An Acquired Vulnerability of Drug-Resistant Melanoma with Therapeutic Potential.

BRAF(V600E) mutant melanomas treated with inhibitors of the BRAF and MEK kinases almost invariably develop resistance that is frequently caused by reactivation of the mitogen activated protein kinase (MAPK) pathway. To identify novel treatment options for such patients, we searched for acquired vulnerabilities of MAPK inhibitor-resistant melanomas. We find that resistance to BRAF+MEK inhibitors is associated with increased levels of reactive oxygen species (ROS). Subsequent treatment with the histone deacetylase inhibitor vorinostat suppresses SLC7A11, leading to a lethal increase in the already-elevated levels of ROS in drug-resistant cells. This causes selective apoptotic death of only the drug-resistant tumor cells. Consistently, treatment of BRAF inhibitor-resistant melanoma with vorinostat in mice results in dramatic tumor regression. In a study in patients with advanced BRAF+MEK inhibitor-resistant melanoma, we find that vorinostat can selectively ablate drug-resistant tumor cells, providing clinical proof of concept for the novel therapy identified here.

Author Correction: Tissue patrol by resident memory CD8+ T cells in human skin.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Tissue patrol by resident memory CD8+ T cells in human skin.

Emerging data show that tissue-resident memory T (TRM) cells play an important protective role at murine and human barrier sites. TRM cells in the epidermis of mouse skin patrol their surroundings and rapidly respond when antigens are encountered. However, whether a similar migratory behavior is performed by human TRM cells is unclear, as technology to longitudinally follow them in situ has been lacking. To address this issue, we developed an ex vivo culture system to label and track T cells in fresh skin samples. We validated this system by comparing in vivo and ex vivo properties of murine TRM cells. Using nanobody labeling, we subsequently demonstrated in human ex vivo skin that CD8+ TRM cells migrated through the papillary dermis and the epidermis, below sessile Langerhans cells. Collectively, this work allows the dynamic study of resident immune cells in human skin and provides evidence of tissue patrol by human CD8+ TRM cells.

Identification of an alternative triglyceride biosynthesis pathway.

Triacylglycerols (TAGs) are the main source of stored energy in the body, providing an important substrate pool for mitochondrial beta-oxidation. Imbalances in the amount of TAGs are associated with obesity, cardiac disease and various other pathologies1,2. In humans, TAGs are synthesized from excess, coenzyme A-conjugated fatty acids by diacylglycerol O-acyltransferases (DGAT1 and DGAT2)3. In other organisms, this activity is complemented by additional enzymes4, but whether such alternative pathways exist in humans remains unknown. Here we disrupt the DGAT pathway in haploid human cells and use iterative genetics to reveal an unrelated TAG-synthesizing system composed of a protein we called DIESL (also known as TMEM68, an acyltransferase of previously unknown function) and its regulator TMX1. Mechanistically, TMX1 binds to and controls DIESL at the endoplasmic reticulum, and loss of TMX1 leads to the unconstrained formation of DIESL-dependent lipid droplets. DIESL is an autonomous TAG synthase, and expression of human DIESL in Escherichia coli endows this organism with the ability to synthesize TAG. Although both DIESL and the DGATs function as diacylglycerol acyltransferases, they contribute to the cellular TAG pool under specific conditions. Functionally, DIESL synthesizes TAG at the expense of membrane phospholipids and maintains mitochondrial function during periods of extracellular lipid starvation. In mice, DIESL deficiency impedes rapid postnatal growth and affects energy homeostasis during changes in nutrient availability. We have therefore identified an alternative TAG biosynthetic pathway driven by DIESL under potent control by TMX1.

Mammalian life depends on two distinct pathways of DNA damage tolerance.

DNA damage threatens genomic integrity and instigates stem cell failure. To bypass genotoxic lesions during replication, cells employ DNA damage tolerance (DDT), which is regulated via PCNA ubiquitination and REV1. DDT is conserved in all domains of life, yet its relevance in mammals remains unclear. Here, we show that inactivation of both PCNA-ubiquitination and REV1 results in embryonic and adult lethality, and the accumulation of DNA damage in hematopoietic stem and progenitor cells (HSPCs) that ultimately resulted in their depletion. Our results reveal the crucial relevance of DDT in the maintenance of stem cell compartments and mammalian life in unperturbed conditions.

A semiparametric Gaussian mixture model for chest CT-based 3D blood vessel reconstruction.

Computed tomography (CT) has been a powerful diagnostic tool since its emergence in the 1970s. Using CT data, 3D structures of human internal organs and tissues, such as blood vessels, can be reconstructed using professional software. This 3D reconstruction is crucial for surgical operations and can serve as a vivid medical teaching example. However, traditional 3D reconstruction heavily relies on manual operations, which are time-consuming, subjective, and require substantial experience. To address this problem, we develop a novel semiparametric Gaussian mixture model tailored for the 3D reconstruction of blood vessels. This model extends the classical Gaussian mixture model by enabling nonparametric variations in the component-wise parameters of interest according to voxel positions. We develop a kernel-based expectation-maximization algorithm for estimating the model parameters, accompanied by a supporting asymptotic theory. Furthermore, we propose a novel regression method for optimal bandwidth selection. Compared to the conventional cross-validation-based (CV) method, the regression method outperforms the CV method in terms of computational and statistical efficiency. In application, this methodology facilitates the fully automated reconstruction of 3D blood vessel structures with remarkable accuracy.

Correction of Ito in human induced pluripotent stem Cell-derived cardiomyocyte carrying DPP6 mutation in early repolarization syndrome by CRISPR/Cas9 genome editing.

Early repolarization syndrome (ERS) is defined as occurring in patients with early repolarization pattern who have survived idiopathic ventricular fibrillation with clinical evaluation unrevealing for other explanations. The pathophysiologic basis of the ERS is currently uncertain. The objective of the present study was to examine the electrophysiological mechanism of ERS utilizing induced pluripotent stem cells (iPSCs) and CRISPR/Cas9 genome editing. Whole genome sequencing was used to identify the DPP6 (c.2561T > C/p.L854P) variant in four families with sudden cardiac arrest induced by ERS. Cardiomyocytes were generated from iPSCs from a 14-year-old boy in the four families with ERS and an unrelated healthy control subject. Patch clamp recordings revealed more significant prolongation of the action potential duration (APD) and increased transient outward potassium current (Ito) (103.97 ± 18.73 pA/pF vs 44.36 ± 16.54 pA/pF at +70 mV, P < 0.05) in ERS cardiomyocytes compared with control cardiomyocytes. Of note, the selective correction of the causal variant in iPSC-derived cardiomyocytes using CRISPR/Cas9 gene editing normalized the Ito, whereas prolongation of the APD remained unchanged. ERS cardiomyocytes carrying DPP6 mutation increased Ito and lengthen APD, which maybe lay the electrophysiological foundation of ERS.

Integration of multidimensional splicing data and GWAS summary statistics for risk gene discovery.

A common strategy for the functional interpretation of genome-wide association study (GWAS) findings has been the integrative analysis of GWAS and expression data. Using this strategy, many association methods (e.g., PrediXcan and FUSION) have been successful in identifying trait-associated genes via mediating effects on RNA expression. However, these approaches often ignore the effects of splicing, which can carry as much disease risk as expression. Compared to expression data, one challenge to detect associations using splicing data is the large multiple testing burden due to multidimensional splicing events within genes. Here, we introduce a multidimensional splicing gene (MSG) approach, which consists of two stages: 1) we use sparse canonical correlation analysis (sCCA) to construct latent canonical vectors (CVs) by identifying sparse linear combinations of genetic variants and splicing events that are maximally correlated with each other; and 2) we test for the association between the genetically regulated splicing CVs and the trait of interest using GWAS summary statistics. Simulations show that MSG has proper type I error control and substantial power gains over existing multidimensional expression analysis methods (i.e., S-MultiXcan, UTMOST, and sCCA+ACAT) under diverse scenarios. When applied to the Genotype-Tissue Expression Project data and GWAS summary statistics of 14 complex human traits, MSG identified on average 83%, 115%, and 223% more significant genes than sCCA+ACAT, S-MultiXcan, and UTMOST, respectively. We highlight MSG's applications to Alzheimer's disease, low-density lipoprotein cholesterol, and schizophrenia, and found that the majority of MSG-identified genes would have been missed from expression-based analyses. Our results demonstrate that aggregating splicing data through MSG can improve power in identifying gene-trait associations and help better understand the genetic risk of complex traits.

Serum electrolyte concentrations and risk of atrial fibrillation: an observational and mendelian randomization study.

BACKGROUND: Atrial fibrillation (AF) is a prevalent arrhythmic condition resulting in increased stroke risk and is associated with high mortality. Electrolyte imbalance can increase the risk of AF, where the relationship between AF and serum electrolytes remains unclear. METHODS: A total of 15,792 individuals were included in the observational study, with incident AF ascertainment in the Atherosclerosis Risk in Communities (ARIC) study. The Cox regression models were applied to calculate the hazard ratio (HR) and 95% confidence interval (CI) for AF based on different serum electrolyte levels. Mendelian randomization (MR) analyses were performed to examine the causal association. RESULTS: In observational study, after a median 19.7 years of follow-up, a total of 2551 developed AF. After full adjustment, participants with serum potassium below the 5th percentile had a higher risk of AF relative to participants in the middle quintile. Serum magnesium was also inversely associated with the risk of AF. An increased incidence of AF was identified in individuals with higher serum phosphate percentiles. Serum calcium levels were not related to AF risk. Moreover, MR analysis indicated that genetically predicted serum electrolyte levels were not causally associated with AF risk. The odds ratio for AF were 0.999 for potassium, 1.044 for magnesium, 0.728 for phosphate, and 0.979 for calcium, respectively. CONCLUSIONS: Serum electrolyte disorders such as hypokalemia, hypomagnesemia and hyperphosphatemia were associated with an increased risk of AF and may also serve to be prognostic factors. However, the present study did not support serum electrolytes as causal mediators for AF development.

Diversifying the anthracycline class of anti-cancer drugs identifies aclarubicin for superior survival of acute myeloid leukemia patients.

The efficacy of anthracycline-based chemotherapeutics, which include doxorubicin and its structural relatives daunorubicin and idarubicin, remains almost unmatched in oncology, despite a side effect profile including cumulative dose-dependent cardiotoxicity, therapy-related malignancies and infertility. Detoxifying anthracyclines while preserving their anti-neoplastic effects is arguably a major unmet need in modern oncology, as cardiovascular complications that limit anti-cancer treatment are a leading cause of morbidity and mortality among the 17 million cancer survivors in the U.S. In this study, we examined different clinically relevant anthracycline drugs for a series of features including mode of action (chromatin and DNA damage), bio-distribution, anti-tumor efficacy and cardiotoxicity in pre-clinical models and patients. The different anthracycline drugs have surprisingly individual efficacy and toxicity profiles. In particular, aclarubicin stands out in pre-clinical models and clinical studies, as it potently kills cancer cells, lacks cardiotoxicity, and can be safely administered even after the maximum cumulative dose of either doxorubicin or idarubicin has been reached. Retrospective analysis of aclarubicin used as second-line treatment for relapsed/refractory AML patients showed survival effects similar to its use in first line, leading to a notable 23% increase in 5-year overall survival compared to other intensive chemotherapies. Considering individual anthracyclines as distinct entities unveils new treatment options, such as the identification of aclarubicin, which significantly improves the survival outcomes of AML patients while mitigating the treatment-limiting side-effects. Building upon these findings, an international multicenter Phase III prospective study is prepared, to integrate aclarubicin into the treatment of relapsed/refractory AML patients.

A Portable Microplasma Optical Emission Spectrometric Device with Online Digestion Function for Field and Sensitive Determination of Lead in Biological Samples.

Accurate detection and screening of Pb in biological samples is helpful to assess the risk associated with lead pollution to human health. However, conventional atomic spectroscopic instruments are bulky and cumbersome, requiring additional sample pretreatment equipment, and difficult to perform field analysis with. Herein, a portable point discharge (PD) microplasma-optical emission spectrometric (OES) device with online digestion function is designed for field and sensitive determination of lead in biological samples. With rice as a model, online digestion of a batch of six 50 mg samples can be achieved in the HNO3 and H2O2 system within 25 min by a temperature control and timing module. Compared to the conventional microwave digestion, the digestion efficiency of this device reaches 97%. Pb in digestion solution is converted into volatile species by hydride generation (HG) and directly introduced into PD-OES for excitation and detection by a self-designed rotatable and telescopic cutoff gas sampling column. Six samples can be successively detected in 2 min, and argon consumption of the whole process is only <800 mL. Under the optimized conditions, the detection limit of Pb is 0.018 mg kg-1 (0.9 µg L-1) and precision is 3.6%. The accuracy and practicability of the present device are verified by measuring several certified reference materials and real biological samples. By virtue of small size (23.5 × 17 × 8.5 cm3), lightweight (2.5 kg), and low energy consumption (24.3 W), the present device provides a convenient tool for field analysis of toxic elements in biological samples.

Dynamic Stomach Model-Capillary Electrophoresis-ICPMS for Evaluation of Release and Transformation Behaviors of Arsenic Species from Microplastics during Digestion.

Microplastics (MPs) can act as carriers of environmental arsenic species into the stomach with food and release arsenic species during digestion, which threatens human health. Herein, an integrated dynamic stomach model (DSM)-capillary electrophoresis-inductively coupled plasma mass spectrometry (CE-ICPMS) is developed for online monitoring of the release and transformation behaviors of arsenic species loaded on MPs (As-MPs) in the simulated human stomach. The 3D-printed DSM with a soft stomach chamber enables the behaviors of gastric peristalsis, gastric and salivary fluid addition, pH adjustment, and gastric emptying (GE) to be controlled by a self-written program after oral ingestion of food with As-MPs. The gastric extract during digestion is introduced into the spiral channel to remove the large particulate impurity and online filtered to obtain the clarified arsenic-containing solution for subsequent speciation analysis of arsenic by CE-ICPMS. The digestion conditions and pretreatment processes of DSM are tracked and validated, and the release rates of As-MPs digested by DSM are compared with those digested by the static stomach model and DSM without GE. The release rate of inorganic arsenic on MPs is higher than that of organic arsenic throughout the gastric digestion process, and 8% of As(V) is reduced to As(III). The detection limits for As(III), DMA, MMA, and As(V) are 0.5-0.9 µg L-1 using DSM-CE-ICPMS, along with precisions of ≤8%. This present method provides an integrated and convenient tool for evaluating the release and transformation of As-MPs during human gastric digestion and provides a reference for exploring the interactions between MPs and metals/metalloids in the human body.

Ubiquitination-dependent degradation of nucleolin mediated by porcine circovirus type 3 capsid protein.

IMPORTANCE: Porcine circovirus type 3 (PCV3) is an emerging pathogen that causes multisystem disease in pigs and poses a severe threat to the swine industry. However, the mechanisms of how PCV3 uses host proteins to regulate its own life cycle are not well understood. In this study, we found that PCV3 capsid protein interacts with nucleolin and degrades it. Degradation of nucleolin by the PCV3 capsid protein requires recruitment of the enzyme RNF34, which is transported to the nucleolus from the cytoplasm in the presence of the PCV3 capsid protein. Nucleolin also decreases PCV3 replication by promoting the release of interferon ß. These findings clarify the mechanism by which nucleolin modulates PCV3 replication in cells, thereby facilitating to provide an important strategy for preventing and controlling PCV3 infection.

The antimicrobial property of JY-1, a complex mixture of Traditional Chinese Medicine, is linked to it abilities to suppress biofilm formation and disrupt membrane permeability.

The substantial increase of infections, caused by novel, sudden, and drug-resistant pathogens, poses a significant threat to human health. While numerous studies have demonstrated the antibacterial and antiviral effects of Traditional Chinese Medicine, the potential of a complex mixture of traditional Chinese Medicine with a broad-spectrum antimicrobial property remains underexplored. This study aimed to develop a complex mixture of Traditional Chinese Medicine (TCM), JY-1, and investigate its antimicrobial properties, along with its potential mechanism of action against pathogenic microorganisms. Antimicrobial activity was assessed using a zone of inhibition assay and the drop plate method. Hyphal induction of Candida albicans was conducted using RPMI1640 medium containing 10% FBS, followed by microscopic visualization. Quantitative real-time PCR (RT-qPCR) was employed to quantify the transcript levels of hyphal-specific genes such as HWP1 and ALS3. The impact of JY-1 on biofilm formation was evaluated using both the XTT reduction assay and scanning electron microscopy (SEM). Furthermore, the cell membrane integrity was assessed by protein and nucleic acid leakage assays. Our results clearly showed that JY-1 significantly inhibits the vegetative growth of Candida spp. and Cryptococcus spp. In addition, this complex mixture is effectively against a wide range of pathogenic bacteria, including Staphylococcus aureus, Vancomycin-resistant enterococci, Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae. More interestingly, JY-1 plays a direct anti-viral role against the mammalian viral pathogen vesicular stomatitis virus (VSV). Further mechanistic studies indicate that JY-1 acts to reduce the expression of hyphal specific genes HWP1 and ALS3, resulting in the suppression of the hyphal formation of C. albicans. The antimicrobial property of JY-1 could be attributed to its ability to reduce biofilm formation and disrupt the cell membrane permeability, a process resulting in microbial cell death and the release of cellular contents. Taken together, our work identified a potent broad-spectrum antimicrobial agent, a complex mixture of TCM which might be developed as a potential antimicrobial drug.

Regulating Excited States by Varying Different Acceptors of D-π-A Emitters for Efficient Non-Doped Blue Electroluminescence with High Luminance and Low Efficiency Roll-Off.

Chromophores with hybridized local and charge-transfer (HLCT) excited state are promising for the realization of high performance blue organic light-emitting diodes (OLEDs). The rational manipulation of HLCT excited state for efficient emitters remains challenging. Herein, we present three donor-π-acceptor (D-π-A) molecules (mPAN, mPANPH, and mPNAPH) with phenanthro[9,10-d]imidazole (PI) and pyridinyl as donor and π-bridge respectively. Changes in various kinds of polycyclic aromatic derivative acceptors (anthracene, 9-phenylanthracene, and 1-phenylnaphthalene) could manipulate the excited states and optoelectronic properties. Theoretical calculations reveal that the S1 state of mPNAPH exhibits HLCT nature while the other two molecules show local excited (LE) state dominated feature. The photophysical properties also demonstrate this characteristic. Therefore, compared with mPAN and mPANPH, mPNAPH has higher photoluminescence quantum yield (PLQY) whether in solutions or neat films. Ultimately, the non-doped devices based on these emitters show high luminance larger than 35000 cd m-2 , and high maximum external quantum efficiencies (EQEmax s) larger than 5 % with low efficiency roll-off. In particular, the mPNAPH-based device displays an excellent performance of pure blue emission at 456 nm with Commission Internationale de L'Eclairage coordinate of (0.15, 0.16) and EQEmax of 6.13 % that benefited from the HLCT state and high-lying reverse intersystem crossing process.

Clinical characteristics of outpatients with influenza-B-associated pneumonia and molecular evolution of influenza B virus in Beijing, China, during the 2021-2022 influenza season.

We analyzed the clinical characteristics of outpatients with influenza-B-associated pneumonia during the 2021-2022 influenza season and analyzed the molecular epidemiology and evolution of influenza B virus. The presence of influenza B virus was confirmed by reverse transcription polymerase chain reaction (RT-PCR). Electronic medical records were used to collect and analyze data of outpatients. The HA and NA genes were phylogenetically analyzed using ClustalW 2.10 and MEGA 11.0. Out of 1569 outpatients who tested positive for influenza B virus, 11.7% (184/1569) developed pneumonia, and of these, 19.0% (35/184) had underlying diseases. Fever, cough, and sore throat were the most common symptoms. Among the complications, acute respiratory distress syndrome (ARDS), acute kidney injury (AKI), and shock accounted for 2.7% (5/184), 4.9% (9/184), and 1.6% (3/184), respectively. Of the outpatients, 2.7% (5/184) were admitted to the hospital, and 0.5% (1/184) of them died. All of the strains from Beijing were identified as belonging to the B/Victoria lineage. The HA and NA gene sequences of 41 influenza B viruses showed high similarity to each other, and all of them belonged to clade 1A.3. Compared with the vaccine strain B/Washington/02/2019, all of the isolates contained N150K, G181E, and S194D mutations. S194D, E195K, and K200R mutations were detected in the 190 helix of the receptor binding region of HA. Co-mutations of H122Q, A127T, P144L, N150K, G181E, S194D, and K200R in HA and D53N, N59S, and G233E in NA were detected in 78.0% (32/41) of the isolates, and 56.3% (18/32) of these were from outpatients with influenza-B-associated pneumonia. Influenza outpatients with underlying diseases were more likely to develop pneumonia. No significant differences were observed in clinical symptoms or laboratory results between outpatients with and without pneumonia, so testing for influenza virus seems to be a good choice. The observed amino acid variations suggest that current vaccines might not provide effective protection.

Association of elevated circulating monocyte-platelet aggregates with hypercoagulability in patients with nephrotic syndrome.

BACKGROUND: Hypercoagulability emerges as a central pathological feature and clinical complication in nephrotic syndrome. Increased platelet activation and aggregability are closely related to hypercoagulability in nephrotic syndrome. Monocyte-platelet aggregates (MPAs) have been proposed to represent a robust biomarker of platelet activation. The aim of this study was to investigate levels of the circulating MPAs and MPAs with the different monocyte subsets to evaluate the association of MPAs with hypercoagulability in nephrotic syndrome. METHODS: Thirty-two patients with nephrotic syndrome were enrolled. In addition, thirty-two healthy age and sex matched adult volunteers served as healthy controls. MPAs were identified by CD14 monocytes positive for CD41a platelets. The classical (CD14 + + CD16-, CM), the intermediate (CD14 + + CD16+, IM) and the non-classical (CD14 + CD16++, NCM) monocytes, as well as subset specific MPAs, were measured by flow cytometry. RESULTS: Patients with nephrotic syndrome showed a higher percentage of circulating MPAs as compared with healthy controls (p < 0.001). The percentages of MPAs with CM, IM, and NCM were higher than those of healthy controls (p = 0.012, p < 0.001 and p < 0.001, respectively). Circulating MPAs showed correlations with hypoalbuminemia (r=-0.85; p < 0.001), hypercholesterolemia (r = 0.54; p < 0.001), fibrinogen (r = 0.70; p < 0.001) and D-dimer (r = 0.37; p = 0.003), but not with hypertriglyceridemia in nephrotic syndrome. The AUC for the prediction of hypercoagulability in nephrotic syndrome using MPAs was 0.79 (95% CI 0.68-0.90, p < 0.001). The sensitivity of MPAs in predicting hypercoagulability was 0.71, and the specificity was 0.78. CONCLUSION: Increased MPAs were correlated with hypercoagulability in nephrotic syndrome. MPAs may serve as a potential biomarker for thrombophilic or hypercoagulable state and provide novel insight into the mechanisms of anticoagulation in nephrotic syndrome.

α4 nicotinic receptors on GABAergic neurons mediate a cholinergic analgesic circuit in the substantia nigra pars reticulata.

Nicotinic acetylcholine receptors (nAChRs) regulate pain pathways with various outcomes depending on receptor subtypes, neuron types, and locations. But it remains unknown whether α4ß2 nAChRs abundantly expressed in the substantia nigra pars reticulata (SNr) have potential to mitigate hyperalgesia in pain states. We observed that injection of nAChR antagonists into the SNr reduced pain thresholds in naïve mice, whereas injection of nAChR agonists into the SNr relieved hyperalgesia in mice, subjected to capsaicin injection into the lower hind leg, spinal nerve injury, chronic constriction injury, or chronic nicotine exposure. The analgesic effects of nAChR agonists were mimicked by optogenetic stimulation of cholinergic inputs from the pedunculopontine nucleus (PPN) to the SNr, but attenuated upon downregulation of α4 nAChRs on SNr GABAergic neurons and injection of dihydro-ß-erythroidine into the SNr. Chronic nicotine-induced hyperalgesia depended on α4 nAChRs in SNr GABAergic neurons and was associated with the reduction of ACh release in the SNr. Either activation of α4 nAChRs in the SNr or optogenetic stimulation of the PPN-SNr cholinergic projection mitigated chronic nicotine-induced hyperalgesia. Interestingly, mechanical stimulation-induced ACh release was significantly attenuated in mice subjected to either capsaicin injection into the lower hind leg or SNI. These results suggest that α4 nAChRs on GABAergic neurons mediate a cholinergic analgesic circuit in the SNr, and these receptors may be effective therapeutic targets to relieve hyperalgesia in acute and chronic pain, and chronic nicotine exposure.

Metabolomic reveals the responses of sludge properties and microbial communities to high nitrite stress in denitrifying phosphorus removal systems.

Nitrite, as an electron acceptor, plays a good role in denitrifying phosphorus removal (DPR); however, high nitrite concentration has adverse affects on sludge performance. We investigated the precise mechanisms of responses of sludge to high nitrite stress, including surface characteristics, intracellular and extracellular components, microbial and metabolic responses. When the nitrite stress reached 90 mg/L, the sludge settling performance was improved, but the activated sludge was aging. FTIR and XPS analysis revealed a significant increase in the hydrophobicity of the sludge, resulting in improve settling performance. However, the intracellular carbon sources synthesis was inhibited. In addition, the components in the tightly bound extracellular polymeric substances (TB-EPS) of sludge were significantly reduced and indicated the disturb of metabolism. Notably, Exiguobacterium emerged as a new genus when face high nitrite stress that could maintaining survival in hostile environments. Moreover, metabolomic analysis demonstrated strong biological response to nitrite stress further supported above results that include the inhibited of carbohydrate and amino acid metabolism. More importantly, some lipids (PS, PA, LysoPA, LysoPC and LysoPE) were significantly upregulated that related enhanced membrane lipid remodeling. The comprehensive analyses provide novel insights into the high nitrite stress responses mechanisms in activated sludge systems.

The Noncanonical Role of ULK/ATG1 in ER-to-Golgi Trafficking Is Essential for Cellular Homeostasis.

ULK1 and ULK2 are thought to be essential for initiating autophagy, and Ulk1/2-deficient mice die perinatally of autophagy-related defects. Therefore, we used a conditional knockout approach to investigate the roles of ULK1/2 in the brain. Although the mice showed neuronal degeneration, the neurons showed no accumulation of P62(+)/ubiquitin(+) inclusions or abnormal membranous structures, which are observed in mice lacking other autophagy genes. Rather, neuronal death was associated with activation of the unfolded protein response (UPR) pathway. An unbiased proteomics approach identified SEC16A as an ULK1/2 interaction partner. ULK-mediated phosphorylation of SEC16A regulated the assembly of endoplasmic reticulum (ER) exit sites and ER-to-Golgi trafficking of specific cargo, and did not require other autophagy proteins (e.g., ATG13). The defect in ER-to-Golgi trafficking activated the UPR pathway in ULK-deficient cells; both processes were reversed upon expression of SEC16A with a phosphomimetic substitution. Thus, the regulation of ER-to-Golgi trafficking by ULK1/2 is essential for cellular homeostasis.