PRP19 transforms into a sensor of RPA-ssDNA after DNA damage and drives ATR activation via a ubiquitin-mediated circuitry.

PRP19 is a ubiquitin ligase involved in pre-mRNA splicing and the DNA damage response (DDR). Although the role for PRP19 in splicing is well characterized, its role in the DDR remains elusive. Through a proteomic screen for proteins that interact with RPA-coated single-stranded DNA (RPA-ssDNA), we identified PRP19 as a sensor of DNA damage. PRP19 directly binds RPA and localizes to DNA damage sites via RPA, promoting RPA ubiquitylation in a DNA-damage-induced manner. PRP19 facilitates the accumulation of ATRIP, the regulatory partner of the ataxia telangiectasia mutated and Rad3-related (ATR) kinase, at DNA damage sites. Depletion of PRP19 compromised the phosphorylation of ATR substrates, recovery of stalled replication forks, and progression of replication forks on damaged DNA. Importantly, PRP19 mutants that cannot bind RPA or function as an E3 ligase failed to support the ATR response, revealing that PRP19 drives ATR activation by acting as an RPA-ssDNA-sensing ubiquitin ligase during the DDR.

Skp2 E3 ligase integrates ATM activation and homologous recombination repair by ubiquitinating NBS1.

The Mre11/Rad50/NBS1 (MRN) complex is thought to be a critical sensor that detects damaged DNA and recruits ATM to DNA foci for activation. However, it remains to be established how the MRN complex regulates ATM recruitment to the DNA foci during DNA double-strand breaks (DSBs). Here we show that Skp2 E3 ligase is a key component for the MRN complex-mediated ATM activation in response to DSBs. Skp2 interacts with NBS1 and triggers K63-linked ubiquitination of NBS1 upon DSBs, which is critical for the interaction of NBS1 with ATM, thereby facilitating ATM recruitment to the DNA foci for activation. Finally, we show that Skp2 deficiency exhibits a defect in homologous recombination (HR) repair, thereby increasing IR sensitivity. Our results provide molecular insights into how Skp2 and the MRN complex coordinate to activate ATM, and identify Skp2-mediatetd NBS1 ubiquitination as a vital event for ATM activation in response to DNA damage.

CRL4(Cdt2)-mediated destruction of the histone methyltransferase Set8 prevents premature chromatin compaction in S phase.

The proper coordination between DNA replication and mitosis during cell-cycle progression is crucial for genomic stability. During G2 and mitosis, Set8 catalyzes monomethylation of histone H4 on lysine 20 (H4K20me1), which promotes chromatin compaction. Set8 levels decline in S phase, but why and how this occurs is unclear. Here, we show that Set8 is targeted for proteolysis in S phase and in response to DNA damage by the E3 ubiquitin ligase, CRL4(Cdt2). Set8 ubiquitylation occurs on chromatin and is coupled to DNA replication via a specific degron in Set8 that binds PCNA. Inactivation of CRL4(Cdt2) leads to Set8 stabilization and aberrant H4K20me1 accumulation in replicating cells. Transient S phase expression of a Set8 mutant lacking the degron promotes premature H4K20me1 accumulation and chromatin compaction, and triggers a checkpoint-mediated G2 arrest. Thus, CRL4(Cdt2)-dependent destruction of Set8 in S phase preserves genome stability by preventing aberrant chromatin compaction during DNA synthesis.

Highly sensitive detection of target ssDNA based on SERS liquid chip using suspended magnetic nanospheres as capturing substrates.

A new approach for sensitive detection of a specific ssDNA (single-stranded DNA) sequence based on the surface enhanced Raman spectroscopy (SERS) liquid chip is demonstrated. In this method, the probe DNA (targeting to one part of target ssDNA) was attached to the nano-SERS-tags (poly(styrene-co-acrylic acid)/(silver nanoparticles)/silica composite nanospheres), and the capture DNA (targeting to the other part of target ssDNA) was attached to the Fe3O4/poly(acrylic acid) core/shell nanospheres. The nano-SERS-tags with probe DNA were first allowed to undergo hybridization with the target ssDNA in solution to achieve the best efficiency. Subsequently, the magnetic composite nanospheres with capture DNA were added as the capturing substrates of the target ssDNA combined with the nano-SERS-tags. Upon attraction with an external magnet, the nanospheres (including the nano-SERS-tags) were deposited together due to the hybridization, and the deposit sediment was then analyzed by SERS. Quantitative detection of target ssDNA was achieved based on the well-defined linear correlation between the SERS signal intensity and the target ssDNA quantity in the range of 10 nM to 10 pM, and the limit of detection was approximately 10 pM. Multiplexed detection of up to three different ssDNA targets in one sample was demonstrated using three different types of nano-SERS-tags under a single excitation laser. The experimental results indicated that the liquid-phase DNA sequencing method, thus named the SERS liquid chip (SLC) method, holds significant promises for specific detection of trace targets of organisms.

Toward designer magnetite/polystyrene colloidal composite microspheres with controllable nanostructures and desirable surface functionalities.

An effective method was developed for synthesizing magnetite/polymer colloidal composite microspheres with controllable variations in size and shape of the nanostructures and desirable interfacial chemical functionalities, using surfactant-free seeded emulsion polymerization with magnetite (Fe(3)O(4)) colloidal nanocrystal clusters (CNCs) as the seed, styrene (St) as the monomer, and potassium persulfate (KPS) as the initiator. The sub-micrometer-sized citrate-acid-stabilized Fe(3)O(4) CNCs were first obtained via ethylene glycol (EG)-mediated solvothermal synthesis, followed by 3-(trimethoxysilyl)propyl methacrylate (MPS) modification to immobilize the active vinyl groups onto the surfaces, and then the hydrophobic St monomers were polymerized at the interfaces to form the polymer shells by seeded emulsion radical polymerization. The morphology of the composite microspheres could be controlled from raspberry- and flower-like shapes, to eccentric structures by simply adjusting the feeding weight ratio of the seed to the monomer (Fe(3)O(4)/St) and varying the amount of cross-linker divinyl benzene (DVB). The morphological transition was rationalized by considering the viscosity of monomer-swollen polymer matrix and interfacial tension between the seeds and polymer matrix. Functional groups, such as carboxyl, hydroxyl, and epoxy, can be facilely introduced onto the composite microspheres through copolymerization of St with other functional monomers. The resultant microspheres displayed a high saturation magnetization (46 emu/g), well-defined core-shell nanostructures, and surface chemical functionalities, as well as a sustained colloidal stability, promising for further biomedical applications.

Mrc1 phosphorylation in response to DNA replication stress is required for Mec1 accumulation at the stalled fork.

DNA replication stress activates a response pathway that stabilizes stalled forks and promotes the completion of replication. The budding yeast Mec1 sensor kinase, Mrc1 mediator, and Rad53 effector kinase are central to this signal transduction cascade in S phase. We report that Mec1-dependent, Rad53-independent phosphorylation of Mrc1 is required to establish a positive feedback loop that stabilizes Mec1 and the replisome at stalled forks. A structure-function analysis of Mrc1 also uncovered a central region required for proper mediator function and association with replisome components. Together these results reveal new insight into how Mrc1 facilitates checkpoint signal amplification at stalled replication forks.

Detecting trace melamine in solution by SERS using Ag nanoparticle coated poly(styrene-co-acrylic acid) nanospheres as novel active substrates.

A systematic study for the preparation of Ag nanoparticle (Ag-NP) coated poly(styrene-co-acrylic acid) (PSA) composite nanospheres by in situ chemical reduction is reported. The experimental results showed that the reaction temperature and the surface coverage of the -COOH determined the surface coverage and grain size of Ag nanoparticles on the PSA nanospheres. The surface enhanced Raman spectroscopy (SERS) sensitivity was investigated using 4-hydroxythiophenol (4-HBT) as the model probe in the solution of composite nanospheres stabilized by polyvinylpyrrolidone (PSA/Ag-NPs/PVP), with the detection limit of about 1 × 10(-6) M. Potential application of the new SERS substrate was demonstrated with the detection of melamine, and the detection limit was about 1 × 10(-3) M. Chemical noises from PVP and other impurities were observed and attributed mainly to the competitive adsorption of PVP on the surfaces of Ag-NPs. After tetrahydrofuran washing of the PSA/Ag-NPs/PVP substrates that removed the PVP and other residuals, the signal/noise levels of SERS were greatly improved and the detection limit of melamine was determined to be 1 × 10(-7) M. This result indicated that the new PSA/Ag-NPs system is highly effective and can be used as the SERS-active substrate for trace analysis of a variety of drugs and food additives.

Epidemiological and spatiotemporal analyses of HIV/AIDS prevalence among older adults in Sichuan, China between 2008 and 2019: A population-based study.

OBJECTIVES: To describe epidemiological trends and spatial distributions of HIV/AIDS among older adults (aged ≥50) in Sichuan Province, China during 2008-19, and provide scientific reference for HIV/AIDS prevention, intervention and treatment. METHODS: Data on HIV/AIDS cases reported in 2008-19 was extracted from the Case Report System. The Cochran-Armitage trend test was used to determine epidemic trends. Spatial autocorrelation and space-time analysis were conducted with ArcGIS10.6 and ArcGIS Pro2.4, respectively. RESULTS: A total of 77854 HIV/AIDS cases among older adults were included in the study. Newly reported cases increased from 320 in 2008 to 22189 in 2019, and the reported incidence rate (number of new reported cases/older adult population) rose from 0.001% to 0.077%. Infections through heterosexual transmission increased from 65.3% to 98.2% of total cases in older adults in this period. Spatial analysis at the county-level showed significant clustering throughout Sichuan, with the main hot spots concentrated in the southeast. Spatiotemporal analysis indicated that most of the southeastern counties/districts were Consecutive Hot Spots. CONCLUSIONS: Older adults have become a key population in the HIV/AIDS epidemic in Sichuan; comprehensive prevention and intervention measures targeted to older adults are urgently needed to control the spread of HIV/AIDS.

Genetic analysis of the kinetochore DASH complex reveals an antagonistic relationship with the ras/protein kinase A pathway and a novel subunit required for Ask1 association.

DASH is a microtubule- and kinetochore-associated complex required for proper chromosome segregation and bipolar attachment of sister chromatids on the mitotic spindle. We have undertaken a genetic and biochemical analysis of the DASH complex and uncovered a strong genetic interaction of DASH with the Ras/protein kinase A (PKA) pathway. Overexpression of PDE2 or deletion of RAS2 rescued the temperature sensitivity of ask1-3 mutants. Ras2 negatively regulates DASH through the PKA pathway. Constitutive PKA activity caused by mutation of the negative regulator BCY1 is toxic to DASH mutants such as ask1 and dam1. In addition, we have discovered two novel subunits of DASH, Hsk2 and Hsk3 (helper of Ask1), which are microproteins of fewer than 75 amino acids, as dosage suppressors of ask1 mutants. These are essential genes that colocalize with DASH components on spindles and kinetochores and are present in the DASH complex. Mutants in hsk3 arrest cells in mitosis with short spindles and broken spindle structures characteristic of other DASH mutants. Hsk3 is critical for the integrity of the DASH complex because in hsk3 mutants the association of Dam1, Duo1, Spc34, and Spc19 with Ask1 is greatly diminished. We propose that Hsk3 acts to incorporate Ask1 into the DASH complex.

Erratum: The anaphase promoting complex impacts repair choice by protecting ubiquitin signalling at DNA damage sites.

[This corrects the article DOI: 10.1038/ncomms15751.].

The anaphase promoting complex impacts repair choice by protecting ubiquitin signalling at DNA damage sites.

Double-strand breaks (DSBs) are repaired through two major pathways, homology-directed recombination (HDR) and non-homologous end joining (NHEJ). While HDR can only occur in S/G2, NHEJ can happen in all cell cycle phases (except mitosis). How then is the repair choice made in S/G2 cells? Here we provide evidence demonstrating that APCCdh1 plays a critical role in choosing the repair pathways in S/G2 cells. Our results suggest that the default for all DSBs is to recruit 53BP1 and RIF1. BRCA1 is blocked from being recruited to broken ends because its recruitment signal, K63-linked poly-ubiquitin chains on histones, is actively destroyed by the deubiquitinating enzyme USP1. We show that the removal of USP1 depends on APCCdh1 and requires Chk1 activation known to be catalysed by ssDNA-RPA-ATR signalling at the ends designated for HDR, linking the status of end processing to RIF1 or BRCA1 recruitment.

[Tobacco cadmium health risk assessment and reduction techniques: A review].

Tobacco is one of the cadmium accumulation and tolerance plants. Decreasing cadmium content of tobacco contributes to environmental safety and human health. Three aspects on tobacco cadmium research were reviewed in this paper, i.e. uptake and distribution of cadmium in tobacco, and health risk assessment of cadmium in tobacco and reduction measures. The current situations and existing challenges in the research field were discussed. The cadmium tolerance mechanisms of tobacco were reviewed, the factors on cadmium uptake were analyzed, and the general distribution of cadmium in tobacco was summarized. From the point of health risk assessment, the lack of cadmium limits in tobacco was identified, the recommended formula to calculate cadmium limits of tobacco based on atmosphere cadmium limits and digestion cadmium limits was provided and the cadmium limits of tobacco were estimated using each formula, and suggestions on cadmium limits in tobacco were presented. At last, we put forward several effective reduction measures to lower cadmium level in tobacco leaves.

The p97-UFD1L-NPL4 protein complex mediates cytokine-induced IκBα proteolysis.

IκBα is an inhibitor of NF-κB, a family of transcription factors that transactivate genes related to inflammation. Upon inflammatory stimuli, IκBα is rapidly degraded via the ubiquitin-proteasome pathway. While it is very clear that the SCF(ß-TRCP) ubiquitin ligase ubiquitinates IκBα upon stimulation, little is known about the postubiquitinational events of IκBα proteolysis. Here, we report that p97, a valosin-containing protein (also called VCP), plays an essential role in the postubiquitinational regulation of IκBα turnover after tumor necrosis factor alpha (TNF-α) or interleukin-1ß (IL-1ß) treatment. The ATPase activity of p97 is essential for its role in IκBα proteolysis. Moreover, we found that UFD1L and NPL4, two cofactors of p97, assist p97 to control the postubiquitinational regulation of IκBα. The p97-UFD1L-NPL4 protein complex specifically associates with ubiquitinated IκBα via the interactions between p97 and the SCF(ß-TRCP) ubiquitin ligase and between the polyubiquitin binding domain of UFD1L and polyubiquitinated IκBα. Furthermore, we observed that the postubiquitinational regulation of IκBα by the p97-UFD1L-NPL4 complex is important for NF-κB activation under stimuli.

[Effects of long-term fertilization on evolution of S forms in a red soil and a black soil].

Sulfur (S) forms in two contrasting soils (a red soil and a black soil) under different long-term fertilization treatments (from 1990 to 2011) from the National Long-term Monitoring Network of Soil Fertility and Fertilizer Effects of China were investigated using a fractionation scheme in order to explore the distribution and transportation of S with different forms in the soils. The soil samples were collected from the topsoil (0-20 cm) and subsoil (20-40 cm) horizons that were treated with no fertilizers (CK), nitrogen, phosphorus, and potassium fertilizers (NPK), or NPK plus organic manures (MNPK) since 1990. The results indicated that when compared with the CK, total S contents in the topsoil layers treated with NPK and MNPK were increased by 42% and 33% for the red soil, and by 6% and 76% for the black soil, respectively, while the total S in the subsoil layer was less affected by the fertilization treatments and obviously lower than in the topsoil layer except for the red soil treated with NPK. The main forms of inorganic S in the red soil and black soil were found to be available S and HCl-extracted S, respectively. The application of NPK and MNPK increased the available S by 447% and 102% in the topsoil layer of the red soil compared with CK, and facilitated the transportation of available S into the lower depth. In contrast, NPK and MNPK only increased the available S by 54% and 93% in the topsoil layer of the black soil, and showed a slight influence on available S in the subsoil. The organic S forms were predominantly composed of ester S and residual S in the two soils. Under long-term fertilization, the residual S significantly increased over 32% and 55% in the topsoil and subsoil layers, respectively, compared with CK. The ester S and carbon-bonded S, which were relatively active, were less affected by the fertilization treatments, but positively related to the level of organic carbon in each soil (P < 0.05). In addition, the results from the long-term experiments indicated that the contribution of S input from atmospheric deposition was significant and should not be neglected.

Aqp5 is a new transcriptional target of Dot1a and a regulator of Aqp2.

Dot1l encodes histone H3 K79 methyltransferase Dot1a. Mice with Dot1l deficiency in renal Aqp2-expressing cells (Dot1l(AC)) develop polyuria by unknown mechanisms. Here, we report that Aqp5 links Dot1l deletion to polyuria through Aqp2. cDNA array analysis revealed and real-time RT-qPCR validated Aqp5 as the most upregulated gene in Dot1l(AC) vs. control mice. Aqp5 protein is barely detectable in controls, but robustly expressed in the Dot1l(AC) kidneys, where it colocalizes with Aqp2. The upregulation of Aqp5 is coupled with reduced association of Dot1a and H3 dimethyl K79 with specific subregions in Aqp5 5' flanking region in Dot1l(AC) vs. control mice. In vitro studies in IMCD3, MLE-15 and 293Tcells using multiple approaches including real-time RT-qPCR, luciferase reporter assay, cell surface biotinylation assay, colocalization, and co-immunoprecipitation uncovered that Dot1a represses Aqp5. Human AQP5 interacts with AQP2 and impairs its cell surface localization. The AQP5/AQP2 complex partially resides in the ER/Golgi. Consistently, AQP5 is expressed in none of 15 normal controls, but in all of 17 kidney biopsies from patients with diabetic nephropathy. In the patients with diabetic nephropathy, AQP5 colocalizes with AQP2 in the perinuclear region and AQP5 expression is associated with impaired cellular H3 dimethyl K79. Taken together, these data for the first time identify Aqp5 as a Dot1a potential transcriptional target, and an Aqp2 binding partner and regulator, and suggest that the upregulated Aqp5 may contribute to polyuria, possibly by impairing Aqp2 membrane localization, in Dot1l(AC) mice and in patients with diabetic nephropathy.

CRL Ubiquitin Ligases and DNA Damage Response.

Cullin/RING ubiquitin ligases (CRL) comprise the largest subfamily of ubiquitin ligases. CRLs are involved in cell cycle regulation, DNA replication, DNA damage response (DDR), development, immune response, transcriptional regulation, circadian rhythm, viral infection, and protein quality control. One of the main functions of CRLs is to regulate the DDR, a fundamental signaling cascade that maintains genome integrity. In this review, we will discuss the regulation of CRL ubiquitin ligases and their roles in control of the DDR.

Tailor-made magnetic Fe3O4@mTiO2 microspheres with a tunable mesoporous anatase shell for highly selective and effective enrichment of phosphopeptides.

Selective enrichment of phosphoproteins or phosphopeptides from complex mixtures is essential for MS-based phosphoproteomics, but still remains a challenge. In this article, we described an unprecedented approach to synthesize magnetic mesoporous Fe(3)O(4)@mTiO(2) microspheres with a well-defined core/shell structure, a pure and highly crystalline TiO(2) layer, high specific surface area (167.1 m(2)/g), large pore volume (0.45 cm(3)/g), appropriate and tunable pore size (8.6-16.4 nm), and high magnetic susceptibility. We investigated the applicability of Fe(3)O(4)@mTiO(2) microspheres in a study of the selective enrichment of phosphopeptides. The experiment results demonstrated that the Fe(3)O(4)@mTiO(2) possessed remarkable selectivity for phosphopeptides even at a very low molar ratio of phosphopeptides/non-phosphopeptides (1:1000), large enrichment capacity (as high as 225 mg/g, over 10 times as that of the Fe(3)O(4)@TiO(2) microspheres), extreme sensitivity (the detection limit was at the fmol level), excellent speed (the enrichment can be completed in less than 5 min), and high recovery of phosphopeptides (as high as 93%). In addition, the high magnetic susceptibility allowed convenient separation of the target peptides by magnetic separation. These outstanding features give the Fe(3)O(4)@mTiO(2) composite microspheres high benefit for mass spectrometric analysis of phosphopeptides.

The E3 ubiquitin ligase Rnf8 stabilizes Tpp1 to promote telomere end protection.

The mammalian shelterin component TPP1 has essential roles in telomere maintenance and, together with POT1, is required for the repression of DNA damage signaling at telomeres. Here we show that in Mus musculus, the E3 ubiquitin ligase Rnf8 localizes to uncapped telomeres and promotes the accumulation of DNA damage proteins 53Bp1 and γ-H2ax. In the absence of Rnf8, Tpp1 is unstable, resulting in telomere shortening and chromosome fusions through the alternative nonhomologous end-joining (A-NHEJ) repair pathway. The Rnf8 RING-finger domain is essential for Tpp1 stability and retention at telomeres. Rnf8 physically interacts with Tpp1 to generate Ubc13-dependent Lys63 polyubiquitin chains that stabilize Tpp1 at telomeres. The conserved Tpp1 residue Lys233 is important for Rnf8-mediated Tpp1 ubiquitylation and localization to telomeres. Thus, Tpp1 is a newly identified substrate for Rnf8, indicating a previously unrecognized role for Rnf8 in telomere end protection.

[Effects of calcium on cadmium bioavailability in lateritic red soil and related mechanisms].

A pot experiment with Cd-polluted lateritic red soils was conducted to study the effects of applying different concentration (0, 40, 100, and 200 mg x kg(-1)) Ca on the rape biomass, its Cd uptake, and the Ca and Cd concentrations in soil solution. Comparing with no Ca application, applying Ca increased the rape dry mass, whether under high or low level Cd pollution. The increment of the dry mass in two cropping seasons was averagely 5.5% (low level Cd pollution) and 17.3% (high level Cd pollution). The Ca concentration both in soil solution and in rape plant increased markedly with increasing Ca application rate. At the Ca application rate 100 mg x kg(-1), the Cd concentration in soil solution increased by 74.5% (low Cd pollution) and 31.0% (high level Cd pollution), while that in rape plant decreased by 4.5% (low Cd pollution) and 13.1% (high level Cd pollution). There was a positive relationship between the Ca/Cd (mass ratio) in soil solution and the Cd concentration in rape plant under both low and high levels Cd pollution. The Ca/Cd (mass ratio) in soil solution affected the bioavailability of soil Cd, and further, affected the Cd up-take by rape.

Identification of MSA1, a cell cycle-regulated, dosage suppressor of drc1/sld2 and dpb11 mutants.

The Dpb11 and Drc1/Sld2 proteins form a complex that is critical for the initiation of DNA replication. In this study we identify MSA1 as a high copy suppressor of a drc1-1 mutant. MSA1 overproduction can also suppress the temperature sensitivity of dpb11-1 and pol2-12 mutants. Reciprocally, msa1 deletion exacerbates the mutant phenotypes of both drc1/sld2 and dpb11 mutants and msa1 deletion alone results in a delay in S phase entry of synchronous cells indicating a positive role for MSA1 in DNA replication. Paradoxically, MSA1 overproduction is deleterious to cdc6-1, cdc7-1, cdc28-1N and cdc14-1 mutants indicating a complex relationship with DNA replication and cell cycle regulatory genes. The Msa1 protein is tightly cell cycle regulated. Msa1 and its paralog, Msa2, both accumulate in highly modified forms just as cells commit to enter S phase and then are rapidly destroyed. MSA1 represents a new cell cycle regulated gene important for S phase entry.