Structural Mass Spectrometry Captures Residue-Resolved Comprehensive Conformational Rearrangements of a G Protein-Coupled Receptor.

G protein-coupled receptor (GPCR) structural studies with in-solution spectroscopic approaches have offered distinctive insights into GPCR activation and signaling that highly complement those yielded from structural snapshots by crystallography or cryo-EM. While most current spectroscopic approaches allow for probing structural changes at selected residues or loop regions, they are not suitable for capturing a holistic view of GPCR conformational rearrangements across multiple domains. Herein, we develop an approach based on limited proteolysis mass spectrometry (LiP-MS) to simultaneously monitor conformational alterations of a large number of residues spanning both flexible loops and structured transmembrane domains for a given GPCR. To benchmark LiP-MS for GPCR conformational profiling, we studied the adenosine 2A receptor (A2AR) in response to different ligand binding (agonist/antagonist/allosteric modulators) and G protein coupling. Systematic and residue-resolved profiling of A2AR conformational rearrangements by LiP-MS precisely captures structural mechanisms in multiple domains underlying ligand engagement, receptor activation, and allostery, and may also reflect local conformational flexibility. Furthermore, these residue-resolution structural fingerprints of the A2AR protein allow us to readily classify ligands of different pharmacology and distinguish the G protein-coupled state. Thus, our study provides a new structural MS approach that would be generalizable to characterizing conformational transition and plasticity for challenging integral membrane proteins.

Integrated control strategy for dual sludge ages in the high-concentration powder carrier bio-fluidized bed (HPB) technology: Enhancing municipal wastewater treatment efficiency.

The high-concentration powder carrier bio-fluidized bed (HPB) technology is an emerging approach that enables on-site upgrading of wastewater treatment plants (WWTPs). HPB technology promotes the formation of biofilm sludge with micron-scale composite powder carriers as the core and suspended sludge mainly composed of flocs surrounding the biofilm sludge. This study proposed a novel integrated strategy for assessing and controlling the sludge ages in suspended/bio-film activated sludge supported by micron-scale composite powder carrier. Utilizing the cyclone unit and the corresponding theoretical model, the proposed strategy effectively addresses the sludge ages contradiction between denitrifying bacteria and polyphosphate-accumulating organisms (PAOs), thereby enhancing the efficiency of municipal wastewater treatment. The sludge age of the suspended (25 d) and bio-film (99 d) sludge, calculated using the model, contribute to the simultaneous removal of nitrogen and phosphorus. Meanwhile, the model further estimates distinct contributions of suspended and bio-film sludge to chemical oxygen demand (COD) and total nitrogen (TN), which are 55% and 42% for COD, 20% and 57% for TN of suspended sludge and bio-film sludge, respectively. This suggests that the contribution of suspended sludge and bio-film sludge to COD and TN removal efficiency can be determined and controlled by the operational conditions of the cyclone unit. Additionally, the simulation values for COD, ammonia nitrogen (NH4+-N), TN and total phosphorus (TP) closely align with the actual values of WWTPs over 70 days (p < 0.001) with the correlation coefficients (R2) of 0.9809, 0.9932, 0.9825, and 0.837, respectively. These results support the theoretical foundation of HPB technology for simultaneous nitrogen and phosphorus removal in sewage treatment plants. Therefore, this model serves as a valuable tool to guide the operation, design, and carrier addition in HPB technology implementation.

ARP2/3 Regulates Fatty Acid Synthesis by Modulating Lipid Droplets' Motility.

The breakdown of lipid droplets (LDs) provides energy and contributes to the proliferation and migration of cancer cells. Recent studies have suggested that motility plays a key role in LD breakdown. However, the molecular mechanisms underlying LD motility were poorly characterized. In this study, we examined the function of microfilament-associated proteins 2 and 3 (ARP2 and ARP3) in regulating LDs' motility in Hela cells. ARP2/3 mediated the LDs' physical contact with F-actin and promoted the recruitment of Myosin Heavy Chain 9 (MYH9). MYH9 regulated the LD content by binding with LDs and ARP2/3. The number of LDs and TG content was increased after MYH9 interfered. The genes related to FA-related genes and neutral lipid synthesis-related genes were significantly increased (p < 0.05) when ARP2 and ARP3 were overexpressed. Bioinformatic analysis indicated that the high expression of ARP2/3 was associated with a poorer prognosis in cervical squamous cell carcinoma (CSCC). This study showed the effect of cytoskeletal filaments on LD metabolism in cancer cells.

Herpes Simplex Virus 1 Infection of Tree Shrews Differs from That of Mice in the Severity of Acute Infection and Viral Transcription in the Peripheral Nervous System.

UNLABELLED: Studies of herpes simplex virus (HSV) infections of humans are limited by the use of rodent models such as mice, rabbits, and guinea pigs. Tree shrews (Tupaia belangeri chinensis) are small mammals indigenous to southwest Asia. At behavioral, anatomical, genomic, and evolutionary levels, tree shrews are much closer to primates than rodents are, and tree shrews are susceptible to HSV infection. Thus, we have studied herpes simplex virus 1 (HSV-1) infection in the tree shrew trigeminal ganglion (TG) following ocular inoculation. In situ hybridization, PCR, and quantitative reverse transcription-PCR (qRT-PCR) analyses confirm that HSV-1 latently infects neurons of the TG. When explant cocultivation of trigeminal ganglia was performed, the virus was recovered after 5 days of cocultivation with high efficiency. Swabbing the corneas of latently infected tree shrews revealed that tree shrews shed virus spontaneously at low frequencies. However, tree shrews differ significantly from mice in the expression of key HSV-1 genes, including ICP0, ICP4, and latency-associated transcript (LAT). In acutely infected tree shrew TGs, no level of ICP4 was observed, suggesting the absence of infection or a very weak, acute infection compared to that of the mouse. Immunofluorescence staining with ICP4 monoclonal antibody, and immunohistochemistry detection by HSV-1 polyclonal antibodies, showed a lack of viral proteins in tree shrew TGs during both acute and latent phases of infection. Cultivation of supernatant from homogenized, acutely infected TGs with RS1 cells also exhibited an absence of infectious HSV-1 from tree shrew TGs. We conclude that the tree shrew has an undetectable, or a much weaker, acute infection in the TGs. Interestingly, compared to mice, tree shrew TGs express high levels of ICP0 transcript in addition to LAT during latency. However, the ICP0 transcript remained nuclear, and no ICP0 protein could be seen during the course of mouse and tree shrew TG infections. Taken together, these observations suggest that the tree shrew TG infection differs significantly from the existing rodent models. IMPORTANCE: Herpes simplex viruses (HSVs) establish lifelong infection in more than 80% of the human population, and their reactivation leads to oral and genital herpes. Currently, rodent models are the preferred models for latency studies. Rodents are distant from primates and may not fully represent human latency. The tree shrew is a small mammal, a prosimian primate, indigenous to southwest Asia. In an attempt to further develop the tree shrew as a useful model to study herpesvirus infection, we studied the establishment of latency and reactivation of HSV-1 in tree shrews following ocular inoculation. We found that the latent virus, which resides in the sensory neurons of the trigeminal ganglion, could be stress reactivated to produce infectious virus, following explant cocultivation and that spontaneous reactivation could be detected by cell culture of tears. Interestingly, the tree shrew model is quite different from the mouse model of HSV infection, in that the virus exhibited only a mild acute infection following inoculation with no detectable infectious virus from the sensory neurons. The mild infection may be more similar to human infection in that the sensory neurons continue to function after herpes reactivation and the affected skin tissue does not lose sensation. Our findings suggest that the tree shrew is a viable model to study HSV latency.

High-Entropy Lithium Niobate Nanocubes for Photocatalytic Water Splitting under Visible Light.

The vast compositional space available in high-entropy oxide semiconductors offers unique opportunities for electronic band structure engineering in an unprecedented large room. In this work, with wide band gap semiconductor lithium niobate (LiNbO3) as a model system, we show that the substitutional addition of high-entropy metal cation mixtures within the Nb sublattice can lead to the formation of a single-phase solid solution featuring a substantially narrowed band gap and intense broadband visible light absorption. The resulting high-entropy LiNbO3 [denoted as Li(HE)O3] crystallizes as well-faceted nanocubes; atomic-resolution imaging and elemental mapping via transmission electron microscopy unveil a distinct local chemical complexity and lattice distortion, characteristics of high-entropy stabilized solid solution phases. Because of the presence of high-entropy stabilized Co2+ dopants that serve as active catalytic sites, Li(HE)O3 nanocubes can accomplish the visible light-driven photocatalytic water splitting in an aqueous solution containing methanol as a sacrificial electron donor without the need of any additional co-catalysts.

Synthetic microbial consortia to enhance the biodegradation of compost odor by biotrickling filter.

Composting generates odorous gases, including ammonia (NH3), hydrogen sulfide (H2S), and volatile organic compounds (VOCs). The Biological Trickling Filter (BTF) is effective for odor treatment, but it may have limitations with hydrophobic VOCs. In this study, a strain of Bacillus subtilis with ammonia-reducing ability, a strain of Bacillus cereus with desulfurization ability and a strain of Schizophyllum commune with the ability to degrade dimethyl disulfide were isolated and screened. The three strains were combined to create synthetic microbial consortia for enhancing odor treatment in the BTF. Compared to the activated sludge control, the BTF with synthetic microbial consortia removed 92.43% ammonia, 92.75% hydrogen sulfide. Furthermore, it demonstrated a significant improvement in the removal rates of p-methyl mercaptan, methyl sulfide, and dimethyl disulfide. High-throughput sequencing was conducted on the fillers of the synthetic microbial consortia-inoculated BTF to analyze the microbial community composition.

Enhanced biological phosphorus removal by high concentration powder carrier bio-fluidized bed (HPB): Phosphorus distribution, cyclone separation, and metagenomics.

This study provides a comparative investigation of phosphorus removal between anaerobic-anoxic-oxic (AAO) and high-concentration powder carrier bio-fluidized bed (HPB) in the same full-scale wastewater treatment plant. The results showed that the total phosphorus removal of HPB was 71.45%-96.71%. Compared with AAO, the total phosphorus removal of HPB can be increased by a maximum of 15.73%. The mechanisms of enhanced phosphorus removal by HPB include the followings. Biological phosphorus removal was significant. The anaerobic phosphorus release capacity of HPB was enhanced and polyphosphate (Poly-P) in the excess sludge of HPB was 1.5 times higher than that of AAO. The relative abundance of Candidatus Accumulibacter was 5 times higher than that of AAO, and oxidative phosphorylation and butanoate metabolism were enhanced. The analysis of phosphorus distribution showed that cyclone separation increased the chemical phosphorus precipitation (Chem-P) in the excess sludge by 16.96% to avoid accumulation in the biochemical tank. The phosphorus adsorbed by extracellular polymeric substance (EPS) in the recycled sludge was stripped, and the EPS bound-P in the excess sludge increased by 1.5 times. This study demonstrated the feasibility of HPB to improve the phosphorus removal efficiency for domestic wastewater.

Adsorption, mobility, and degradation of the pesticide propaquizafop in five agricultural soils in China.

Propaquizafop is a fatty acid synthetic herbicide used to control annual and perennial grasses. To understand the potential environmental risks of propaquizafop to crops and food safety, the adsorption, mobility, and degradation of propaquizafop in five different soils were studied. At an initial concentration of 5 mg L-1 propaquizafop, its adsorption equilibrium was reached within 24 h, and the adsorption rates were between 46.98 and 57.76%. The Elovich kinetic model provided the best fit for the kinetic model, with R2 values between 0.9882 and 0.9940. For the isothermal adsorption tests, the Freundlich model was used to better fit the adsorption characteristics of propaquizafop in different soils, with R2 values between 0.9748 and 0.9885. Increasing the concentration of Ca2+ was beneficial for propaquizafop adsorption. In the soil thin-layer chromatography tests, the Rf of propaquizafop in the five soil samples ranged from 0.076 to 0.123. The results of the soil column leaching tests showed that propaquizafop did not migrate in the five soil columns; it was not detected in the leachate of each soil column, and propaquizafop in the soil columns only existed in the 0-5 cm soil layer. The results of soil thin-layer chromatography and soil column leaching tests showed that propaquizafop is a pesticide with a weak migration ability. Under the same environmental conditions, the degradation rate of propaquizafop in different soils followed the order LF fluvo-aquic soil (T1/2 = 1.41 d) > CS red loam (T1/2 = 2.76 d) > SX paddy soil (T1/2 = 3.52 d) > CC black soil (T1/2 = 5.74 d) > BS ginseng soil (T1/2 = 7.75 d). Considering the effects of soil moisture, incubation temperature, and microorganisms on propaquizafop degradation in the soil, temperature was found to have the greatest influence on its degradation rate.

Modified integrated fixed-film activated sludge process: Advanced nitrogen removal for low-C/N domestic wastewater.

Actual low-C/N domestic wastewater was treated using the high-concentration powder carrier bio-fluidized bed (HPB) process comparing diatomite and Fe-C as the carriers. The total nitrogen removal efficiencies were increased from 50.08% to 65.40% and 78.58%, respectively. The diatomite HPB process increased the relative abundance of autotrophic N-cycle bacteria to more than twofold and the sludge size. Therefore, the contributions for nitrogen removal by anammox and simultaneous nitrification-denitrification were increased. The Fe-C HPB process improved the nitrogen removal efficiency mainly by increasing the biodegradability and activities of electron transfer system and key enzymes. The key device (hydrocyclone separator) of the HPB process significantly improved the recovery efficiency of the carriers. It also improved the capacity of microbial aggregations for adsorbing pollutants. Furthermore, it reduced the relative abundance of filamentous bacteria. This study demonstrated the feasibility and mechanism of the HPB process for improving the nitrogen removal efficiency for low-C/N wastewater.

Effect of transjugular intrahepatic portosystemic shunt combined with 125I particle implantation on portal vein tumor thrombus in hepatocellular carcinoma.

OBJECTIVE: The efficacy of transjugular intrahepatic portosystemic shunt (TIPS) combined with 125I particle implantation in the treatment of portal vein tumor thrombus (PVTT) in hepatocellular carcinoma was discussed and analyzed in this study. METHODS: A total of 127 patients with primary hepatocellular carcinoma (PHC) complicated with PVTT admitted to our hospital from March 2017 to June 2018 were enrolled. The patients were classified into an observation group (n=69) and a control group (n=58) in the light of the different treatment methods. The control group patients were treated with TIPS alone, and the observation group patients received 125I particle implantation on the basis of TIPS in the control group. Subsequently, the clinical therapeutic efficacy, perioperative indicators, postoperative complications, quality of life and survival of patients before and after treatment were compared between the two groups. RESULTS: The remission rate in the observation group was remarkably higher than that of the control group (P<0.05), and the difference in the overall response rate (ORR) of the two groups of patients was not statistically significant (P>0.05). The AFP, PLT, WBC and the diameter of the main portal vein in the two groups dropped substantially compared to those before treatment (P<0.05), and the AFP and the diameter of the main portal vein in the observation group were notably lower than those in the control group (P<0.05). After treatment, the ALT, AST and TBiL of the two groups were remarkably higher than those before treatment (P<0.05), and these indicators in the observation group were apparently higher than those in the control group (P<0.05). There was no significant difference in the incidence of postoperative gastrointestinal bleeding, fever, granulocytopenia and abnormal hepatic dysfunction between the observation group and the control group (P>0.05). The functional assessment of cancer therapy-hepatobiliary (FACT-Hep) scores of the two groups 6 months after operation was substantially lower than pre-op scores (P<0.05), and the observation group had apparently lower postoperative scores than the control group (P<0.05). The progression-free survival (PFS) and overall survival (OS) in the observation group were critically superior to those in the control group (P<0.05). CONCLUSION: TIPS combined with 125I particle implantation in the treatment of PHC patients with PVTT can help improve patients' clinical treatment efficacy after surgery while prolonging their postoperative survival. The treatment is safe and worthy of clinical promotion.

Pt1 enhanced C-H activation synergistic with Ptn catalysis for glycerol cascade oxidation to glyceric acid.

The selective oxidation of glycerol to glyceric acid, an important value-added reaction from polyols, is a typical cascade catalytic process. It is still of great challenge to simultaneously achieve high glycerol activity and glyceric acid selectivity, suffering from either deep oxidation and C-C cleavage or poor oxidation efficiency from glyceraldehyde to glyceric acid. Herein, this work, inspired by nature, proposes a cascade synergistic catalysis strategy by atomic and low-coordinated cluster Pt on well-defined Cu-CuZrOx, which involves enhanced C-H activation on atomic Pt1 and O-H activation on cluster Ptn in the oxidation of glycerol to glyceraldehyde, and cluster Ptn for C=O activation followed by O-H insertion and atomic Pt1 for C-H activation in the tandem oxidation of glyceraldehyde to glyceric acid. The enhanced C-H activation in the cascade process by atomic Pt1 is revealed to be essential for the high glycerol activity (90.0±0.1%) and the glyceric acid selectivity (80.2±0.2%).

CTCF interacts with the lytic HSV-1 genome to promote viral transcription.

CTCF is an essential chromatin regulator implicated in important nuclear processes including in nuclear organization and transcription. Herpes Simplex Virus-1 (HSV-1) is a ubiquitous human pathogen, which enters productive infection in human epithelial and many other cell types. CTCF is known to bind several sites in the HSV-1 genome during latency and reactivation, but its function has not been defined. Here, we report that CTCF interacts extensively with the HSV-1 DNA during lytic infection by ChIP-seq, and its knockdown results in the reduction of viral transcription, viral genome copy number and virus yield. CTCF knockdown led to increased H3K9me3 and H3K27me3, and a reduction of RNA pol II occupancy on viral genes. Importantly, ChIP-seq analysis revealed that there is a higher level of CTD Ser2P modified RNA Pol II near CTCF peaks relative to the Ser5P form in the viral genome. Consistent with this, CTCF knockdown reduced the Ser2P but increased Ser5P modified forms of RNA Pol II on viral genes. These results suggest that CTCF promotes HSV-1 lytic transcription by facilitating the elongation of RNA Pol II and preventing silenced chromatin on the viral genome.

Histone Lysine Methylation in TGF-ß1 Mediated p21 Gene Expression in Rat Mesangial Cells.

Transforming growth factor beta1- (TGF-ß1-) induced p21-dependent mesangial cell (MC) hypertrophy plays a key role in the pathogenesis of chronic renal diseases including diabetic nephropathy (DN). Increasing evidence demonstrated the role of posttranscriptional modifications (PTMs) in the event; however, the precise regulatory mechanism of histone lysine methylation remains largely unknown. Here, we examined the roles of both histone H3 lysine 4 and lysine 9 methylations (H3K4me/H3K9me) in TGF-ß1 induced p21 gene expression in rat mesangial cells (RMCs). Our results indicated that TGF-ß1 upregulated the expression of p21 gene in RMCs, which was positively correlated with the increased chromatin marks associated with active genes (H3K4me1/H3K4me2/H3K4me3) and negatively correlated with the decreased levels of repressive marks (H3K9me2/H3K9me3) at p21 gene promoter. TGF-ß1 also elevated the recruitment of the H3K4 methyltransferase (HMT) SET7/9 to the p21 gene promoter. SET7/9 gene silencing with small interfering RNAs (siRNAs) significantly abolished the TGF-ß1 induced p21 gene expression. Taken together, these results reveal the key role of histone H3Kme in TGF-ß1 mediated p21 gene expression in RMC, partly through HMT SET7/9 occupancy, suggesting H3Kme and SET7/9 may be potential renoprotective agents in managing chronic renal diseases.

Clinicopathological characteristics of non-diabetic renal disease in patients with type 2 diabetes mellitus in a northeastern Chinese medical center: a retrospective analysis of 273 cases.

OBJECTIVE: To investigate the clinical and histopathological features of non-diabetic renal disease (NDRD) superimposed on diabetic nephropathy (DN) in northeastern Chinese patients with type 2 diabetes mellitus (T2D), and compare the changes with those of pure DN and isolated NDRD. METHODS: Single-center retrospective analysis based on medical records of 273 patients (172 men, mean age: 51.1 ± 12.4 years) with T2D who underwent renal biopsy between February 2000 and October 2015. All patients were diagnosed as cases of pure DN, isolated NDRD or NDRD superimposed on DN. RESULTS: Out of the 273 T2D patients, 68 (24.9 %) had DN, 175 (64.1 %) had NDRD, and 30 (11.0 %) had NDRD superimposed on DN. Idiopathic membranous nephropathy (IMN, 29.7 %) was the most common NDRD followed by IgA nephropathy (IgAN, 22.9 %), and hypertensive renal arteriolar sclerosis was the most common lesion in patients diagnosed as NDRD superimposed on DN. Patients with NDRD had a shorter duration of diabetes and lower frequencies of diabetic retinopathy (DR, 6.9 %) and renal failure (28.0 %), which is consistent with higher estimated glomerular filtration rates (eGFR) and lower systolic blood pressure (SBP). No significant between-group differences were observed with respect to proteinuria and hematuria. CONCLUSION: Renal biopsy is strongly recommended for T2D patients to distinguish DN, NDRD and NDRD superimposed on DN, especially in patients with no signs of DR. This approach may help in early diagnosis and treatment of NDRD and improve renal outcomes in northeastern Chinese T2D patients.

Involvement of Histone Lysine Methylation in p21 Gene Expression in Rat Kidney In Vivo and Rat Mesangial Cells In Vitro under Diabetic Conditions.

Diabetic nephropathy (DN), a common complication associated with type 1 and type 2 diabetes mellitus (DM), characterized by glomerular mesangial expansion, inflammation, accumulation of extracellular matrix (ECM) protein, and hypertrophy, is the major cause of end-stage renal disease (ESRD). Increasing evidence suggested that p21-dependent glomerular and mesangial cell (MC) hypertrophy play key roles in the pathogenesis of DN. Recently, posttranscriptional modifications (PTMs) have uncovered novel molecular mechanisms involved in DN. However, precise regulatory mechanism of histone lysine methylation (HKme) mediating p21 related hypertrophy associated with DN is not clear. We evaluated the roles of HKme and histone methyltransferase (HMT) SET7/9 in p21 gene expression in glomeruli of diabetic rats and in high glucose- (HG-) treated rat mesangial cells (RMCs). p21 gene expression was upregulated in diabetic rats glomeruli; chromatin immunoprecipitation (ChIP) assays showed decreased histone H3-lysine9-dimethylation (H3K9me2) accompanied with enhanced histone H3-lysine4-methylation (H3K4me1/3) and SET7/9 occupancies at the p21 promoter. HG-treated RMCs exhibited increased p21 mRNA, H3K4me level, SET7/9 recruitment, and inverse H3K9me, which were reversed by TGF-ß1 antibody. These data uncovered key roles of H3Kme and SET7/9 responsible for p21 gene expression in vivo and in vitro under diabetic conditions and confirmed preventive effect of TGF-ß1 antibody on DN.

Selective recruitment of host factors by HSV-1 replication centers.

Herpes simplex virus type 1 (HSV-1) enters productive infection after infecting epithelial cells, where it controls the host nucleus to make viral proteins, starts viral DNA synthesis and assembles infectious virions. In this process, replicating viral genomes are organized into replication centers to facilitate viral growth. HSV-1 is known to use host factors, including host chromatin and host transcription regulators, to transcribe its genes; however, the invading virus also encounters host defense and stress responses to inhibit viral growth. Recently, we found that HSV-1 replication centers recruit host factor CTCF but exclude γH2A.X. Thus, HSV-1 replication centers may selectively recruit cellular factors needed for viral growth, while excluding host factors that are deleterious for viral transcription or replication. Here we report that the viral replication centers selectively excluded modified histone H3, including heterochromatin mark H3K9me3, H3S10P and active chromatin mark H3K4me3, but not unmodified H3. We found a dynamic association between the viral replication centers and host RNA polymerase II. The centers also recruited components of the DNA damage response pathway, including 53BP1, BRCA1 and host antiviral protein SP100. Importantly, we found that ATM kinase was needed for the recruitment of CTCF to the viral centers. These results suggest that the HSV-1 replication centers took advantage of host signaling pathways to actively recruit or exclude host factors to benefit viral growth.