A Complex of U1 snRNP with Cleavage and Polyadenylation Factors Controls Telescripting, Regulating mRNA Transcription in Human Cells.

Full-length transcription in the majority of human genes depends on U1 snRNP (U1) to co-transcriptionally suppress transcription-terminating premature 3' end cleavage and polyadenylation (PCPA) from cryptic polyadenylation signals (PASs) in introns. However, the mechanism of this U1 activity, termed telescripting, is unknown. Here, we captured a complex, comprising U1 and CPA factors (U1-CPAFs), that binds intronic PASs and suppresses PCPA. U1-CPAFs are distinct from U1-spliceosomal complexes; they include CPA's three main subunits, CFIm, CPSF, and CstF; lack essential splicing factors; and associate with transcription elongation and mRNA export complexes. Telescripting requires U1:pre-mRNA base-pairing, which can be disrupted by U1 antisense oligonucleotide (U1 AMO), triggering PCPA. U1 AMO remodels U1-CPAFs, revealing changes, including recruitment of CPA-stimulating factors, that explain U1-CPAFs' switch from repressive to activated states. Our findings outline this U1 telescripting mechanism and demonstrate U1's unique role as central regulator of pre-mRNA processing and transcription.

THZ1 Reveals Roles for Cdk7 in Co-transcriptional Capping and Pausing.

The Cdk7 subunit of TFIIH phosphorylates RNA polymerase II (Pol II) during initiation, and, while recent studies show that inhibition of human Cdk7 negatively influences transcription, the mechanisms involved are unclear. Using in vitro transcription with nuclear extract, we demonstrate that THZ1, a covalent Cdk7 inhibitor, causes defects in Pol II phosphorylation, co-transcriptional capping, promoter proximal pausing, and productive elongation. THZ1 does not affect initiation but blocks essentially all Pol II large subunit C-terminal domain (CTD) phosphorylation. We found that guanylylation of nascent RNAs is length dependent and modulated by a THZ1-sensitive factor present in nuclear extract. THZ1 impacts pausing through a capping-independent block of DSIF and NELF loading. The P-TEFb-dependent transition into productive elongation was also inhibited by THZ1, likely due to loss of DSIF. Capping and pausing were also reduced in THZ1-treated cells. Our results provide mechanistic insights into THZ1 action and how Cdk7 broadly influences transcription and capping.

Functional association of Gdown1 with RNA polymerase II poised on human genes.

Most human genes are loaded with promoter-proximally paused RNA polymerase II (Pol II) molecules that are poised for release into productive elongation by P-TEFb. We present evidence that Gdown1, the product of the POLR2M gene that renders Pol II responsive to Mediator, is involved in Pol II elongation control. During in vitro transcription, Gdown1 specifically blocked elongation stimulation by TFIIF, inhibited the termination activity of TTF2, and influenced pausing factors NELF and DSIF, but did not affect the function of TFIIS or the mRNA capping enzyme. Without P-TEFb, Gdown1 led to the production of stably paused polymerases in the presence of nuclear extract. Supporting these mechanistic insights, ChIP-Seq demonstrated that Gdown1 mapped over essentially all poised polymerases across the human genome. Our results establish that Gdown1 stabilizes poised polymerases while maintaining their responsiveness to P-TEFb and suggest that Mediator overcomes a Gdown1-mediated block of initiation by allowing TFIIF function.

Experimental demonstration of building and operating QoS-aware survivable vSD-EONs with transparent resiliency.

Software-defined elastic optical networks (SD-EONs) provide operators more flexibility to customize their optical infrastructures dynamically. By leveraging infrastructure-as-a-service (IaaS), virtual SD-EONs (vSD-EONs) can be realized to further enhance the adaptivity of SD-EONs and shorten the time-to-market of new services. In this paper, we design and demonstrate the building and operating of quality-of-service (QoS) aware survivable vSD-EONs that are equipped with transparent data plane (DP) resiliency. Specifically, when slicing a vSD-EON, our network hypervisor (NHV) chooses to use "1:1" virtual link (VL) protection or on-demand VL remapping as the DP restoration scheme, according to the service-level agreement (SLA) between the vSD-EON's operator and the infrastructure provider (InP). Then, during an actual substrate link (SL) failure, the NHV realizes automatic DP restoration that is transparent to the controllers of vSD-EONs. We build a network testbed to demonstrate the creation of QoS-aware survivable vSD-EONs, the activation of lightpaths in the vSD-EONs to support upper-layer applications, and the automatic and simultaneous QoS-aware DP restorations during an SL failure. The experimental results indicate that our vSD-EON slicing system can build QoS-aware survivable vSD-EONs on-demand, operate them to set up lightpaths for carrying real application traffic, and facilitate differentiated DP restorations during SL failures to recover the vSD-EONs' services according to their SLAs.

Regulation of RNA polymerase II termination by phosphorylation of Gdown1.

Gdown1 is a substoichiometric subunit of RNA polymerase II (Pol II) that has been recently demonstrated to be involved in stabilizing promoter-proximal paused Pol II. It was shown to inhibit termination of Pol II by transcription termination factor 2 (TTF2) as well as block elongation stimulation by transcription factor IIF (TFIIF). Here, using in vitro transcription assays, we identified two functional domains in Gdown1. Although both are required to maintain a tight association with Pol II, the N- and C-terminal domains are responsible for blocking TTF2 and TFIIF, respectively. A highly conserved LPDKG motif found in the N-terminal domain of Gdown1 is also highly conserved in TTF2. Deletion of this motif eliminated the TTF2 inhibitory activity of Gdown1. We identified a phosphorylated form of Gdown1 with altered mobility in SDS-PAGE that appears during mitosis. A kinase in HeLa nuclear extract that caused the shift was partially purified. In vitro, Gdown1 phosphorylated by this kinase demonstrated reduced activity in blocking both TTF2 and TFIIF because of its reduced affinity for Pol II. Mass spectrometry identified Ser-270 as the site of this phosphorylation. An S270A mutation was not phosphorylated by the partially purified kinase, and an S270E mutation partially mimicked the properties of phospho-Gdown1. Gdown1 Ser-270 phosphorylation occurs predominately during mitosis, and we suggest that this would enable TTF2 to terminate all Pol II even if it is associated with Gdown1.

Functional interactions of the RNA polymerase II-interacting proteins Gdown1 and TFIIF.

Gdown1, the substoichiometric 13th subunit of RNA polymerase II (pol II), has an important role in pausing during the initial stage of transcript elongation. However, Gdown1 quantitatively displaces the essential initiation factor TFIIF from free pol II and elongating pol II. Thus, it is not clear how or even if pol II can initiate in the presence of Gdown1. Using an in vitro transcription system with purified factors and pol II lacking Gdown1, we found that although Gdown1 is strongly inhibitory to transcription when prebound to pol II, a fraction of complexes do remain active. Surprisingly, when Gdown1 is added to complete preinitiation complexes (PICs), it does not inhibit initiation or functionally associate with the PICs. Gdown1 does associate with pol II during the early stage of transcript elongation but this association is competitive with TFIIF. By phosphorylating TFIIF, PICs can be assembled that do not retain TFIIF. Gdown1 also fails to functionally associate with these TFIIF-less PICs, but once polymerase enters transcript elongation, complexes lacking TFIIF quantitatively bind Gdown1. Our results provide a partial resolution of the paradox of the competition between Gdown1 and TFIIF for association with pol II. Although Gdown1 completely displaces TFIIF from free pol II and elongation complexes, Gdown1 does not functionally associate with the PIC. Gdown1 can enter the transcription complex immediately after initiation. Modification of TFIIF provides one pathway through which efficient Gdown1 loading can occur early in elongation, allowing downstream pausing to be regulated.

Thermal adaptation of dihydrofolate reductase from the moderate thermophile Geobacillus stearothermophilus.

The thermal melting temperature of dihydrofolate reductase from Geobacillus stearothermophilus (BsDHFR) is ~30 °C higher than that of its homologue from the psychrophile Moritella profunda. Additional proline residues in the loop regions of BsDHFR have been proposed to enhance the thermostability of BsDHFR, but site-directed mutagenesis studies reveal that these proline residues contribute only minimally. Instead, the high thermal stability of BsDHFR is partly due to removal of water-accessible thermolabile residues such as glutamine and methionine, which are prone to hydrolysis or oxidation at high temperatures. The extra thermostability of BsDHFR can be obtained by ligand binding, or in the presence of salts or cosolvents such as glycerol and sucrose. The sum of all these incremental factors allows BsDHFR to function efficiently in the natural habitat of G. stearothermophilus, which is characterized by temperatures that can reach 75 °C.

Loop interactions during catalysis by dihydrofolate reductase from Moritella profunda.

Dihydrofolate reductase (DHFR) is often used as a model system to study the relation between protein dynamics and catalysis. We have studied a number of variants of the cold-adapted DHFR from Moritella profunda (MpDHFR), in which the catalytically important M20 and FG loops have been altered, and present a comparison with the corresponding variants of the well-studied DHFR from Escherichia coli (EcDHFR). Mutations in the M20 loop do not affect the actual chemical step of transfer of hydride from reduced nicotinamide adenine dinucleotide phosphate to the substrate 7,8-dihydrofolate in the catalytic cycle in either enzyme; they affect the steady state turnover rate in EcDHFR but not in MpDHFR. Mutations in the FG loop also have different effects on catalysis by the two DHFRs. Despite the two enzymes most likely sharing a common catalytic cycle at pH 7, motions of these loops, known to be important for progression through the catalytic cycle in EcDHFR, appear not to play a significant role in MpDHFR.

Effect of dimerization on dihydrofolate reductase catalysis.

Dihydrofolate reductase (DHFR) from the hyperthermophile Thermotoga maritima (TmDHFR) forms a very stable homodimer, while DHFRs from other organisms are monomers. We investigated the effect of dimerization on DHFR catalysis by preparing a dimeric variant, Xet-3, of DHFR from Escherichia coli (EcDHFR). Introducing residues located at the TmDHFR dimer interface into EcDHFR increases the melting temperature to ∼60 °C, approximately 9 °C higher than that measured for EcDHFR. The steady-state and pre-steady-state rate constants measured for Xet-3 were similar to those of dimeric TmDHFR but significantly lower than those of the parent EcDHFR. This reduction in the degree of catalytic competence is likely a consequence of the loss of flexibility of catalytically important loop regions of EcDHFR on dimerization and a compromise of the electrostatic environment of the active site. In contrast, the reduced catalytic ability of TmDHFR relative to that of EcDHFR is not simply a consequence of reduced loop flexibility in the dimeric enzyme. Our studies demonstrate that EcDHFR is not a good model for understanding the properties of other DHFRs, including TmDHFR.

Peripheral T-cell receptor repertoire dynamics in small cell lung cancer.

Background: Identifying a circulating biomarker predictive of immune checkpoint inhibitor (ICI) benefit in patients with small cell lung cancer (SCLC) remains an unmet need. Characteristics of peripheral and intratumoral T-cell receptor (TCR) repertoires have been shown to predict clinical outcomes in non-small cell lung cancer (NSCLC). Recognizing a knowledge gap, we sought to characterize circulating TCR repertoires and their relationship with clinical outcomes in SCLC. Methods: SCLC patients with limited (n=4) and extensive (n=10) stage disease were prospectively enrolled for blood collection and chart review. Targeted next-generation sequencing of TCR beta and alpha chains of peripheral blood samples was performed. Unique TCR clonotypes were defined by identical CDR3, V gene, and J gene nucleotide sequences of the beta chain and subsequently used to calculate TCR diversity indices. Results: Patients with stable versus progressive and limited versus extensive stage disease did not demonstrate significant differences in V gene usage. Kaplan-Meier curve and log-rank analysis did not identify a statistical difference in progression-free survival (PFS) (P=0.900) or overall survival (OS) (P=0.200) between high and low on-treatment TCR diversity groups, although the high diversity group exhibited a trend toward increased OS. Conclusions: We report the second study investigating peripheral TCR repertoire diversity in SCLC. With a limited sample size, no statistically significant associations between peripheral TCR diversity and clinical outcomes were observed, though further study is warranted.

Millimeter-Wave and Short-Range Wireless Communication Antenna Based on High-Conductivity Graphene-Assembled Film.

Millimeter-wave and short-range wireless communication is an important part of the Internet of Things due to its advantages of high transmission speed and large data capacity. In this paper, two antenna arrays operating at typical millimeter-wave bands (45 and 60 GHz) based on graphene-assembled films (GAF) are proposed for short-range wireless communication application. The 45 GHz graphene-assembled film antenna array is in the form of a magnetoelectric dipole antenna with a strip slot coupling to achieve bidirectional radiation, which offers an operating bandwidth of 40-49.5 GHz with a realized gain of 11.8 dBi. The 60 GHz graphene-assembled film antenna utilizes a microstrip discontinuous radiation array to achieve radiation with an operating bandwidth of 59-64 GHz, reaching the peak realized gain of 14.92 dBi over the working frequency. Finally, we proposed an experimental validation to verify the transmission performance of both antenna arrays in an actual conference room. The results show that the signal drops slowly in the room with drop rates of 0.064 dB/cm (at 45 GHz) and 0.071 dB/cm (at 60 GHz), while it steeply dropped through the wall with the drop rates of 2.3 and 3.13 dB/cm, more than 35-fold difference in signal drop rates in the room and through the wall. It has been confirmed that the proposed antenna arrays can successfully realize fast indoor short-range wireless communication while also preventing signal leakage through walls, thereby enhancing the security of information. In summary, this is the first time that we have applied graphene-based materials to millimeter-wave and short-range wireless communications, revealing the significant potential of carbon-based materials in high-frequency communication systems.

Semiquantitative microscopic pore characterizations of the metamorphic rock reservoir in the central paleo-uplift belt, Songliao Basin.

Currently, metamorphic rock is a common target for natural gas exploration, and reservoirs are the key factors restricting natural gas exploration and development in metamorphic rocks. The deep metamorphic rock gas reservoir in the central paleo-uplift of the northern Songliao Basin has good exploration and development potential. In this study, we use a combination of qualitative descriptions and quantitative analysis to comprehensively analyze the pore characteristics of the reservoir and explore the factors controlling the pore characteristics of the metamorphic rock reservoir in the central paleo-uplift belt of the Songliao Basin. The metamorphic rock reservoir in the central paleo-uplift belt contains three types of lithologies: chlorite schist, mica schist and mylonite, each with different protoliths and metamorphic histories. The results of high-pressure mercury intrusion and nitrogen adsorption indicate that the pore size distributions of the schist and mylonite differ. Compared with the mylonite, the schist has larger reservoir space, more heterogeneity, smaller pore size, larger specific surface area and larger adsorbed gas storage capacity. This paper also studies the formation process of the reservoir and divides it into four stages. Finally, this article discusses in detail the factors controlling the microscopic pore characteristics of metamorphic rock reservoirs in the central paleo-uplift belt; the metamorphic rock protolith is the most important controlling factor.

NLRP3 and Gut Microbiota Homeostasis: Progress in Research.

The inflammasome is a platform for inflammatory signaling, and the NLRP3 inflammasome recognizes stimuli in vitro and in vivo, and releases inflammatory cytokines that trigger inflammation and pyroptosis. In the gut, the NLRP3 inflammasome is a key sensor for protecting the body from damage and exogenous pathogens. It plays a fundamental role in maintaining the stability of the gut's immune system. We focus on the role of NLRP3 as a key node in maintaining the homeostasis of gut microbiota which has not been fully highlighted in the past; gut microbiota and innate immunity, as well as the NLRP3 inflammasome, are discussed in this article.

Hydrogen sulfide against preeclampsia exposure-induced oxidative mitochondrial damage in HTR-8/SVneo cells.

Extravillous trophoblast invasion disorder caused by oxidative stress is involved in the pathogenesis of preeclampsia (PE). In order to identify whether hydrogen sulfide (H2S) can prevent oxidative stress injury in extravillous trophoblasts. HTR-8/SVneo cells were detected by H2S inhibiting H2O2 induced oxidative mitochondrial damage. Reactive oxygen species (ROS) were detected, as well as malondialdehyde (MDA), catalase (CAT), and superoxide dismutase (SOD). JC-1 detected the potential of the mitochondrial membrane in this experiment. Then to detect the expression level of the apoptosis-inducing protein B-cell lymphoma-2 (Bcl-2) associated X protein (Bax), caspase 3, p53, p-p53, the apoptosis-inhibiting protein Bcl-2, PRAP, and the mitochondria fission protein Drp1, p-Drp1. CCK-8 assay, it was demonstrated that cell proliferation in the NaHS group was significantly higher than that in the Mod group, indicating that H2S may induce cell proliferation. Transwell assay elucidated that cell invasion in the NaHS group was recovered compared to the Mod group. ROS concentration no matter in cells or mitochondria was decreased by NaHS, which we could get from the comparison between the Mod group, PAG group, and NaHS group. The concentration of MDA was significantly lower in the NaHS group, and the concentration of SOD was extremely high in the NaHS group. Utilized JC-1 to detect mitochondrial membrane potential and found that cells from the NaHS group had a stable potential while cells from the Mod group and PAG group partly lost their potential, which could demonstrate that NaHS could maintain mitochondrial membrane potential. The western blot results revealed that p-Drp1 had a significant decline in the NaHS group, which means mitochondria fission was decreased in the NaHS group. The expression level of Bax and caspase 3 was significantly lower than in the Mod group and PAG group, and the expression level of Bcl-and PRAP was significantly higher in the NaHS group. That could prove that NaHS protect HTR-8/SVneo cell by inhibiting cell apoptosis. These promising results show that H2S elicits its effects on cell apoptosis by decreasing ROS concentration, maintaining mitochondrial membrane stability, and promoting apoptosis-inhibiting protein expression in cells.

Combination of Type I and Type II MET Tyrosine Kinase Inhibitors as Therapeutic Approach to Prevent Resistance.

MET-targeted therapies are clinically effective in MET-amplified and MET exon 14 deletion mutant (METex14) non-small cell lung cancers (NSCLCs), but their efficacy is limited by the development of drug resistance. Structurally distinct MET tyrosine kinase inhibitors (TKIs) (type I/II) have been developed or are under clinical evaluation, which may overcome MET-mediated drug resistance mechanisms. In this study, we assess secondary MET mutations likely to emerge in response to treatment with single-agent or combinations of type I/type II MET TKIs using TPR-MET transformed Ba/F3 cell mutagenesis assays. We found that these inhibitors gave rise to distinct secondary MET mutant profiles. However, a combination of type I/II TKI inhibitors (capmatinib and merestinib) yielded no resistant clones in vitro The combination of capmatinib/merestinib was evaluated in vivo and led to a significant reduction in tumor outgrowth compared with either MET inhibitor alone. Our findings demonstrate in vitro and in vivo that a simultaneous treatment with a type I and type II MET TKI may be a clinically viable approach to delay and/or diminish the emergence of on target MET-mediated drug-resistance mutations.

Metaplastic breast carcinoma: a retrospective study of 26 cases.

Metaplastic breast carcinoma is a rare invasive breast cancer. Metaplastic breast carcinoma is mainly characterized by an epithelial or mesenchymal cell population mixed with adenocarcinoma. We collected 26 cases of metaplastic breast carcinoma in the First Affiliated Hospital of Bengbu Medical College from 2008 to 2014. Tumor size, tumor grade, vascular invasion, ER/PR status, histologic classification, and HER2/neu status were assessed for all cases and the literature was reviewed. Clinicopathologic characteristics of patients diagnosed with metaplastic breast carcinomas and its key points of differential diagnosis were discussed. All patients were female, with the median age of 50 years. The mean tumor size was 3.2 cm. 4 subtypes of metaplastic breast carcinomas were documented. Fibromatosis-like metaplastic carcinomas are typically characterized by wavy, intertwined, gentle spindle cells. When the tumor components are almost squamous cell carcinoma components and the primary squamous cell carcinoma of other organs and tissues are excluded, we can diagnose breast squamous cell carcinoma. In spindle cell carcinoma, atypical spindle cells are arranged in many ways and are usually accompanied by inflammatory cell infiltrate. Cancer with interstitial differentiation has mixed malignant epithelial and mesenchymal differentiation, and the mesenchymal components are diverse. Most tumors are triple negative. At present, surgical resection combined with chemotherapy or radiation therapy is the most effective and acceptable method for treating metaplastic breast carcinoma.

Catalysis by dihydrofolate reductase from the psychropiezophile Moritella profunda.

The influence of temperature and pH on the stability and catalytic activity of dihydrofolate reductase (MpDHFR) from the cold-adapted deep-sea bacterium Moritella profunda was studied. The thermal melting temperature was found to be ∼38 °C and was not affected by pH, while activity measurements demonstrated that its stability was maximal at pH 7 and was reduced dramatically below pH 6 or above pH 8. The steady-state rate constant (k(cat)) was maximal at neutral pH and higher temperatures, while the Michaelis constants (K(M)) for both substrate and cofactor were optimal at lower temperatures and at elevated or reduced pH. For both temperature and pH, any change in k(cat) was therefore offset by a similar change in K(M). Both the activation enthalpy and entropy of the MpDHFR-catalysed reaction were lower than those of DHFR from E. coli leading overall to a very small difference in activation free energy and therefore similar steady-state rate constants at the same temperature. The chemical step of the reaction is not rate limiting at pH 7, but becomes progressively more rate limiting as the pH increases. These results demonstrate adaptation of MpDHFR to its environment and show compromises between enthalpic and entropic contributions to the reaction, and between k(cat) and K(M).

Isolation and purification of macrocyclic components from Penicillium fermentation broth by high-speed counter-current chromatography.

In this paper, high-speed counter-current chromatography (HSCCC), assisted with ESI-MS, was first successfully applied to the preparative separation of three macrolide antibiotics, brefeldin A (12.6 mg, 99.0%), 7'-O-formylbrefeldin A (6.5 mg, 95.0%) and 7'-O-acetylbrefeldin A (5.0 mg, 92.3%) from the crude extract of the microbe Penicillium SHZK-15. Considering the chemical nature and partition coefficient (K) values of the three target compounds, a two-step HSCCC isolation protocol was developed in order to obtain products with high purity. In the two-step method, the crude ethyl acetate extract was first fractionated and resulted in two peak fractions by HSCCC using solvent system n-hexane/ethyl acetate/methanol/water (HEMWat) (3:7:5:5 v/v/v/v), then purified using solvent systems HEMWat (3:5:3:5 v/v/v/v) and HEMWat (7:3:5:5 v/v/v/v) for each fraction. The purities and structures of the isolated compounds were determined by HPLC, X-ray crystallography, ESI-MS and NMR. The results demonstrated that HSCCC is a fast and efficient technique for systematic isolation of bioactive compounds from the microbes.

Expression of AGGF1 and Twist1 in hepatocellular carcinoma and their correlation with vasculogenic mimicry.

BACKGROUND: The most common reason for hepatocellular carcinoma (HCC) treatment failure is recurrence and metastasis. AGGF1 (a promoting gene of tumor metastasis), vasculogenic mimicry (VM, new blood supply formation in malignant tumors), and Twist1 (an evolutionarily conserved basic helix-loop-helix transcription factor) are all valuable factors for metastasis and prognosis in diverse common human cancers. However, the correlation of AGGF1, Twist1, and VM in HCC is still unclear. In this study, we analyzed the correlations among these factors as well as their correlation with clinicopathologic data and survival in HCC. METHODS: Immunohistochemical (IHC) analysis was used to detect the expression of AGGF1 and Twist1 in 111 archival surgical specimens of human HCC. Furthermore, clinical data were collected. RESULTS: Levels of VM, AGGF1 and Twist1 were significantly higher in HCC tissues than in normal hepatic tissues. Levels of VM, AGGF1, and Twist1 were positively associated with AFP, HBsAg, size, capsular invasion, Child-Pugh classification level, and tumor node metastasis (TNM) stage, and negatively associated with patients' overall survival (OS). In multivariate analysis, high levels of VM, AGGF1, Twist1, AFP, Child-Pugh classification level, as well as TNM stage were independently correlated with lower OS in patients with HCC. CONCLUSION: VM and the expression of AGGF1 and Twist1 may represent promising metastatic and prognostic biomarkers, as well as therapeutic targets for HCC.