modified Gompertz model and the modified Logistic model were applied to simulate recovery performance. The modified Boltzmann model was found to be appropriate for predicting recovery performance of the phenol-inhibited reactor, while the modified Logistic model effectively simulated the recovery performance of the sulfide suppressed reactor.

[Construction of antisense human tankyrase-1 RNA retroviral vector and its inhibition on tongue cancer cells].

OBJECTIVE: To investigate the inhibition of telomerase activity and cellular proliferation in tongue cancer TCCA-8113 cell lines by antisense human tankyrase-1 RNA treatment, and explore the possibility of the tankyrase-1 as a target of gene therapy for tongue cancer. METHODS: The replication deficient retrovirus expressing tankyrase-1 antisense RNA was constructed to infect the TCCA-8113 cells. Tankyrase-1 expression was examined by RT-PCR. Telomerase activity was assayed by telomerase repeat amplification protocol (TRAP). Cell proliferation was investigated by cellular growth curve. Cellular apoptosis was detected by flow cytometry method and invert microscope. RESULTS: Tankyrase-1 expression and telomerase activity of tongue cancer TCCA-8113 cells were significantly inhibited. There was G(1)-S phase arrest when TCCA-8113 cells were treated with antisense tankyrase-1 transduction. Cellular proliferation was arrested, and cellular apoptosis occurred after antisense tankyrase transduction. CONCLUSIONS: The transduction of antisense tankyrase-1 by retroviral vector can significantly inhibit the tankyrase-1 expression and telomerase activity of tongue cancer TCCA-8113 cell lines, and arrest the cellular proliferation and promote cellular apoptosis. The tankyrase may be a potential target of gene therapy for tongue cancer.

[Survivin antisense oligonucleotide induces human Hep-2 cell apoptosis].

OBJECTIVE: Survivin highly overexpresses in the most of human tumors, and it may play an important role in the development of tumor. The aim of this study was to explore the effects of survivin antisense oligonucleotide (ASODN) on the proliferation and the apoptosis of human Hep-2 cell. METHODS: Hep-2 cells were transfected with survivin ASODN mediated by lipofectamine, MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5 diphenyl tetrazolium bromide] method was used to observe the cell growth inhibitory rate, the expressions of survivin mRNA and protein were detected by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot respectively. Flow cytometry was used to examine cell apoptosis rate. Kinase activity test was used to detect the changes of caspase-3 activity. RESULTS: Survivin ASODN obviously inhibited the cell growth of Hep-2 cells after transfection. After transfected with survivin ASODN the expressions of survivin mRNA and protein of Hep-2 cells were down-regulated, and apoptosis rate was significantly increased. The activity of caspase-3 increased highly in Hep-2 cells transfected with survivin ASODN, which showed time-dependent. CONCLUSIONS: Survivin ASODN could inhibit the proliferation of Hep-2 cell and induced apoptosis through down-regulating the the expressions of survivin mRNA and protein.