Membrane-Based Preparation Process and Antioxidant and Anti-AGEs Activities of a Novel Propolis Ultrafiltrate.

Propolis is one functional supplement with hundreds of years of usage. However, it's rarely consumed directly for its resinous property. Herein, a pre-treated process which can remove the impurity while preserve its bioactivities is needed to maximise its therapeutic opportunities. In the present study, a membrane-based ultrafiltration process was developed on a KM1812-NF experimental instrument. Using Brazilian green propolis as testing material, all experimental steps and parameters were sequentially optimized. In addition, a mathematical model was developed to fit the process. As a result, the optimum solvent was 60 % ethanol adjusted to pH 8-9, while the optimum MWCO (molecular weight cut-off) value of membrane was 30 KDa. The membrane filtration dynamic model fitted with the function y=(ax+b)/(1+cx+dx2 ). The resulting propolis ultrafiltrate from Brazilian green propolis, termed P30K, contains the similar profile of flavonoids and phenolic acids as raw propolis. Meanwhile, the ORAC (oxygen radical absorbance capacity) value of P30K is 11429.45±1557.58 µM TE/g and the IC50 value of inhibition of fluorescent AGEs (advanced glycation end products) formation is 0.064 mg/mL. Our work provides an innovative alternative process for extraction of active compounds from propolis and reveals P30K as an efficient therapeutic antioxidant.

Dual Proton/Silver-Catalyzed Serial (5 + 2)-Cycloaddition and Nazarov Cyclization of (E)-2-Arylidene-3-hydroxyindanones with Conjugated Eneynes: Synthesis of Indanone-Fused Benzo[cd]azulenes.

A one-pot step-economic tandem process involving (5 + 2)-cycloaddition and Nazarov cyclization reactions has been reported for the facile synthesis of indanone-fused benzo[cd]azulenes from (E)-2-arylidene-3-hydroxyindanones and conjugated eneynes. This highly regio- and stereoselective bisannulation reaction is enabled by dual silver and Brønsted acid catalysis and opens up a new avenue for the construction of important bicyclo[5.3.0]decane skeletons.

[Effects of electroacupuncture at "Zhongliao" and "Xialiao" on serotonin signaling system and short-chain fatty acids in slow transit constipation rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) at "Zhongliao" (BL33) and "Xialiao" (BL34) on the 5-hydroxytryptamine (5-HT) signaling system in colon tissue and short-chain fatty acids in feces of rats with slow transit constipation (STC), so as to explore the underlying mechanisms of EA in the treatment of STC. METHODS: A total of 32 SD rats were randomly divided into normal, model, drug control and EA groups, with 8 rats in each group. The STC model was established by intragastric administration of loperamide for 14 days. The EA stimulation (2 Hz/15 Hz) was performed at bilateral BL33 and BL34 for 30 min, once a day for 14 days. The first black stool de-fecation time and fecal water content were detected after treatment. The expressions of 5-hydroxytryptamine 4 receptor (5-HT4R), tryptophan hydroxylase 1 (TPH1) and 5-HT transporter (SERT) in colon tissues were detected by Western blot. The contents of substance P (SP) and vasoactive intestinal peptide (VIP) in serum were detected by ELISA. The contents of 5-HT in colon tissue and short chain fatty acid (SCFA) in feces were detected by mass spectrometry. RESULTS: Compared with the normal group, the fecal water content, the expressions of 5-HT, 5-HT4R, TPH1 and SERT in colon tissue, the content of serum SP were significantly decreased (P<0.05), the first black stool de-fecation time, and the content of serum VIP was significantly increased (P<0.05), the contents of SCFA in feces were significantly decreased except isobutyric acid (P<0.05) in the model group. Compared with the model group, the fecal water content, the expressions of 5-HT, 5-HT4R, TPH1 and SERT in colon tissues, the contents of acetic acid and butyrate in feces were significantly increased (P<0.05) in the EA and drug control groups, the first black stool defecation time was decreased (P<0.05) in the EA and drug control groups, and the content of serum SP was increased and the content of serum VIP was decreased (P<0.05) in the EA group. Compared with the drug control group, the content of serum VIP was significantly decreased (P<0.05), and the expressions of TPH1 and SERT in colon tissue were significantly increased (P<0.05) in the EA group. CONCLUSION: EA at BL33 and BL34 can promote intestinal motility by intervening multiple links of 5-HT signaling system in treating STC.

Meta-analysis on the epidemiology of gastroesophageal reflux disease in China.

BACKGROUND: No large-scale epidemiological survey on the prevalence of gastroesophageal reflux disease (GERD) in China has been conducted. China has a large population and a complex geographical environment. It is important to understand the prevalence and spatial distribution of GERD in China. AIM: To explore the prevalence and the spatial, temporal, and population distributions of GERD in the natural Chinese population. METHODS: We searched Chinese and English databases for literature on the prevalence of GERD in the natural Chinese population. The prevalence of GERD was pooled using a random-effects meta-analysis model. Subgroup analysis was performed according to time, region, and population. We used ArcGIS software to draw statistical maps and trend analysis charts. Spatial autocorrelation analysis was carried out using Geoda software. Spearman correlation analysis was used to assess the spatial distribution relationship between GERD and upper digestive tract tumours. RESULTS: Altogether, 70 studies involving 276014 individuals from 24 provinces of China were included. The overall pooled prevalence of GERD was 8.7% (95%CI: 7.5%-9.9%) in mainland China. Over the past two decades, the prevalence of GERD in China has increased from 6.0% to 10.6%. GERD was more common in people aged 40-60, with body mass index ≥ 24, and of Uygur ethnicity. The prevalence was higher in the west and east than in the centre, and there may be a local spatial autocorrelation between the Qinghai-Tibet Plateau and the southeast. GERD was correlated with gastric (r = 0.421, P = 0.041) and oesophageal tumours (r = 0.511, P = 0.011) in spatial distribution. CONCLUSION: GERD is becoming common in China. The prevalence differs by region and population. The development of appropriate strategies for the prevention and treatment of GERD is needed.

TERRA and RAD51AP1 promote alternative lengthening of telomeres through an R- to D-loop switch.

Alternative lengthening of telomeres (ALT), a telomerase-independent process maintaining telomeres, is mediated by break-induced replication (BIR). RAD52 promotes ALT by facilitating D-loop formation, but ALT also occurs through a RAD52-independent BIR pathway. Here, we show that the telomere non-coding RNA TERRA forms dynamic telomeric R-loops and contributes to ALT activity in RAD52 knockout cells. TERRA forms R-loops in vitro and at telomeres in a RAD51AP1-dependent manner. The formation of R-loops by TERRA increases G-quadruplexes (G4s) at telomeres. G4 stabilization enhances ALT even when TERRA is depleted, suggesting that G4s act downstream of R-loops to promote BIR. In vitro, the telomeric R-loops assembled by TERRA and RAD51AP1 generate G4s, which persist after R-loop resolution and allow formation of telomeric D-loops without RAD52. Thus, the dynamic telomeric R-loops formed by TERRA and RAD51AP1 enable the RAD52-independent ALT pathway, and G4s orchestrate an R- to D-loop switch at telomeres to stimulate BIR.

Protocol to stimulate and delineate alternative lengthening of telomeres in human U2OS cells.

Alternative lengthening of telomeres (ALT) is a telomerase-independent but recombination-dependent pathway that maintains telomeres. Here, we describe a protocol to stimulate the formation of ALT-associated PML bodies (APBs) and ALT activity by tethering PML-IV to telomeres in human U2OS cells. Through immunofluorescence, in situ hybridization, and microscopy, we analyze dynamics of telomere clustering, visualize recruitment of DNA repair proteins to APBs, and measure telomere DNA synthesis during ALT. This protocol provides a unique approach to delineate the ALT pathway. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2021).

Spatial-temporal Distribution and Influencing Factors of Helicobacter pylori Infection in Chinese Mainland, 2001-2020: A Systematic Review and Meta-Analysis.

BACKGROUND: The spatial-temporal distribution of Helicobacter pylori infection in China is poorly understood. We aimed to study the spatial-temporal distribution of H. pylori infection in Chinese mainland and to explore its influencing factors. MATERIALS AND METHODS: We searched the relevant literature from 2001 to 2021 and applied meta-analysis to obtain the pooled prevalence estimates of all studies and subgroups. Then, we used the pooled prevalence as the dependent variable for the following analysis, including time series analysis, statistical mapping, spatial autocorrelation analysis, and influencing factor analysis based on generalized additive model and panel data model. RESULTS: A total of 726 articles and 3,407,392 people were included. The pooled prevalence was 43.7% (95% confidence interval: 42.7%-44.8%). The prevalence decreased in the past 20 years, with high in the eastern and western regions and low in the central region. Qinghai Tibet Plateau and Guizhou Plateau were the high incidence areas of this disease. The intake of vegetable oil, aquatic products, meat, milk, per capita gross domestic product, and annual average humidity were significantly correlated with H. pylori. CONCLUSIONS: The prevalence of H. pylori is decreasing in Chinese mainland, but still high in underdeveloped areas. Appropriate strategies for the prevention need greater attention.

Effect of cooking methods on the edible, nutritive qualities and volatile flavor compounds of rabbit meat.

BACKGROUND: Rabbit meat is a good edible meat source with high nutritional values. Cooking has a significant impact on the edible properties, nutritional qualities and flavor characteristics of meat. Studying the effect of cooking methods on rabbit meat qualities could encourage more understanding and acceptance of rabbit meat by consumers, and could also provide some reference for rabbit meat processing. Therefore, the effects of boiling, sous-vide cooking, steaming, microwaving, roasting, frying and pressure cooking on the edible, nutritive and volatile qualities of rabbit meat were investigated. RESULTS: The sous-vide cooked rabbit meat sample showed higher moisture content, water-holding capacity and lower cooking losses than other samples, but the results of roasted rabbit meat sample were the opposite, and scanning electron microscopy observations also verified the results. There was no significant difference in 2-thiobarbituric acid reactive substance (TBARS) value in the cooked samples except for roasting. Microwaving, roasting and frying exhibited stronger antioxidant activity than the other cooked samples after in vitro digestion. A total of 38 volatiles were identified in the cooked meat samples, and the samples were well divided into four groups by principal component analysis, and 13 volatiles were considered discriminatory variables for the cooked rabbit meat. CONCLUSION: The physicochemical characteristics of cooked meat differed significantly between the processing methods. Roasted meat showed lower TBARS value and stronger antioxidant activity after simulated digestion compared to the other meats. However, pressure cooked meat detected the most volatile components while roasting the least. © 2022 Society of Chemical Industry.

Ding's herbal enema treats dextran sulfate sodium-induced colitis in mice by regulating the gut microbiota and maintaining the Treg/Th17 cell balance.

Ding's herbal enema (DHEP) is a traditional Chinese medicinal therapy that has been used to treat ulcerative colitis (UC) in China. The present study determined the molecular mechanism of the effect of DHEP in UC treatment. C57BL/6J mice were treated with 3.5% (w/v) dextran sulfate sodium (DSS) for 7 days to establish an animal model of colitis. The mice were divided into five groups (n=5): Control, vehicle, DHEP, mesalazine and ß-sitosterol. After oral administration for 7 days, the body weight, disease activity index, histopathology and inflammatory factors were analyzed. The fractions of CD4+Foxp3+ regulatory T (Treg) cells and CD4+IL-17A+ T helper (Th) cells were determined by flow cytometry. Gut microbiota composition was analyzed by next-generation sequencing. The results revealed that DHEP and ß-sitosterol could significantly alleviate the symptoms of DSS-induced UC. Furthermore, the levels of IL-6, cyclooxygenase-2, TNF-α and p65 were reduced after administration of DHEP. Additionally, the data indicated that DHEP could increase the abundance of seven operational taxonomic units (OTUs) and decrease the abundance of 12 OTUs in the gut microbiota. The content of short-chain fatty acids in the colon remodeled the balance of Treg/Th17 cells in DSS-induced UC in mice. The present study preliminarily defined the mechanism of action of DHEP in UC that may be associated with the regulation of the gut microbiota composition, and maintenance of the balance between Treg and Th17 cells. Furthermore, ß-sitosterol exhibited the same effects with DHEP and it could be a possible substitute for DHEP in UC treatment.

NR4A1 regulates expression of immediate early genes, suppressing replication stress in cancer.

Deregulation of oncogenic signals in cancer triggers replication stress. Immediate early genes (IEGs) are rapidly and transiently expressed following stressful signals, contributing to an integrated response. Here, we find that the orphan nuclear receptor NR4A1 localizes across the gene body and 3' UTR of IEGs, where it inhibits transcriptional elongation by RNA Pol II, generating R-loops and accessible chromatin domains. Acute replication stress causes immediate dissociation of NR4A1 and a burst of transcriptionally poised IEG expression. Ectopic expression of NR4A1 enhances tumorigenesis by breast cancer cells, while its deletion leads to massive chromosomal instability and proliferative failure, driven by deregulated expression of its IEG target, FOS. Approximately half of breast and other primary cancers exhibit accessible chromatin domains at IEG gene bodies, consistent with this stress-regulatory pathway. Cancers that have retained this mechanism in adapting to oncogenic replication stress may be dependent on NR4A1 for their proliferation.

Toxicity of CuS/CdS semiconductor nanocomposites to liver cells and mice liver.

Semiconductor nanomaterials not only bring great convenience to peoples lives but also become a potential hazard to human health. The purpose of this study was to evaluate the toxicity of CuS/CdS nanocomposites in hepatocytes and mice liver. The CuS/CdS semiconductor nanocomposites were synthesized by a biomimetic synthesis - ion exchange strategy. Nanosize was confirmed by high-resolution transmission electron microscopy and dynamic light scattering. The composition and physical properties were measured by powder X-ray diffraction, Fourier transform infrared spectra, atomic absorption spectroscopy, thermogravimetry-differential scanning calorimetry and zeta potential analysis. The results revealed that CuS/CdS nanocomposites had 8.7 nm diameter and negative potential. Ion exchange time could adjust the ratio of CuS and CdS in nanocomposites. The toxicological study revealed that CuS/CdS nanocomposites could be internalized into liver cells, inhibited endogenous defense system (e.g. GSH and SOD), induced the accumulation of oxidation products (e.g. ROS, GSSG and MDA), and caused hepatocyte apoptosis. The in vivo experiments in Balb/c mice showed that the experimental dose (4 mg/kg) didn't cause observable changes in mice behavior, physical activity and pathological characteristics, but the continuous accumulation of Cd2+ in the liver and kidney might be responsible for its long-term toxicity.

RNA transcripts stimulate homologous recombination by forming DR-loops.

Homologous recombination (HR) repairs DNA double-strand breaks (DSBs) in the S and G2 phases of the cell cycle1-3. Several HR proteins are preferentially recruited to DSBs at transcriptionally active loci4-10, but how transcription promotes HR is poorly understood. Here we develop an assay to assess the effect of local transcription on HR. Using this assay, we find that transcription stimulates HR to a substantial extent. Tethering RNA transcripts to the vicinity of DSBs recapitulates the effects of local transcription, which suggests that transcription enhances HR through RNA transcripts. Tethered RNA transcripts stimulate HR in a sequence- and orientation-dependent manner, indicating that they function by forming DNA-RNA hybrids. In contrast to most HR proteins, RAD51-associated protein 1 (RAD51AP1) only promotes HR when local transcription is active. RAD51AP1 drives the formation of R-loops in vitro and is required for tethered RNAs to stimulate HR in cells. Notably, RAD51AP1 is necessary for the DSB-induced formation of DNA-RNA hybrids in donor DNA, linking R-loops to D-loops. In vitro, RAD51AP1-generated R-loops enhance the RAD51-mediated formation of D-loops locally and give rise to intermediates that we term 'DR-loops', which contain both DNA-DNA and DNA-RNA hybrids and favour RAD51 function. Thus, at DSBs in transcribed regions, RAD51AP1 promotes the invasion of RNA transcripts into donor DNA, and stimulates HR through the formation of DR-loops.

Coinheritance of OLFM2 and SIX6 variants in a Chinese family with juvenile-onset primary open-angle glaucoma: A case report.

BACKGROUND: Juvenile-onset primary open-angle glaucoma (JOAG), characterized by severe elevation of intraocular pressure and optic neuropathy prior to the age of 40, is a rare subtype of primary open-angle glaucoma. Several genetic mutations have been associated with JOAG. CASE SUMMARY: The proband patient was a young male, diagnosed with primary open-angle glaucoma at the age of 27. The patient and his unaffected parents who have been excluded from classic genetic mutations for primary open-angle glaucoma were included to explore for other possible genetic variants through whole genome sequencing and bioinformatics analysis. In this trio, we found two heterozygous variants inherited from the parents in the proband: c.281G>A, p.Arg94His in OLFM2 and c.177C>G, p.Ile59Met in SIX6. Both genetic mutations are predicted through bioinformatics analysis to replace evolutionary conserved amino acids, therefore rendering a pathogenic effect on proteins. In contrast, very low frequencies for these genetic mutations were recorded in most common control databases. CONCLUSION: This is the first report on coinherited mutations of OLFM2 and SIX6 in a JOAG family, which shows the complexity of JOAG inheritance. Large-scale clinical screening and molecular functional investigations on these coinherited mutations are imperative to improve our understanding of the development of JOAG.

Alternative lengthening of telomeres is a self-perpetuating process in ALT-associated PML bodies.

Alternative lengthening of telomeres (ALT) is mediated by break-induced replication (BIR), but how BIR is regulated at telomeres is poorly understood. Here, we show that telomeric BIR is a self-perpetuating process. By tethering PML-IV to telomeres, we induced telomere clustering in ALT-associated PML bodies (APBs) and a POLD3-dependent ATR response at telomeres, showing that BIR generates replication stress. Ablation of BLM helicase activity in APBs abolishes telomere synthesis but causes multiple chromosome bridges between telomeres, revealing a function of BLM in processing inter-telomere BIR intermediates. Interestingly, the accumulation of BLM in APBs requires its own helicase activity and POLD3, suggesting that BIR triggers a feedforward loop to further recruit BLM. Enhancing BIR induces PIAS4-mediated TRF2 SUMOylation, and PIAS4 loss deprives APBs of repair proteins and compromises ALT telomere synthesis. Thus, a BLM-driven and PIAS4-mediated feedforward loop operates in APBs to perpetuate BIR, providing a critical mechanism to extend ALT telomeres.

Simultaneous directed evolution of coupled enzymes for efficient asymmetric synthesis of l-phosphinothricin.

The traditional strategy to improve the efficiency of an entire coupled enzyme system relies on separate direction of the evolution of enzymes involved in their respective enzymatic reactions. This strategy can lead to enhanced single-enzyme catalytic efficiency but may also lead to loss of coordination among enzymes. This study aimed to overcome such shortcomings by executing a directed evolution strategy on multiple enzymes in one combined group that catalyzes the asymmetric biosynthesis of l-phosphinothricin. The genes of a glutamate dehydrogenase from Pseudomonas moorei (PmGluDH) and a glucose dehydrogenase from Exiguobacterium sibiricum (EsGDH), along with other gene parts (promoters, ribosomal binding sites (RBSs), and terminators) were simultaneously evolved. The catalytic efficiency of PmGluDH was boosted by introducing the beneficial mutation A164G (from 1.29 s-1mM-1 to 183.52 s-1mM-1), and the EsGDH expression level was improved by optimizing the linker length between the RBS and the start codon of gdh. The total turnover numbers of the bioreaction increased from 115 (GluDH WTNADPH) to 5846 (A164GNADPH coupled with low expression of EsGDH), and to 33950 (A164GNADPH coupled with high expression of EsGDH). The coupling efficiency was increased from ∼30% (GluDH_WT with low expression of GDH) to 83.3% (GluDH_A164G with high expression of GDH). In the batch production of l-phosphinothricin utilizing whole-cell catalysis, the strongest biocatalytic reaction exhibited a high space-time yield (6410 g·L-1·d-1) with strict stereoselectivity (>99% enantiomeric excess).Importance: The traditional strategy to improve multienzyme-catalyzed reaction efficiency may lead to enhanced single-enzyme catalytic efficiency but may also result in loss of coordination among enzymes. We describe a directed evolution strategy of an entire coupled enzyme system to simultaneously enhance enzyme coordination and catalytic efficiency. The simultaneous evolution strategy was applied to a multienzyme-catalyzed reaction for the asymmetric synthesis of l-phosphinothricin, which not only enhanced the catalytic efficiency of GluDH but also improved the coordination between GluDH and GDH. Since this strategy is enzyme-independent, it may be applicable to other coupled enzyme systems for chiral chemical synthesis.

Comparative review and the recent progress in detection technologies of meat product adulteration.

Meat adulteration, mainly for the purpose of economic pursuit, is widespread and leads to serious public health risks, religious violations, and moral loss. Rapid, effective, accurate, and reliable detection technologies are keys to effectively supervising meat adulteration. Considering the importance and rapid advances in meat adulteration detection technologies, a comprehensive review to summarize the recent progress in this area and to suggest directions for future progress is beneficial. In this review, destructive meat adulteration technologies based on DNA, protein, and metabolite analyses and nondestructive technologies based on spectroscopy were comparatively analyzed. The advantages and disadvantages, application situations of these technologies were discussed. In the future, determining suitable indicators or markers is particularly important for destructive methods. To improve sensitivity and save time, new interdisciplinary technologies, such as biochips and biosensors, are promising for application in the future. For nondestructive techniques, convenient and effective chemometric models are crucial, and the development of portable devices based on these technologies for onsite monitoring is a future trend. Moreover, omics technologies, especially proteomics, are important methods in laboratory detection because they enable multispecies detection and unknown target screening by using mass spectrometry databases.

LncRNA-AK149641 regulates the secretion of tumor necrosis factor-α in P815 mast cells by targeting the nuclear factor-kappa B signaling pathway.

Long noncoding RNAs play important roles in various biological processes. However, not much is known about their roles in inflammatory response. Mast cells, involved in innate and adaptive immunity, are one of the major effector cells in allergic inflammatory reactions and contribute to the pathogenesis of disorders, including asthma. In the present study, we aimed to verify and elucidate the function and possible role of a novel lncRNA, called lncRNA-AK149641, in the mechanism of lipopolysaccharide (LPS)-induced inflammatory response in P815 mast cells. The results showed that downregulating lncRNA-AK149641 decreased secretion of tumor necrosis factor-α into the supernatants of LPS-stimulated mast cells. Mechanistically, the activity of nuclear factor-kappa B (NF-κB) decreased after downregulating lncRNA-AK149641, as shown by western blot and electrophoretic mobility shift assays. Moreover, RNA binding protein immunoprecipitation (RIP) verified that lncRNA-AK149641 was able to bind to NF-κB in the nucleus. In conclusion, we demonstrated that lncRNA-AK149641 regulated LPS-induced inflammatory response in mast cells through the NF-κB signaling pathway.

Analysis of Clinical Efficacy of Traditional Chinese Medicine in Recovery Stage of Stroke: A Systematic Review and Meta-Analysis.

BACKGROUND: We provide an updated meta-analysis with detailed information on a combination of TCM and routine treatment. METHODS: Retrieve appropriate articles with no language restrictions on keywords until 8 July 2019 in an electronic database. All trajectories are screened according to certain criteria. The quality of certified research was also evaluated. We made a detailed record of the results of the measurement. Meta-analysis was carried out by using the Revman 5.3 software. RESULTS: Sixty-seven RCTs were included, and 6594 subjects were analyzed. Compared with routine treatment, the total effective rate (TER) of TCM combined with routine treatment was improved, and the recovery of stroke was also significantly accelerated. Regulation of blood lipids by notably shrinking the contents of TC, TG, and LDL and enhancing the levels of HDL. The levels of serum hs-CRP, WHV, and WLV decreased significantly, indicating that the expression of thrombomodulin was decreased after the comprehensive treatment of traditional Chinese medicines (TCMs). The combination of TCM treatment could enhance the protection of neural function by decreasing the NIHSS scoring while increasing the BI scoring. Paeoniae Radix Rubra, Angeticae Sinensis Radix, etc., can effectively improve the clinical symptoms of stroke convalescent patients and promote the recovery of neurological function. ACU of Baihui, Renzhong, etc., can improve the clinical rehabilitation effect of patients. However, our findings must be handled with care because of the small sample size and low quality of clinic trials cited. Other rigorous and large-scale RCTs are in need to confirm these results. CONCLUSION: A combination of TCM and routine treatment in the treatment of stroke could improve TER, and it is beneficial to the rehabilitation of patients in the recovery period of apoplexy. These effects can be mediated by a combination of several mechanisms. Nevertheless, due to the limitations of this study, these results should be handled with caution.

Synthetic lethality by targeting the RUVBL1/2-TTT complex in mTORC1-hyperactive cancer cells.

Despite considerable efforts, mTOR inhibitors have produced limited success in the clinic. To define the vulnerabilities of mTORC1-addicted cancer cells and to find previously unknown therapeutic targets, we investigated the mechanism of piperlongumine, a small molecule identified in a chemical library screen to specifically target cancer cells with a hyperactive mTORC1 phenotype. Sensitivity to piperlongumine was dependent on its ability to suppress RUVBL1/2-TTT, a complex involved in chromatin remodeling and DNA repair. Cancer cells with high mTORC1 activity are subjected to higher levels of DNA damage stress via c-Myc and displayed an increased dependency on RUVBL1/2 for survival and counteracting genotoxic stress. Examination of clinical cancer tissues also demonstrated that high mTORC1 activity was accompanied by high RUVBL2 expression. Our findings reveal a previously unknown role for RUVBL1/2 in cell survival, where it acts as a functional chaperone to mitigate stress levels induced in the mTORC1-Myc-DNA damage axis.

CRL4Cdt2 ubiquitin ligase regulates Dna2 and Rad16 (XPF) nucleases by targeting Pxd1 for degradation.

Structure-specific endonucleases (SSEs) play key roles in DNA replication, recombination, and repair. SSEs must be tightly regulated to ensure genome stability but their regulatory mechanisms remain incompletely understood. Here, we show that in the fission yeast Schizosaccharomyces pombe, the activities of two SSEs, Dna2 and Rad16 (ortholog of human XPF), are temporally controlled during the cell cycle by the CRL4Cdt2 ubiquitin ligase. CRL4Cdt2 targets Pxd1, an inhibitor of Dna2 and an activator of Rad16, for degradation in S phase. The ubiquitination and degradation of Pxd1 is dependent on CRL4Cdt2, PCNA, and a PCNA-binding degron motif on Pxd1. CRL4Cdt2-mediated Pxd1 degradation prevents Pxd1 from interfering with the normal S-phase functions of Dna2. Moreover, Pxd1 degradation leads to a reduction of Rad16 nuclease activity in S phase, and restrains Rad16-mediated single-strand annealing, a hazardous pathway of repairing double-strand breaks. These results demonstrate a new role of the CRL4Cdt2 ubiquitin ligase in genome stability maintenance and shed new light on how SSE activities are regulated during the cell cycle.