A RORγt+ cell instructs gut microbiota-specific Treg cell differentiation.

The mutualistic relationship of gut-resident microbiota and the host immune system promotes homeostasis that ensures maintenance of the microbial community and of a largely non-aggressive immune cell compartment1,2. The consequences of disturbing this balance include proximal inflammatory conditions, such as Crohn's disease, and systemic illnesses. This equilibrium is achieved in part through the induction of both effector and suppressor arms of the adaptive immune system. Helicobacter species induce T regulatory (Treg) and T follicular helper (TFH) cells under homeostatic conditions, but induce inflammatory T helper 17 (TH17) cells when induced Treg (iTreg) cells are compromised3,4. How Helicobacter and other gut bacteria direct T cells to adopt distinct functions remains poorly understood. Here we investigated the cells and molecular components required for iTreg cell differentiation. We found that antigen presentation by cells expressing RORγt, rather than by classical dendritic cells, was required and sufficient for induction of Treg cells. These RORγt+ cells-probably type 3 innate lymphoid cells and/or Janus cells5-require the antigen-presentation machinery, the chemokine receptor CCR7 and the TGFß activator αv integrin. In the absence of any of these factors, there was expansion of pathogenic TH17 cells instead of iTreg cells, induced by CCR7-independent antigen-presenting cells. Thus, intestinal commensal microbes and their products target multiple antigen-presenting cells with pre-determined features suited to directing appropriate T cell differentiation programmes, rather than a common antigen-presenting cell that they endow with appropriate functions.

BCR selection and affinity maturation in Peyer's patch germinal centres.

The antigen-binding variable regions of the B cell receptor (BCR) and of antibodies are encoded by exons that are assembled in developing B cells by V(D)J recombination1. The BCR repertoires of primary B cells are vast owing to mechanisms that create diversity at the junctions of V(D)J gene segments that contribute to complementarity-determining region 3 (CDR3), the region that binds antigen1. Primary B cells undergo antigen-driven BCR affinity maturation through somatic hypermutation and cellular selection in germinal centres (GCs)2,3. Although most GCs are transient3, those in intestinal Peyer's patches (PPs)-which depend on the gut microbiota-are chronic4, and little is known about their BCR repertoires or patterns of somatic hypermutation. Here, using a high-throughput assay that analyses both V(D)J segment usage and somatic hypermutation profiles, we elucidate physiological BCR repertoires in mouse PP GCs. PP GCs from different mice expand public BCR clonotypes (clonotypes that are shared between many mice) that often have canonical CDR3s in the immunoglobulin heavy chain that, owing to junctional biases during V(D)J recombination, appear much more frequently than predicted in naive B cell repertoires. Some public clonotypes are dependent on the gut microbiota and encode antibodies that are reactive to bacterial glycans, whereas others are independent of gut bacteria. Transfer of faeces from specific-pathogen-free mice to germ-free mice restored germ-dependent clonotypes, directly implicating BCR selection. We identified somatic hypermutations that were recurrently selected in such public clonotypes, indicating that affinity maturation occurs in mouse PP GCs under homeostatic conditions. Thus, persistent gut antigens select recurrent BCR clonotypes to seed chronic PP GC responses.

Author Correction: c-MAF-dependent regulatory T cells mediate immunological tolerance to a gut pathobiont.

In this Letter, the 'Competing interests' statement should have stated: 'D.R.L. consults for and has equity in Vedanta Biosciences.' The original Letter has not been corrected.

c-MAF-dependent regulatory T cells mediate immunological tolerance to a gut pathobiont.

Both microbial and host genetic factors contribute to the pathogenesis of autoimmune diseases. There is accumulating evidence that microbial species that potentiate chronic inflammation, as in inflammatory bowel disease, often also colonize healthy individuals. These microorganisms, including the Helicobacter species, can induce pathogenic T cells and are collectively referred to as pathobionts. However, how such T cells are constrained in healthy individuals is not yet understood. Here we report that host tolerance to a potentially pathogenic bacterium, Helicobacter hepaticus, is mediated by the induction of RORγt+FOXP3+ regulatory T (iTreg) cells that selectively restrain pro-inflammatory T helper 17 (TH17) cells and whose function is dependent on the transcription factor c-MAF. Whereas colonization of wild-type mice by H. hepaticus promoted differentiation of RORγt-expressing microorganism-specific iTreg cells in the large intestine, in disease-susceptible IL-10-deficient mice, there was instead expansion of colitogenic TH17 cells. Inactivation of c-MAF in the Treg cell compartment impaired differentiation and function, including IL-10 production, of bacteria-specific iTreg cells, and resulted in the accumulation of H. hepaticus-specific inflammatory TH17 cells and spontaneous colitis. By contrast, RORγt inactivation in Treg cells had only a minor effect on the bacteria-specific Treg and TH17 cell balance, and did not result in inflammation. Our results suggest that pathobiont-dependent inflammatory bowel disease is driven by microbiota-reactive T cells that have escaped this c-MAF-dependent mechanism of iTreg-TH17 homeostasis.

CRISPR/Cas9-induced structural variations expand in T lymphocytes in vivo.

CRISPR/Cas9 has been adapted to disrupt endogenous genes in adoptive T-lymphocyte therapy to prevent graft-versus-host disease. However, genome editing also generates prevalent deleterious structural variations (SVs), including chromosomal translocations and large deletions, raising safety concerns about reinfused T cells. Here, we dynamically monitored the progression of SVs in a mouse model of T-cell receptor (TCR)-transgenic T-cell adoptive transfer, mimicking TCR T therapeutics. Remarkably, CRISPR/Cas9-induced SVs persist and undergo clonal expansion in vivo after three weeks or even two months, evidenced by high enrichment and low junctional diversity of identified SVs post infusion. Specifically, we detected 128 expanded translocations, with 20 615 as the highest number of amplicons. The identified SVs are stochastically selected among different individuals and show an inconspicuous locus preference. Similar to SVs, viral DNA integrations are routinely detected in edited T cells and also undergo clonal expansion. The persistent SVs and viral DNA integrations in the infused T cells may constantly threaten genome integrity, drawing immediate attention to the safety of CRISPR/Cas9-engineered T cells mediated immunotherapy.

Development of a specific and sensitive diagnostic PCR for rapid molecular authentication of the medicinal plant Portulaca oleracea.

Adulteration by Bacopa monnieri (BM) in Portulaca oleracea (PO) plants frequently occurs; it decreases the efficacy of traditional Chinese medicine (TCM) and leads to fraud in the herbal marketplace. In this study, a diagnostic PCR assay was established for the rapid authentication of PO and BM in the herbal market. The sequence divergences in internal transcribed spacer 2 (ITS2) between PO and its adulterant species were used to design diagnostic PCR primers. The specific designed primer sets were evaluated and show that the diagnostic PCR assay can be used to verify the authenticity of PO and BM. The detection limits of the primer set for PO and BM identification were 10 pg and 1 pg, respectively. The reactivity of diagnostic PCR was 0.1% PO genomic DNA and 0.01% BM genomic DNA in the test sample during DNA amplification. In addition, multiplex PCR (mPCR) for PO and BM identification was also established. The samples were more susceptible to the effect of steaming in authentication by singleplex PCR and mPCR than boiling and drying treatment. Furthermore, commercial samples from the market were used to demonstrate the applicability of the developed diagnostic PCR for PO authentication and diagnose BM adulteration, and the investigation found that approximately 72.2% (13/18) of PO plants in the herbal market were adulterated. In conclusion, the diagnostic PCR assay was successfully developed and its specificity, sensitivity and reactivity for PO and BM authentication were proven. These developed PCR-based molecular methods can be applied as an identification tool for PO authenticity and can be practically applied for inspection of BM adulteration in the herbal market in the future.

Genetic Authentication of the Medicinal Plant Portulaca oleracea Using a Quick, Precise, and Sensitive Isothermal DNA Amplification Assay.

Portulaca oleracea (PO) is a commonly known medicinal crop that is an important ingredient for traditional Chinese medicine (TCM) due to its use as a vegetable in the diet. PO has been recorded to be frequently adulterated by other related species in the market of herbal plants, distorting the PO plant identity. Thus, identification of the botanical origin of PO is a crucial step before pharmaceutical or functional food application. In this research, a quick assay named "loop-mediated isothermal amplification (LAMP)" was built for the specific and sensitive authentication of PO DNA. On the basis of the divergences in the internal transcribed spacer 2 (ITS2) sequence between PO and its adulterant species, the LAMP primers were designed and verified their specificity, sensitivity, and application for the PO DNA authentication. The detection limit of the LAMP assay for PO DNA identification specifically was 100 fg under isothermal conditions at 63 °C for 30 min. In addition, different heat-processed PO samples can be applied for use in PO authentication in the LAMP assay. These samples of PO were more susceptible to the effect of steaming in authentication by PCR than boiling and drying treatment. Furthermore, commercial PO samples pursued from herbal markets were used to display their applicability of the developed LAMP analysis for PO postharvest authentication, and the investigation found that approximately 68.4% of PO specimens in the marketplace of herbal remedies were adulterated. In summary, the specific, sensitive, and rapid LAMP assay for PO authentication was first successfully developed herein, and its practical application for the inspection of adulteration in PO samples from the herbal market was shown. This LAMP assay created in this study will be useful to authenticate the botanical origin of PO and its commercial products.

Comprehensive analysis of PTEN-related ceRNA network revealing the key pathways WDFY3-AS2 - miR-21-5p/miR-221-3p/miR-222-3p - TIMP3 as potential biomarker in tumorigenesis and prognosis of kidney renal clear cell carcinoma.

Kidney renal clear cell carcinoma (KIRC) is one of the most common malignancies, and there is still a lack of effective biomarkers for early detection and prognostic prediction. In here, we compared the characteristics of RNA sequencing data sets of KIRC samples based on the tumor suppressor gene phosphatase and tensin homolog (PTEN). The 1016 long noncoding RNAs, 48 microRNAs (miRNAs), and 2104 messenger RNAs associated with PTEN were identified and these genes were differentially expressed between tumor and paracancerous tissues. The most relevant pathway was found to be WDFY3-AS2 - miR-21-5p/miR-221-3p/miR-222-3p - TIMP3 according to the rules of competing endogenous RNA (ceRNA) regulation. WDFY3-AS2 and TIMP3 expression were positively correlated and reduced in KIRC samples, while miR-21-5p, miR-221-3p, and miR-222-3p were relatively highly expressed. The relatively low expression of WDFY3-AS2 and TIMP3 in KIRC were associated with poor prognosis in KIRC patients, while higher expression of miR-21-5p, miR-221-3p, and miR-222-3p predicted reduced survival (p < 0.05). Univariate and multivariate Cox regression analysis showed that lower expression of WDFY3-AS2 and TIMP3 was significantly related to tumor grade, tumor size, lymph node metastasis, distant metastasis, and TNM stage. The expression of TIMP3 in KIRC tissues was also verified by immunohistochemistry, and the results were consistent with our analytical data. In summary, this study constructed a new model with clinical predictive value and identified the WDFY3-AS2/TIMP3 pathway that was closely associated with the prognosis of KIRC, which could serve as a promising biomarker for the diagnosis and treatment of KIRC.

Differential ultrastructural alterations in the Vglut2 glutamatergic input to the substantia nigra pars compacta/pars reticulata following nigrostriatal dopamine loss in a progressive mouse model of Parkinson's disease.

Loss of nigrostriatal dopamine (DA) in Parkinson's disease results in over-activation/bursting of the subthalamic nucleus (STN). The STN projects to the substantia nigra (SN) pars compacta (SNpc) and pars reticulata (SNpr). The vesicular glutamate transporter 2 (Vglut2) is localized within at least STN terminals synapsing within the SN, but it is not known if there are differential changes in the Vglut2+ input to the SNpc versus SNpr following DA loss. The goal/rationale of this current study was to determine whether there were differential changes in the density/levels of glutamate immuno-gold labeling within Vglut2+ nerve terminals synapsing in the SNpc/SNpr and in the proportion of Vglut2+ terminals contacting tyrosine hydroxylase (TH) positively(+) or negatively(-) labeled dendrites following DA loss. Within the SNpc, there was a significant increase (51.3%) in the density of nerve terminal glutamate immuno-gold labeling within Vglut2+ terminals synapsing on TH(-) dendrites following MPTP versus the vehicle (VEH) group. There was a significant decrease (16%) in the percentage of Vglut2+ terminals contacting TH(+) labeled dendrites in the MPTP- versus VEH-treated group within the SNpc. Within the SNpr, there was a significant decrease in the density of glutamate immuno-gold labeling in Vglut2+ terminals contacting TH(+) (71.5%) and TH(-) (55.5%) labeled dendrites, suggesting an increase in glutamate release. There was no change in the percentage of Vglut2+ terminals contacting TH(+) or TH(-) dendrites in the SNpr. We conclude that there is a differential effect following DA loss on the glutamate input from Vglut2+ terminals synapsing within the SNpr versus SNpc.

Circ_RPL23A acts as a miR-1233 sponge to suppress the progression of clear cell renal cell carcinoma by promoting ACAT2.

Clear cell renal cell carcinoma (ccRCC) is a prevalent urological carcinoma with high metastatic risk. Circular RNAs (circRNAs) have been identified as effective diagnostic and therapeutic biomarkers for ccRCC. This research aims to disclose the effect and regulatory mechanism of circRNA ribosomal protein L23a (circ_RPL23A) in ccRCC. We performed quantitative real-time polymerase chain reaction (qRT-PCR) to examine circ_RPL23A, microRNA-1233 (miR-1233) and acetyl-coenzyme A acetyltransferase 2 (ACAT2). Cell cycle progression, apoptosis, cell viability, invasion and migration, which were respectively conducted by using flow cytometry, 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyl tetrazolium bromide (MTT), transwell assays. The levels of ACAT2 protein and cell cycle proteins, proliferation-associated protein, and epithelial-mesenchymal transition (EMT) associated proteins were measured by western blot. Target relationship was analyzed via dual-luciferase reporter assay and RNA pull down assay. The animal model was used to study how circ_RPL23A affects in vivo. Circ_RPL23A was lower expressed in ccRCC tissues and cells. The elevated circ_RPL23A suppressed cell cycle progression, proliferation, migration and invasion but promoted apoptosis in ccRCC cells. MiR-1233 was a target of circ_RPL23A and direct targeted to ACAT2. Besides, circ_RPL23A exerted its anti-tumor effect by sponging miR-1233, and then relieved the inhibition effect of miR-1233 on ACAT2. Overexpression of circ_RPL23A also curbed ccRCC tumor growth in vivo. Circ_RPL23A inhibited ccRCC progression by upregulating ACAT2 expression by competitively binding miR-1233, which might provide an in-depth cognition for ccRCC pathogenesis and circ_RPL23A might be a promising biomarker in ccRCC diagnosis and treatment.

Focused specificity of intestinal TH17 cells towards commensal bacterial antigens.

T-helper-17 (TH17) cells have critical roles in mucosal defence and in autoimmune disease pathogenesis. They are most abundant in the small intestine lamina propria, where their presence requires colonization of mice with microbiota. Segmented filamentous bacteria (SFB) are sufficient to induce TH17 cells and to promote TH17-dependent autoimmune disease in animal models. However, the specificity of TH17 cells, the mechanism of their induction by distinct bacteria, and the means by which they foster tissue-specific inflammation remain unknown. Here we show that the T-cell antigen receptor (TCR) repertoire of intestinal TH17 cells in SFB-colonized mice has minimal overlap with that of other intestinal CD4(+) T cells and that most TH17 cells, but not other T cells, recognize antigens encoded by SFB. T cells with antigen receptors specific for SFB-encoded peptides differentiated into RORγt-expressing TH17 cells, even if SFB-colonized mice also harboured a strong TH1 cell inducer, Listeria monocytogenes, in their intestine. The match of T-cell effector function with antigen specificity is thus determined by the type of bacteria that produce the antigen. These findings have significant implications for understanding how commensal microbiota contribute to organ-specific autoimmunity and for developing novel mucosal vaccines.

Identification of nitrogen-sources in an aquifer beneath a municipal solid waste landfill in the vicinity of multiple pollutant sources.

Nitrogen contamination of groundwater has become a global issue and has aroused considerable concern among authorities. However, it is difficult to trace nitrogen sources in settings where a municipal solid waste (MSW) landfill site co-exists with intensive agriculture and other human activities. Therefore, a field investigation that combined a statistical analysis (factor analysis: FA) and hydrochemical analysis was designed and undertaken to identify nitrogen-pollutant sources in the shallow groundwater beneath an MSW landfill near to an agricultural area and human settlement. The results of the case study showed that nitrate was the specific pollutant produced by agricultural non-point-sources (Pbi = 15.5) and domestic pollution sources (Pbi = 41.0). The total phosphorus (Pbi = 37.2) and organic matter (Pbi = 16.6) were the specific pollutants released by the aquaculture and animal husbandry point-sources, and chloride (Pbi = 75.4) and organic matter (Pbi = 16.1) were the specific pollutants produced by the landfill. In the investigated area, the domestic pollution sources and agricultural non-point-sources were the most likely sources of nitrate contamination in the shallow aquifer. However, the landfill source and the aquaculture and animal husbandry point sources were the most likely sources of ammonium contamination. The combined method used in this study could successfully identify the nitrogen pollution sources in the shallow groundwater beneath an MSW landfill located in the vicinity of multiple pollutant sources. The method could be used to improve the control of nitrogen contamination and the management of groundwater quality.

Effects of sleep disruption on stress, nigrostriatal markers, and behavior in a chronic/progressive MPTP male mouse model of parkinsonism.

Sleep complaints are an early clinical symptom of neurodegenerative disorders. Patients with Parkinson's disease (PD) experience sleep disruption (SD). The objective of this study was to determine if preexisting, chronic SD leads to a greater loss of tyrosine hydroxylase (TH) within the striatum and the substantia nigra following chronic/progressive exposure with the neurotoxin, 1-methyl-2-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Male mice underwent chronic SD for 4 weeks, then injected with vehicle (VEH) or increasing doses of MPTP for 4 weeks. There was a significant decrease in the plasma corticosterone levels in the MPTP group, an increase in the SD group, and a return to the VEH levels in the SD+MPTP group. Protein expression levels for TH in the striatum (terminals) and substantia nigra pars compacta (dopamine [DA] cell counts) revealed up to a 78% and 38% decrease, respectively, in the MPTP and SD+MPTP groups compared to their relevant VEH and SD groups. DA transporter protein expression increased in the striatum in the MPTP versus VEH group and in the SN/midbrain between the SD+MPTP and the VEH group. There was a main effect of MPTP on various gait measures (e.g., braking) relative to the SD or VEH groups. In the SD+MPTP group, there were no differences compared to the VEH group. Thus, SD, prior to administration of MPTP, has effects on serum corticosterone and gait but more importantly does not potentiate greater loss of TH within the nigrostriatal pathway compared to the MPTP group, suggesting that in PD patients with SD, there is no exacerbation of the DA cell loss.

Effect of the loam inter-layer on the migration and breakthrough of benzene under constant flow in the unsaturated zone: column experiments.

The reliable prediction of transport and attenuation of dissolved-phase contamination in the unsaturated zone is a complex and multi-process problem. Based on the adsorption properties of soil samples to solutes, the soil column test and laboratory analysis were carried out in this study. The effects of the loam inter-layer on the migration and breakthrough of the characteristic pollutant benzene and non-absorbent Br- were studied. The results showed that the relatively high clay content of the inter-layer significantly changed the BTC (breakthrough curve). It not only delayed the migration time of benzene into the aquifer but also to some extent produced an attenuation effect, effectively reducing the content of the characteristic pollutants through the unsaturated zone. The dispersion coefficient was obtained through the measured Br-. The theoretical values were calculated and compared with the experimental data by using a one-dimensional unsaturated solute transport equation. The result was basically consistent, which proved the validity and reliability of the model. Through the BTC of benzene, the retardation factor was obtained and used to describe the influence of the loam inter-layer on the migration and breakthrough, which could provide the basis for the accurate modeling of groundwater remediation projects.

Photochemical Vapor Generation of Tellurium: Synergistic Effect from Ferric Ion and Nano-TiO2.

Photochemical vapor generation (PVG) is emerging as a promising analytical tool for Te determination, thanks to its efficient matrix separation, and simple and green procedure. However, the low PVG generation efficiency of Te is the bottleneck for its wide application in environmental samples containing trace Te. Herein, we reported a high efficient PVG for Te determination by synergistic effect of ferric ion and nano-TiO2. The analytical sensitivity was enhanced approximately 15-fold for Te(IV) in the presence of both ferric ions and nano-TiO2, comparing to conventional PVG. Besides, the use of nano-TiO2 can provide Te(VI) and Te(IV) an equal and high PVG efficiency in the presence of ferric ions, owned to the high photocatalytic performance of TiO2 under short-wavelength UV irradiation (254 and 185 nm). Under the optimized experimental conditions, a detection limit of 1.0 ng L-1 was obtained. The precision of replicate measurements was 2.3% (RSD, n = 7) at 0.5 µg L-1 for Te(IV). The methodology was validated by successful determination of Te in surface waters and two standard reference sediment samples. To our best knowledge, this is the first report of the synergistic enhancement of transitional metal ions and nano-TiO2 in PVG, which possesses potential for highly sensitive determination of vapor-forming elements.

GADD45α is involved in the apoptosis of lymphocytes induced by riboflavin and ultraviolet light.

BACKGROUND: Riboflavin plus ultraviolet (UV) pathogen reduction technology (RF-PRT) is an effective method for inactivating the residual white blood cells (WBCs) in blood components. The RF-PRT system for platelets is known to activate many signaling pathways, including p38 and NF-κB. Nevertheless, proteomic studies in WBCs after riboflavin plus UV treatment requires further analysis. STUDY DESIGN AND METHODS: ABO/D-matched lymphocytes were pooled, split, and treated with RF-PRT or UV light or left untreated. After treatment, cell apoptosis was measured. In addition, cell proliferation and the cycle distribution were evaluated upon stimulation with phytohemagglutinin. The changes in the protein expression levels of growth arrest and DNA damage-inducible (GADD)45α, p38, and c-Jun N-terminal kinase (JNK) were determined by Western blotting. The effect of GADD45α, p38, and JNK on apoptosis was assessed. RESULTS: RF-PRT significantly inhibited proliferation and induced G1 arrest in lymphocytes. Furthermore, the percentage of apoptotic cells was increased in RF-PRT-treated lymphocytes compared to UV-treated cells or untreated cells, associated with the up regulation of GADD45α expression. Consistent with these observations, the inhibition of GADD45α expression partially counteracted the effects of riboflavin plus UV treatment. The p38 and JNK signaling pathways were activated by GADD45α in RF-PRT-treated lymphocytes. CONCLUSIONS: These data revealed that RF-PRT effectively inhibited proliferation and induced apoptosis of lymphocytes by promoting GADD45α expression, which subsequently activates p38 and JNK signaling pathways.

H3.3-H4 tetramer splitting events feature cell-type specific enhancers.

Previously, we reported that little canonical (H3.1-H4)(2) tetramers split to form "hybrid" tetramers consisted of old and new H3.1-H(4) dimers, but approximately 10% of (H3.3-H4)2 tetramers split during each cell cycle. In this report, we mapped the H3.3 nucleosome occupancy, the H3.3 nucleosome turnover rate and H3.3 nucleosome splitting events at the genome-wide level. Interestingly, H3.3 nucleosome turnover rate at the transcription starting sites (TSS) of genes with different expression levels display a bimodal distribution rather than a linear correlation towards the transcriptional activity, suggesting genes are either active with high H3.3 nucleosome turnover or inactive with low H3.3 nucleosome turnover. H3.3 nucleosome splitting events are enriched at active genes, which are in fact better markers for active transcription than H3.3 nucleosome occupancy itself. Although both H3.3 nucleosome turnover and splitting events are enriched at active genes, these events only display a moderate positive correlation, suggesting H3.3 nucleosome splitting events are not the mere consequence of H3.3 nucleosome turnover. Surprisingly, H3.3 nucleosomes with high splitting index are remarkably enriched at enhancers in a cell-type specific manner. We propose that the H3.3 nucleosomes at enhancers may be split by an active mechanism to regulate cell-type specific transcription.

Metal ion-assisted photochemical vapor generation for the determination of lead in environmental samples by multicollector-ICPMS.

A novel and sensitive approach for the accurate and precise determination of Pb in environmental samples is presented using transition metal ion-assisted photochemical vapor generation (PVG) for sample introduction with multicollector inductively coupled plasma mass spectrometry (MC-ICPMS) detection. A significant improvement in PVG efficiency of lead is achieved in the presence of transition metal ions (Co(2+) and Ni(2+)) in solutions of 5% (v/v) formic acid. The determination of Pb in digests of sediment or soil samples is readily achieved due to coexisting transition metal ions which facilitate the PVG reaction. The method detection limit of 0.005 ng g(-1) (3σ) using external calibration is comparable to that obtained using hydride generation (HG) ICPMS. However, PVG methodology is simpler, results in lower blanks, and avoids unstable reagents. The accuracy of the proposed method was demonstrated by analysis of several environmental certified reference materials (CRMs; SLRS-5 and SRM1640a river water CRMs and MESS-3, MESS-4, and SRM2702 sediments) with satisfying results. High precision of determination (<0.4% RSD) of Pb in river water and sediments was realized on the basis of isotope dilution calibration.

A model for mitotic inheritance of histone lysine methylation.

Histone lysine methylation has been implicated in epigenetic regulation of transcription. Using stable-isotope labelling and quantitative mass spectrometry, we analysed the dynamics of histone lysine methylation. Here we report that histone methylation levels are transiently reduced during S phase and are gradually re-established during subsequent cell cycle stages. However, despite the recovery of overall methylation levels before the next S phase, the methylation levels of parental and newly incorporated histones differ significantly. In addition, histone methylation levels are maintained at steady states by both restriction of methyltransferase activity and the active turnover of methyl groups in cells undergoing an extended G1/S phase arrest. Finally, we propose a 'buffer model' that unifies the imprecise inheritance of histone methylation and the faithful maintenance of underlying gene silencing.