Due to the stringent population bottleneck that occurs during sexual HIV-1 transmission, systemic infection is typically established by a limited number of founder viruses. Elucidation of the precise forces influencing the selection of founder viruses may reveal key vulnerabilities that could aid in the development of a vaccine or other clinical interventions. Here, we utilize deep sequencing data and apply a genetic distance-based method to investigate whether the mode of sexual transmission shapes the nascent founder viral genome. Analysis of 74 acute and early HIV-1 infected subjects revealed that 83% of men who have sex with men (MSM) exhibit a single founder virus, levels similar to those previously observed in heterosexual (HSX) transmission. In a metadata analysis of a total of 354 subjects, including HSX, MSM and injecting drug users (IDU), we also observed no significant differences in the frequency of single founder virus infections between HSX and MSM transmissions. However, comparison of HIV-1 envelope sequences revealed that HSX founder viruses exhibited a greater number of codon sites under positive selection, as well as stronger transmission indices possibly reflective of higher fitness variants. Moreover, specific genetic “signatures” within MSM and HSX founder viruses were identified, with single polymorphisms within gp41 enriched among HSX viruses while more complex patterns, including clustered polymorphisms surrounding the CD4 binding site, were enriched in MSM viruses. While our findings do not support an influence of the mode of sexual transmission on the number of founder viruses, they do demonstrate that there are marked differences in the selection bottleneck that can significantly shape their genetic composition. This study illustrates the complex dynamics of the transmission bottleneck and reveals that distinct genetic bottleneck processes exist dependent upon the mode of HIV-1 transmission. While the global spread of HIV-1 has been fueled by sexual transmission the genetic determinants underlying the transmission bottleneck remains poorly understood. Here we characterized founder virus population diversity from next generation sequencing data in a cohort of 74 acute and early HIV-1 infected individuals. We observe that the risk of multi-variant infection in men-who-have-sex-with-men (MSM) is not greater than that observed for heterosexuals (HSX), contrary to reports of higher rates of multiple founder virus infections in higher-risk MSM transmissions. These findings were further supported through a metadata analysis of 354 acute and early HIV-1 subjects. We did, however, observe differences between HSM and MSM founder viruses, including a higher selection barrier in HSX transmission with founder viruses being more cohort consensus-like that may be reflective of increased replicative fitness. We also identified a number of residues within Envelope that behave in a risk-dependent manner and could be key for HIV-1 transmission. These novel insights improve our understanding of the HIV-1 transmission bottleneck and underscore the differential selective pressures that founder viruses within the two major transmission risk groups are subjected to. The global spread of HIV-1 has been fueled predominantly by HSX transmission, with MSM representing a second major risk group –6. WhileA number of genetic, immunologic and phenotypic signatures of founder viruses, predominantly located within the envelope glycoprotein (Env), have been identified that affect HIV-1 entry , 21–28. To date, attributes particular to HIV-1 founder viruses have been identified by comparing these sequences to larger datasets of contemporaneous chronic viruses , 25–27, env founder virus sequences from 127 subjects (Dataset 1) for which the multiplicity of infection had previously been determined [in silico sequencing reads with multiple replicates generated resulting in a dataset of 7.9 million env reads. Calculation of the APHD score for each subject from each replicate demonstrated a lack of significant difference between the simulated data and actual data suggesting that modeling sample variation and incorporating the error profiles associated with 454 sequencing has not had an undue effect on the classification of subjects. We conclude, therefore, that the APHD approach is suitable for discriminating between homogeneous and heterogeneous infections, and represents an alternative approach to discriminate between single and multiple founder viruses.To adapt the application of more sensitive and higher-throughput next-generation deep sequencing data, where the shorter sequencing reads makes the inference of haplotype reconstruction difficult, we implemented a Hamming distance-based genetic approach, termed here the average (over read pairs) pairwise hamming distance (APHD). This method calculates the number of mismatches between reads in a sliding window of a defined size to analyze the level of genetic diversity within the viral quasispecies. To validate this approach we applied it to previously published SGA/S-derived termined , 19  or HSX (n = 2) exposure including source plasma donors (SPD) (n = 6), or through percutaneous exposure (n = 1) or injecting drug use , see . To redugag (IQR: 285–794), 402 for pol (IQR: 203–679) and 600 for the 3′ genomic half (IQR: 359–862). Only 4 subjects had a 3’ genomic half sequencing depth < 200-fold with the remainder of subjects harboring sufficient sequence coverage to detect variants at 1%. Utilizing env sequences the APHD scores derived from the 74 acute HIV-1 infected subjects ranged from 0.0002 to 1.580 across different Fiebig stages  were established by a single founder virus.To provide a more comprehensive and informative genome-wide analysis of multiple founder viruses, we PCR amplified the entire protein-coding region of HIV-1 in conjunction with 454 deep sequencing. While sequence coverage varied between samples and amplicons there was no significant difference in sequence coverage between amplicons with a median sequencing depth of 501 for g stages . The logg stages . NotablyP = 0.167, Chi-Square test; P = 0.177, Chi-Square test). Taken together, these findings compiled from a number of different studies indicate that at the time of sampling there are no detectable differences in terms of multiplicity of infection between different modes of transmission.Given that our findings were contrary to the prevailing understanding that higher risk infections such as MSM transmission frequently exhibit multiple founder viruses and that the results of such studies exploring the multiplicity of infection in HSX, MSM and IDU vary widely  7, 14, , 47–49, Given that we observed no differences in the number of founder viruses between MSM and HSX infection we sought to investigate whether other differences in the founder viruses may exist between the two modes of transmission. To do so we assembled a collection of 131 founder viruses (Dataset 3) derived from acute clade B HIV-1 infected subjects with a defined HSX (55 subjects) or MSM (76 subjects) exposure , 34, 44,P = 3 x 10−5, To determine whether other differences exist between HSX and MSM founder viruses we next compared their ‘transmission index’, which was shown to be predictive of which sequence in the donor will establish infection in the recipient . In thisRecently, Gnanakaran and colleagues reported on a number of sequence motifs in HIV-1 Env associated with founder viruses . In partq value <0.2 and R633 with a q value <0.3  or <0.4  or <0.4  were significant at P<0.05 when using the opposite variable (i.e. MSM), the models are nonetheless distinct and did identify different associations at the q-value cutoffs. From this analysis 7 sites (8 residues) were statistically associated with HSX founder viruses at a .4 K617, . All of q = 0.086; Four of these sites  were found to overlap in both analyses and thus were found to be under significant, but opposing, selection in both HSX and MSM founder viruses. For example, at position Q389, the presence of proline is positively associated with HSX founder viruses while conversely the absence of proline is associated with MSM founder viruses. Specifically, only 1% (1 subject) of MSM sequences contain a proline at position Q389 while 20% (11 subjects) of HSX sequences contain this proline substitution. This Q389 signature site was only seen as significant when taken into account its association with a strongly covarying and highly conserved proline at residue P417  around the CD4 binding loop with at least 6 of the 8 MSM signature sites identified in gp120. Namely I165, T283, N362, Q389, E429, and G471, have previously been shown to alter HIV-1 replication, CD4 dependence and/or lie proximal to the CD4 biding site –69. The P = 0.026, Chi-Square test). Thus, the higher frequency of K343E could be driven in part by its higher frequency in chronic circulating strains. Conversely, for 8 of the signature sites (9 residues)  there was no evidence supporting an influence of differences in chronic residue frequencies impacting the transmission of these variants . Therefore, the presence of proline appears to be strongly selected for at transmission during HSX infection. Although it is difficult to decipher the origin of these selection events, since higher chronic frequencies could be similarly driven by differences selected at the time of transmission, at least 9 residues are strongly supportive of selection occurring upon transmission followed by regression during chronic infection. Thus, taken together we hypothesize that some of these variants may modestly improve overall fitness and hence be selected for during transmission and conversely selected against  over the course of ensuing chronic infection.Finally, we sought to determine whether the aforementioned HSX and MSM signature sites described in variants . For exaenv (such as gag and pol) to assess the complexity of founder virus populations with the caveat that the sensitivity may vary. More importantly, these data demonstrated that the majority (83%) of MSM infections in our cohort exhibited a single founder virus—levels similar to those typically characteristic of HSX infections. Further characterization of these data demonstrated that HSX founder viruses do, however, appear to be under different selective pressures than MSM founder viruses, with HSX founder viruses subject to broader, albeit weaker, diversifying selective pressure than their MSM counterparts. Distinct genetic footprints were also found to be specific to HSX and MSM founder virus populations, supporting discrete selection pressures exhibited by each mode of sexual transmission.In the present study we build upon a large body of work characterizing acute HIV-1 infection by shedding new light on the genetic properties of founder viruses distinguished by the two primary risk groups responsible for driving the global HIV-1 pandemic. Development of an average pairwise Hamming distance (APHD) approach, benchmarked to SGA/S data as the gold standard method, enabled us to distinguish between single and multiple founder virus infections using deep sequencing data. Application of this approach can also be extended to other genomic regions other than P = 0.003). Importantly, although differences in the number of founder viruses could be attributable to compartmentalization in the source, examination of viruses in a cohort of Zambian transmission pairs found no evidence for preferential selection in the donor genital tract [The increasing use of next-generation sequencing has led to the development of specialized computational tools to reconstruct the viral haplotypes that constitute the quasispecies within a single host –74, and al tract . Thus, oWhile it is clear that various bottlenecks can limit the number of HIV-1 founder viruses successfully transmitted from a diverse, chronically infected donor (as recently reviewed in ), factorGiven that the majority of our HSX subjects were men (79% of our 131 founder virus dataset), and that within MSM the risk from unprotected receptive anal intercourse is >10 fold higher than for insertive anal intercourse , our stuGiven previous studies have espoused differences in variable loop length and potential N-linked glycosylation site count in the transmitted virus for HIV-1 subtypes A and C , 24, 32,in vitro [In contrast, nearly half of the residues associated with MSM risk were located in gp120 with six residues  clustered around the CD4-binding pocket with the potential to influence CD4 binding . In addiin vitro . Within in vitro , while fin vitro . Thus, mP = 0.66). The reported higher transmission indices for HSX founder viruses also continued to be significant when compared with MSM founder viruses . Thus, the overall study findings are unlikely to have been influenced by the inclusion of this subject group alone.A limitation of this study is the inclusion of subjects designated as source plasma donors (SPD). These subjects had limited behavioral information available but as part of routine blood-banking practice underwent extensive questioning for HIV-1 risk behaviors and denied having sex for money, homosexual activity or i.v. drug use. Nevertheless, self-reporting of risk behaviors among paid plasma donors is imperfect and it is plausible that some subjects whom were designated as belonging to the HSX risk group as previously categorized , 34 may While HIV-1 acquisition in MSM may be immunologically and virologically distinct from that of heterosexual exposure, we observe no differences in the number of HIV-1 founder viruses. Although the severe HIV-1 transmission bottleneck has a stochastic component in which any reasonably fit CCR5-tropic virus may be capable of establishing productive infection, our data do argue that any selection bias may be comparatively relaxed with ano-rectal MSM transmission, potentially due to the greater frequency of target cells at the site of transmission and the distinct kinetics of virus dissemination –90. ConvPlasma samples were obtained from subjects with acute or early HIV-1 infection enrolled in HIV-1 cohorts in Berlin, Germany, and Massachusetts, California, North Carolina, and South Carolina, USA. The clinical and sociodemographic characteristics of the study participants can be found in HIV-1 was PCR amplified and 454 sequenced using a nested RT-PCR with 3 amplicons overlapping the genome. Briefly, viral RNA was isolated from 1ml of plasma using the QiAmp Viral RNA Mini Kit  and RT-PCR of near full-length HIV-1 genomes performed using nested-PCR primers specific for gag, pol and 3′ half of the viral genome see . Pooled Following alignment of cleaned reads to the consensus assembly sequence a sliding window approach was used to collect all reads that covered a window of 120bp with a step size of 21bp. We then calculated the pairwise Hamming distance (HD)  for all reads and averaged this value over all windows to obtain the average pairwise Hamming distance (APHD). To formalize the criteria and evaluate the APHD approach for its discriminative ability we obtained SGA/S sequence data from studies where each sample had been previously designated as infection by a single virus or infection by multiple viruses , 19. To To differentiate between single and multiple founder viruses using 454 sequencing reads intra-patient HIV-1 genetic diversity was first characterized as previously described, with nucleotide-phasing information used to distinguish true variants from sequencing errors see . The meacDNA was serially diluted and amplified as previously described , 17, 112A number of previous published datasets were used throughout this study and are numbered accordingly. Briefly, Dataset 1 comprising the SGA/S data collected from 127 acute individuals sampled at varying times post-infection and clinically staged as described by Fiebig et al  were usep-value is obtained). In this model the main assumption is that a single founder virus evolves under neutral evolution, generating a star-like phylogeny, with a distribution of mutations conforming to a Poisson distribution [PoissonFitter was used to test the hypothesis that a single virus establishes infection . Poissonribution , 14.q-value cutoff, all associations identified using one variable were significant at P<0.05 when using the opposite variable. The q-value is the minimal false discovery rate that adjusts for multiple tests [q-value threshold is context specific and depends on how the results will be interpreted. In the present study, we typically report all tests where q is <0.2  but sometimes report higher q values to include sites in a hypothesis-raising framework. The associations identified in this study are referred to as adapted and nonadapted forms. Adapted forms (commonly referred in many studies as escape variants) are amino acids significantly enriched for in the presence of the risk behavior in question. Nonadapted forms  are amino acids significantly depleted in the presence of the risk behavior.We used the phylogenetically corrected logistic regression to identify sites positively or negatively associated with MSM or HSX state , 55. Brile tests . The appTo distinguish the pattern of selection between MSM and HSX founder viruses we used a comparative codon-based phylogenetic framework test implemented in HyPhy that formally tests whether selective pressures are intensified or relaxed relative to a subset of branches . In thisIn the context of heterosexual linked transmission pairs, we previously trained a model that estimates the probability that any particular amino acid will be transmitted from a donor to a recipient . AlthougAll statistical analysis was performed using JMP Pro, version 12 (SAS Institute). Descriptive measures were used to summarize the data. Continuous variables were summarized using median and inter quartile range (IQR); categorical variables were summarized using frequency and percent (%). Chi-square and Mann-Whitney tests were used to compare categorical and continuous variables between the study groups, respectively.S1 Text(DOC)Click here for additional data file.S1 Fig(A) Heatmap from the 454 sequencing data illustrating a diverse number of sites throughout the 3′ half of the genome showing up to 40% codon diversity. (B) The APHD plots showing a mean APHD of 0.306 (red line) and standard deviation (dotted black line) demonstrating a high level of diversity across the 3′ half of the HIV-1 genome. (C) SGA sequences displaying a phylogeny (left) revealing infection by at least four viruses with inter-lineage recombinants. Founder virus lineages are color-coded while recombinant sequences are shown by green symbols. Highlighter plots (right) compare sequences for each subject’s sequence set to an intrasubject consensus (uppermost sequence) and depict the pattern of nucleotide base mutations. Subject 882283 was viral RNA positive but Western blot negative (Fiebig stage II/III of infection).(TIF)Click here for additional data file.S2 Fig(A) Heatmap from the 454 sequencing data illustrating a diverse number of sites throughout the 3′ half of the genome showing up to 40% codon diversity. (B) The APHD plots showing a mean APHD of 0.593 (red line) and standard deviation (dotted black line) demonstrating a high level of diversity across the 3′ half of the HIV-1 genome. (C) SGA sequences displaying a phylogeny (left) revealing infection by at least four viruses with inter-lineage recombinants. Founder virus lineages are color-coded while recombinant sequences are shown by green symbols. Highlighter plots (right) compare sequences for each subject’s sequence set to an intrasubject consensus (uppermost sequence) and depict the pattern of nucleotide base mutations. Subject 702865 was viral RNA positive but Western blot indeterminate (Fiebig stage IV of infection).(TIF)Click here for additional data file.S3 Fig(A) Heatmap from the 454 sequencing data illustrating a diverse number of sites throughout the 3′ half of the genome showing up to 40% codon diversity. (B) The APHD plots showing a mean APHD of 1.718 (red line) and standard deviation (dotted black line) demonstrating a high level of diversity across the 3′ half of the HIV-1 genome. (C) SGA sequences displaying a phylogeny (left) revealing infection by at least four viruses with inter-lineage recombinants. Founder virus lineages are color-coded while recombinant sequences are shown by green symbols. Highlighter plots (right) compare sequences for each subject’s sequence set to an intrasubject consensus (uppermost sequence) and depict the pattern of nucleotide base mutations. Subject 574194 was viral RNA positive but Western blot positive with 3 bands (Fiebig stage V of infection).(TIF)Click here for additional data file.S4 FigSGA sequences derived from this study (red) were compared to sequences derived from Keele et al.  in which(TIF)Click here for additional data file.S5 Fig(A) Sites that show the same frequency trend in chronic and acute infection are depicted. (B) Sites that show the opposite trend in chronic and acute infection are shown. Sites that showed a statistically significant difference between the chronic stage of infection at a P value of less than 0.05 (Chi-Square test) are indicated by a single asterisk (*).Frequency comparison of previously found signatures sites in our panel of HSX and MSM founder viruses compared to a dataset of chronic HSX and MSM viruses. Chronic viruses comprised 462 SGA/S sequences derived from 24 chronically infected subjects who reported heterosexual as their risk factor for HIV-1 infection and 867 SGA/S sequences from 35 chronically infected MSM subjects. Sites and amino acids examined are listed on the x-axis with the frequency of that amino acid at that position shown on the y-axis. Acute HSX (red bars), acute MSM (blue bars), chronic HSX (red striped bars) and chronic MSM (blue striped bars). (TIF)Click here for additional data file.S6 Fig(A) Distribution of point mutation errors across the HIV-1 genome from 3 independent sequencing runs of an HIV-1 NL4-3 plasmid control. (B) Effect of PCR amplification on point mutation variant mismatches across three independent PCR and sequencing runs.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Prospective surveillance of invasive mold diseases (IMDs) in haematology patients should be standard of care but is hampered by the absence of a reliable laboratory prompt and the difficulty of manual surveillance. We used a high throughput technology, natural language processing (NLP), to develop a classifier based on machine learning techniques to screen computed tomography (CT) reports supportive for IMDs.We conducted a retrospective case-control study of CT reports from the clinical encounter and up to 12-weeks after, from a random subset of 79 of 270 case patients with 33 probable/proven IMDs by international definitions, and 68 of 257 uninfected-control patients identified from 3 tertiary haematology centres. The classifier was trained and tested on a reference standard of 449 physician annotated reports including a development subset (n = 366), from a total of 1880 reports, using 10-fold cross validation, comparing binary and probabilistic predictions to the reference standard to generate sensitivity, specificity and area under the receiver-operating-curve (ROC).For the development subset, sensitivity/specificity was 91% (95%CI 86% to 94%)/79% (95%CI 71% to 84%) and ROC area was 0.92 (95%CI 89% to 94%). Of 25 (5.6%) missed notifications, only 4 (0.9%) reports were regarded as clinically significant.CT reports are a readily available and timely resource that may be exploited by NLP to facilitate continuous prospective IMD surveillance with translational benefits beyond surveillance alone. Despite the high health and economic burden Aspergillus and hyaline molds is positive in 50% or fewer of cases For surveillance in general, the primary screening method should have a high sensitivity in order to minimise the burden of case finding while maximising case capture. However, for epidemiological surveillance of IMDs the ideal screening method is undefined. Laboratory-based surveillance is subject to significant underreporting because IMDs may be diagnosed with or without microbiological confirmation corresponding to probable/proven and possible categories respectively Although the optimal screening method for epidemiological surveillance of IMDs is undefined, the high frequency of pulmonary involvement makes chest computed tomography (CT) imaging an attractive target for screening. Chest CT is routinely performed when IMD is suspected as it is a non-invasive test that is widely available with results reported within hours rather than days and pulmonary involvement is present in 90% to 100% of patients with IA We hypothesized that epidemiological surveillance of IMDs that is cost-effective and sustainable could be facilitated by technology. Natural language processing (NLP) is a computational method of analyzing human language that has detected several medical conditions This was a retrospective case-control cohort study of patients from three tertiary adult university-affiliated hospitals . AH and RMH operate statewide haematopoietic stem cell transplant (HSCT) services which collectively perform approximately100 allogeneic transplants/year. De-identified patient records were used and the human research ethics committees at each site who granted permission for the study, waived patient consent.Case and uninfected control patients from 2003 to 2011 inclusive were identified from previously completed clinical mycology studies CT reports from haematology/HSCT patients with (i.e. cases) and without IMDs (i.e. controls) were manually downloaded from each hospital as de-identified text files. CT reports, from performance of the diagnostic scan and for 12 weeks thereafter  performed during the clinical encounter were included. The clinical encounter was defined from admission to either discharge, death or transfer. Clinical information was extracted from aforementioned study datasets and hospital records. IMDs were classified according to consensus definitions by expert reviewers A randomly selected convenience subset of reports from case and control patients were annotated at sentence and scan level by three infectious diseases physicians . The primary reviewer (MAR) annotated all case and control reports. Secondary review of case reports was undertaken by two physicians . The secondary reviewers served to validate the primary reviewer’s analysis through measures of agreement. Pre-specified annotation guidelines were refined with differences in opinion resolved by discussion at face-to-face meetings. An iterative process of annotation ensued with reports from case patients annotated twice or three times over, while those from control patients  were annotated once by the primary reviewer (MAR).For annotation, each sentence within a report and each report was treated as an independent observation, meaning that for sentence level annotation, sentences rather than specific words were coded according to the following contextual features: specificity for IMD ; certainty , directionality/change ; temporality ; alternative processes  and clinical alerts (i.e. urgent follow-up required). Scan level annotation referred to classification of the entire report as being either supportive or not for IMD with equivocal scans subsequently merged with supportive reports. Supportive reports had specific features of IMD including halo, nodule, cavity, focal mass, wedge-shaped lesions, bony sinus erosions. Negative reports had none of the above features while equivocal reports included infiltrate, consolidation, ground glass change, effusions or uncertainty by the reporting radiologist.Importantly, the reference standard was informed by the opinion of clinician experts rather than radiologists given that the guiding principle was to flag reports of concern to end-users irrespective of the final diagnostic outcome.Reports were classified in a binary fashion as being supportive or not supportive of IMD (i.e. positive/negative). A multi-class classification approach at sentence level was used: each sentence allocated one of a number of classes, with all sentence-level classifications subsequently informing the report level decision.Text was analyzed as groups of words (“bag-of-words”), phrases (“bag-of-phrases”) and concepts (“bag-of-concepts”) We adopted a supervised machine learning approach experimenting with several machine learning algorithms including Support Vector Machines (SVM), Naïve Bayes, Random Forests, and Bayesian Nets, as implemented in the Weka 3.6.0 Physician annotated reports were divided into development (n = 366) and held-out sets (n = 83), the latter annotated at scan level only. We used 10-fold cross validation on the development set with the optimal classifier identified from experiments, tested against the held-out set which served as an additional validation step. Cross-validation is an accepted method within the machine learning domain that maximizes limited gold standard data while minimizing the risk of overfitting associated with training on the test set The results of each manually annotated report was compared to the binary and probabilistic predictions of the classifier allowing calculation of sensitivity, specificity and receiver operating curve (ROC).Hypothetical IMD prevalence rates of 5%, 10% and 20% were used to estimate positive and negative predictive values . Inter-annotator agreement was assessed using Cohen’s kappa coefficient, a chance corrected index of agreement 9 cells/L) was present in 82% and 75% of clinical encounters among case and control patients respectively and was prolonged (median 18 and 19 days respectively). A history of HSCT was present in 46% and 52% of case and control patients, being allogeneic in 86% and 77% of patients respectively.A total of 147 patients were included in the annotated subset of 449 reports; 79 (54%) had IMDs and 68 (46%) were control patients . NeutropScedosporium and Rhizopus species identified in 10/33 (30%).IMDs were probable/proven in 33/79 (42%) and possible infections in 46/79 (58%). Sinus and/or pulmonary disease occurred in 91% of case-patients. IA comprised 20/33 (61%) of microbiologically confirmed cases with rare molds including Overall, 1880 reports were retrieved from 527 patients (51% with IMDs) . A totalInter-annotator agreement between primary reviewer and the two secondary reviewers combined, was fair at sentence level for distinguishing certainty from equivocal/negative labels (K = 0.64) but improved at report level for the same classification (0.83) given that K levels ≥0.75 represent excellent agreement Classifier performance among development and held-out sets respectively, was as follows : sensitiOf the 25 missed cases , 4  were significant as shown in Review of 45 false positive reports revealed several sources of systematic error described in Mold infections are not well suited to prospective detection using manual methods of epidemiological surveillance due to the absence of an easily identifiable electronic prompt but may be rendered amenable to real-time detection using NLP of CT reports. Our classifier flagged reports suggestive of IMD from a variety of anatomic sites but overwhelmingly the sino-pulmonary tract (92%), the site most commonly involved by IA For screening purposes, sensitivity is favoured over specificity especially for uncommon events like IMDs because missed cases are less tolerable than the resources spent following up false notifications In the absence of a gold standard for IMD reporting we relied upon peer review. Sentence level agreement between the primary and secondary annotator pair was fair (K = 0.64) despite measures mitigating unreliability including pre-specified guidelines, multiple experts and repeated consultation to resolve differences Our classifier has several limitations. Its poor PPV was not unexpected as PPV is highly conditional upon disease prevalence and, for uncommon events like IMDs, will be low despite a high sensitivity, as we observed Further improvements in specificity may be achieved by omitting progress reports given their overrepresentation among false notifications. Experts often annotated progress reports negative because diagnostic radiological features may not always be re-iterated in a progress report. Non-specific pulmonary lesions such as atelectasis or scarring could be disregarded with the creation of handcrafted rules. Pulmonary nodules of questionable significance are more challenging to address, as size of lesions was not taken into account by the classifier. The development dataset did not exclusively comprise proven/probable-IMDs (despite these representing a higher degree of certainty) for several reasons: possible IMDs constitute a substantial burden in clinical practice and may predominate (up to 90%) in some centres The classifier is not a diagnostic adjunct but rather a screening tool designed to facilitate IMD case finding by exploiting routinely available clinical data Epidemiological surveillance for IMDs is needed for many reasons, including antifungal stewardship


Psychological stress during pregnancy increases the risk of childhood wheeze and asthma. However, the transmitting mechanisms remain largely unknown. Since epigenetic alterations have emerged as a link between perturbations in the prenatal environment and an increased disease risk we used whole genome bisulfite sequencing (WGBS) to analyze changes in DNA methylation in mothers and their children related to prenatal psychosocial stress and assessed its role in the development of wheeze in the child. We evaluated genomic regions altered in their methylation level due to maternal stress based of WGBS data of 10 mother-child-pairs. These data were complemented by longitudinal targeted methylation and transcriptional analyses in children from our prospective mother-child cohort LINA for whom maternal stress and wheezing information was available (n = 443). High maternal stress was associated with an increased risk for persistent wheezing in the child until the age of 5. Both mothers and children showed genome-wide alterations in DNA-methylation specifically in enhancer elements. Deregulated neuroendocrine and neurotransmitter receptor interactions were observed in stressed mothers and their children. In children but not in mothers, calcium- and Wnt-signaling required for lung maturation in the prenatal period were epigenetically deregulated and could be linked with wheezing later in children’s life. The psychosocial health of a pregnant woman can significantly impact the growing fetus and predispose offspring toward increased risk of developing obstructive airway diseases later in life. Although maternal stress during pregnancy has been linked to the development of repeated wheeze, asthma, or airway hyper-responsiveness1234678211In the current study we assessed the perceived stress level of women during the course of their pregnancy and were able to confirm previous findings showing a positive association of the prenatal maternal stress levels with an increase in risk for the child to develop persistent wheezing. Our central aim was to assess possible biological mechanisms by which prenatal stress translates into lung dysfunction in the child.It is hypothesized that transient effects of environmental exposure early in life, in this case prenatal maternal stress, can be preserved by epigenetic mechanisms (e.g. DNA methylation) that silence or activate disease relevant signaling pathways in a persistent way and thereby may modify disease susceptibilityFurther information and details are available in the Lifestyle and environmental factors and their Influence on Newborns Allergy risk), which originally included 629 mother–child pairs  recruited between May 2006 and December 2008 in Leipzig, Germanyet al.This study is based on a subset of the prospective mother-child cohort LINA 17th week of gestation were subjected to whole genome bisulfite sequencing . Using the bsseq v0.10 package for R statistical software v3.0.0 we followed a slightly adapted approachet al.24Whole blood samples of 5 mother-child pairs with a low stress score compared to 5 pairs with a high stress score were evaluated. Bisulfite converted DNA from cord blood and venous blood of mothers obtained at the 36Methylation differences of selected differentially methylated regions (DMRs) were assessed using the MassARRAY system , a mass spectrometry based approach for targeted methylation analysisEqual parameter distribution in our sub- and the entire LINA cohort was tested using Chi-square. To assess whether maternal stress, or DNA methylation and gene expression changes observed in our subcohort contribute to an increased risk for the child to develop persistent wheeze logistic regression models were implemented adjusting for known confounding factors of lung disease in early childhoodAfter logarithmic transformation of DNA methylation and gene expression values adjusted mean ratios (MR), i.e. the ratio of the geometric mean, were calculated to evaluate their relationship to the maternal stress level. Confounding factors used were: gender of the child, smoking during pregnancy, ETS exposure after birth (where applicable), age of the mother at time of birth, birth week, mode of delivery, maternal medication during pregnancyThe median total stress score of mothers in our subcohort was 1.85 . Total stress score grouping into quartiles resulted in n = 110, 221 and 112 mothers with a low, medium and high stress level respectively .Urinary homovanillic acid concentration was significantly elevated in mothers with high compared to mothers reporting low stress. This effect sustained even after adjusting for maternal medication a possible influencing factor of homovanillic acid production . A total of 2306 stress-dependent differentially methylated regions  that may destroy the CpG context and thereby directly induce differential DNA methylation, SNPs may also create or disrupt transcription factor binding sites (TFBS) affecting the level of DNA methylationThe total number of DMRs was similar in mothers and children. In children the number of hypermethylated DMRs was significantly higher than in mothers -preferentially overlapped with functional regulatory regions . The majAnalysis of DMR overlap with tissue/-cell type specific enhancer regions of the ENCODE roadmapIn a subsequent analysis we evaluated the overlap of DMRs with TFBS . Using E3233NR3C1), the main regulator of the glucocorticoid (cortisol in humans) mediated stress response, were significantly enriched for an overlap with DMRs in children  enriched only in children for stress-induced DMRs . Further35NMUR1), a GPCR known to be involved in hypothalamic-pituitary-adrenal axis (HPA) response37NMUR1 DMR significantly increased dependent on the maternal stress score , although with a much smaller effect size than observed by WGBS. Comparing methylation levels in the same children obtained by WGBS and MassARRAY respectively reveals that this is most likely related to a much smaller dynamic range of the validation method in this region . Correspondingly, a significant decrease in the expression of NMUR1 in children born to highly stressed compared to those born to lowly stressed mothers was observed , In children, we determined a hypermethylated ngDMR in the gene of neuromedin U receptor 1 . Applying linear regression models adjusted for possible confounders of interleukin concentrations observed in cord blood revealed a significant relationship between NMUR1 methylation and IL-4, and IL-6 concentrations at time of birth only in those children developing late or persistent wheeze later in their life’s .Together with the perturbed protein secretion of IL-4, IL-5, and IL-6 the differential methylation in the GNA11 gene coding for the alpha subunit of the G protein of the G11 class. GNA11 is assigned to the KEGG “Calcium signaling pathway” linking neuroendocrine/-transmitter signaling to calcium release . The GNA11 gDMR contained eight subsequent CpGs in a poised enhancer region  (GNA11 gDMR (adj. OR = 1.41 (1.01–1.98)) with a concomitant lower level of GNA11 gene expression (adj. OR = 0.48 (0.24–0.95)) when compared to non-symptomatic children (Mean methylation of this = 0.042) . High le= 0.042) . Among pchildren .CACNB4. The hypermethylated ngDMR located in a weak enhancer region in the CACNB4 gene likely interacts with its host gene promoter based on ENCODE ChIA-PET data (Table E3). Multiple regression analysis revealed a significant relationship of CACNB4 methylation and maternal stress score at time of birth  and NFATC3. While no differential transcription was observed for PPP3CA (data not shown), PPP3R1 was significantly elevated in four-year-old children dependent on maternal stress . A positive correlation of the stress score with NFATC3 expression was only observed in late and persistently wheezing children. For both PPP3R1 and NFATC3 no correlation of maternal stress score and transcription was observed in children without any respiratory symptoms  To elucidate the functional impact of these DMRs, we determined transcriptional changes of their downstream targets. For the Wnt/Ca2+ -signaling pathway we evaluated transcription of symptoms .CTNNB1 (beta-catenin) showed a stress-dependent increase of transcription already at time of birth , in four-year-old children expression of both CTNNB1 and AXIN2  were significantly elevated in children of highly stressed mothers  compared to those of low stressed mothers. Furthermore, elevated CTNNB1 expression levels at time of birth were associated with an increased risk for the child to develop late or persistent wheeze later in life (adj. OR = 1.78 (1.28–2.89)). At year four differential transcription related to late or persistent wheeze was no longer observed neither for CTNNB1 nor for AXIN2 .While Maternal distress during pregnancy has been shown to persistently affect the health of the child in different ways. Premature birth, low birth weight42454615051Given that the prenatal period is susceptible to external stimuli that can shape the epigenetic landscape and thereby determine disease susceptibility later in life the aim of the current study was to evaluate global DNA methylation changes relating prenatal maternal stress to the increased risk for the child to develop wheezing.We studied differential DNA methylation at time of birth by WGBS of 10 mother-child-pairs complemented by longitudinal targeted methylation and transcriptional analysis in over 300 children. Our WGBS approach offered us the advantage of a global evaluation of DNA methylation changes not restricted to a selected set of CpGs as in commonly used DNA methylation arrays. This is noteworthy since only 5% of our identified DMRs showed an overlap with CpG sites covered by the Infinium HumanMethylation450 BeadChip array, which would have left the vast majority of the maternal-stress-dependent DMRs undiscovered.Both mothers and children showed genome-wide perturbations in DNA-methylation affecting genomic regulatory elements in particular enhancer elements. Although this preferential deregulation in DNA methylation has already been described in disease states especially in cancer53A variety of different biological pathways showed an enrichment of DMRs with a minimal overlap between mothers and children. DNA methylation is thought to be subjected to age-dependent changes55NR3C1 (“glucocorticoid receptor”) as the key mediator of this response has been associated with different types of stressful life events. However, the extent and direction of the alteration in DNA methylation vary dependent on the kind of stress experienced596062NR3C1 with maternal depression or pregnancy related anxiety however no differential methylation was observed related to maternal stress assessment64NR3C1 related DMR. However, we found a significant enrichment of DMRs overlapping with binding sites of NR3C1. This suggests that perturbations in cortisol mediated signaling might also occur independent of a direct effect of maternal stress on NR3C1 methylation in the child.Stressful life events have been widely related to changes in the cortisol mediated stress responseIt has been accepted that perturbations in the mammalian stress response contribute to an aberrant immune response and may adversely affect lung development in prenatally exposed children13PPP3R1) results in immaturity of the lungCalcineurin/NFAT signaling is activated by GPCR-mediated intracellular calcium increase. In knockout mice it has been shown that calcineurin/NFAT signaling controls lung maturation prior to birth and deletion of the calcium binding subunit calcineurin b  observed at birth.The hypermethylated DMR in an enhancer of NMUR1 we identified in children of highly stressed mothers was confirmed in the entire LINA cohort. Although the observed methylation difference in the larger sample subset was rather small, this seems to be related to a much narrower distribution of methylation values observed by MassARRAY compared to WGBS. The around 5% higher methylation found in prenatally stressed children might be related to a deregulated subset of T cells producing Th2 cytokines. GNA11) and G alpha iGNA11 and PLCB4 “phospholipase C beta 4” showed a hypermethylated enhancer regions related to prenatal maternal stress. For GNA11 this was associated with decreased expression in both prenatally stressed and children with wheezing symptoms. Similar observations for the calcium channel coding CACNB4 gene further suggest an involvement of calcium signaling in impaired lung function.Activation of NMUR1/2, coupled to G alpha q/11 .


In order to study angiogenesis driven by other growth factors, we developed a basal medium to use on a co-culture cord formation system of adipose derived stem cells (ADSCs) and endothelial colony forming cells (ECFCs). We found that cord formation driven by different angiogenic factors led to unique phenotypes that could be differentiated and combination studies indicate dominant phenotypes elicited by some growth factors. VEGF-driven cords were highly covered by smooth muscle actin, and bFGF-driven cords had thicker nodes, while EGF-driven cords were highly branched. Multiparametric analysis indicated that when combined EGF has a dominant phenotype. In addition, because this assay system is run in minimal medium, potential proangiogenic molecules can be screened. Using this assay we identified an inhibitor that promoted cord formation, which was translated into in vivo tumor models. Together this study illustrates the unique roles of multiple anti-angiogenic agents, which may lead to improvements in therapeutic angiogenesis efforts and better rational for anti-angiogenic therapy.Vascular endothelial growth factor (VEGF) plays a dominant role in angiogenesis. While inhibitors of the VEGF pathway are approved for the treatment of a number of tumor types, the effectiveness is limited and evasive resistance is common. One mechanism of evasive resistance to inhibition of the VEGF pathway is upregulation of other pro-angiogenic factors such as fibroblast growth factor (FGF) and epidermal growth factor (EGF). Numerous  Angiogenesis, the formation of new blood vessels from existing vessels, is a complex multistep process involving numerous growth factors. These steps include initiation, tip formation and sprouting, migration, proliferation, lumen formation, anastamosis, and maturation Inhibitors targeting the VEGF pathway have had some success in the clinic; however, the effects of anti-angiogenic therapy tend to result in transitory improvements measured in months. These treatments result in tumor stasis and shrinkage with some resulting in increased survival. Inevitably, however, the tumors return to growth and progression in many patients. A number of possible evasive resistance pathways have been proposed Numerous in vitro and in vivo assays have been developed to examine the various steps in the angiogenic process including sprouting and tip formation, migration, differentiation, proliferation, lumen formation, and tube or cord formation 2. The next day, the media was removed and 4–5K ECFCs were plated on the ADSC monolayer, incubated at 37°C, 5% CO2 for 3–6 hours to allow ECFC attachment before the addition of growth factors and/or inhibitors (2–5X) to achieve the indicated final concentrations. The differences in cell counts reflect differences observed with different cell counters. For validation experiments, a modified assay to increase pericyte association was used whereby 15K ADSCs and 3K ECFCs were plated in a 384-well plate. When indicated cell bound growth factors were removed from the ADSC monolayer by a 60 minute treatment with 500 µg/mL sodium heparin (Sigma) in basal medium prior to ECFC addition. Co-cultures were grown for 3 days, at which time they were fixed, stained, and imaged as described below.Human adipose derived stem cells (ADSCs) and endothelial colony forming cells (ECFCs) purchased from Lonza  were cultured as previously described Multiple doses of growth factors were used to determine the optimal dose to use for subsequent studies (data not shown). Vascular endothelial growth factor  was used at 10–20 ng/mL, hepatocyte growth factor  at 100 ng/mL basic fibroblast growth factor  at 50 ng/mL, and epidermal growth factor  at 20 ng/mL. To block VEGF and HGF signaling, antibodies to VEGF  and HGF  were used. To examine whether additional growth factors were dependent on each other to induce cords, inhibitors to VEGFR2 (Ramucirumab), bFGF , or EGFR (Gefitinib) were used in basal, VEGF, bFGF, and EGF driven cords. Finally, a TGF-β inhibitor (LY2157299), an inactive control (LY596144), and a multikinase inhibitor  were tested to examine increases in cord or tumor vessels.Following completion of the assay, the ADSC/ECFC co-culture was fixed and permeabilized with either 70% ice cold ethanol or 80% ethanol  for 10–20 minutes. Cells were stained essentially as previously described Table S1) to differentiate and assess the different growth factor dependent morphologies. Clustering of the different descriptors were performed in an unsupervised manner as to not place particular meaning on each individual parameter, but rather to utilize these characteristics as unique phenotypes.Cord formation images were capture with either a Cellomics Arrayscan VTI or an Acumen eX3 microplate cytometer. Images taken on the Arrayscan were read at a 5X magnification and the tube formation bio-application was used to detect CD31 staining. Total tube area was calculated from 9 fields for each well with 3–4 wells for each treatment. SMA index was calculated from the intensity of the SMA staining and related to the number of cords as previously described Basal medium, media collected from ADSC monocultures, media collected from ADSC/ECFC co-cultures, and media collected from cocultures treated with VEGF were analyzed for growth factors using Luminex beads according to the manufacturers' protocol (R&D Systems and Millipore).6 cells) were mixed 1∶1 with Matrigel (BD Biosciences) and injected subcutaneously into the flank of CD1 nu/nu male mice. Tumors were allowed to grow to ∼150 mm3 (∼7–10 days) prior to treatment. Animals were orally treated by oral gavage with twice a day (BID) dosing of vehicle, a TGF-β inhibitor , an inactive control , or a multi-tyrosine kinase inhibitor targeting VEGF  for 5 days prior to fixation and staining. Tumors were fixed in Zn Tris, processed, and embedded in paraffin. Four micron sections were cut and stained with a CD31 antibody  and Alexa Fluor 488 conjugated secondary antibodies. Each tumor was imaged at 10× with an inverted fluorescence microscope looking at multiple fields residing completely within the tumor that were well-separated and non-overlapping. The number of fields imaged ranged between 2 to 11 fields and depended upon the size of tumor and amount of non-necrotic tissue present. The following total number of fields was imaged for each group: non-treated (43), LY2157299 (45), LY596144 (31), and Sutent (45). The field data for total area of blood vessels (using CD31) as a ratio of total tissue area (using Hoechst 33342) for individual tumors was averaged to derive the individual tumor vessel density values. The individual tumor values within a group were averaged to derive the group vessel density values expressed with standard errors and P-values. Four to eight animals were analyzed in each treatment group.A549 NSCLC tumor cells  Figure S2). Quantitative validation was performed on a similar assay ran on the Acumen eX3 in a 384 well format .Studies indicate that co-cultures of ADSCs and ECFCs lead to robust cord formation with associated SMA positive pericyte-like structures in response to VEGF . Exogenous bFGF and EGF, two growth factors that had no detectable expression in the basal system, were able to induce cords in the ADSC/ECFC co-culture system  from whole well images of the various growth factor driven cords  blocked basal (IC50 = 0.32 µg/ml), VEGF (IC50 = 0.96 µg/ml), and EGF (IC50 = 0.14 µg/ml) driven cords, but not bFGF driven cords (IC50>10 µg/ml) Figure 6Development of a medium for the ECFC/ADSC cord formation assay which lacks serum or growth factors allows testing of potential pro-angiogenic small molecules without interference from angiogenic growth factors. It is known that TGF-β inhibits endothelial cell proliferation Neo-vascularization is a complex process with coordinated cellular signaling between precursor and mature forms of endothelial cells with stromal cells  coupled with morphological changes including endothelial tube formation, anastomosis, endothelial sprouting, and endothelial vessel stabilization by pericyte recruitment/differentiation. While vascular endothelial growth factor, VEGF, is recognized as the dominant pro-angiogenic factor, other factors such as fibroblast growth factor, epidermal growth factor, hepatocyte growth factor, angiopoietins, thrombomodulin, notch ligands, and transforming growth factor- β are involved as well. Endothelial cord formation in co-cultures of mature endothelial and smooth muscle cells are dependent on addition of VEGF, bFGF, and EGF (16) but do not inform on pericyte differentiation. Development of a basal media co-culture system of progenitor endothelial and stromal cells provide an opportunity to study the roles of multiple angiogenic factors on endothelial cord formation and pericyte differentiation. We found that HGF, bFGF, EGF, and VEGF can all drive cord formation in this system, but each has a unique phenotype that can be differentiated using multiparametric analysis. In general, we find that VEGF has small nodes with many branched and connected tubes and is associated with high levels of SMA. In contrast, bFGF driven cords have thick nodes that are not associated with SMA and EGF has thin nodes with less branching and intermediate levels of SMA. When growth factors were combined, we found that EGF drives a dominate phenotype, while other growth factors have a mixed phenotype when combined (ie VEGF + bFGF). Together these results demonstrate the unique roles of different growth factors in inducing angiogenesis and while different growth factors can induce angiogenesis, the blood vessels that form may not be the same. In addition, we show that use of a basal medium allows one to interrogate these individual roles of the growth factors and allows for the identification of other proangiogenic factors and drugs such as an inhibitor of TGF-β receptor.Evaluation of growth factors in the ADSC and ECFC co-cultures revealed expression of VEGF, HGF, TGF-β and Ang1 by the feeder layer (ADSCs). Addition of ECFCs decreased the level of VEGF in the medium. This may be a reflection of internalization and degradation of the ligand by endothelial cells bFGF and EGF signaling have been shown to play a role in revascularization of tumors after development of resistance to anti-VEGF/VEGFR2 therapy In addition to bFGF and EGF driving different phenotypic cords, we were able to demonstrate that their ability to induce cord formation is independent of VEGF. Numerous growth factors have been shown to drive angiogenesis, but many of these induce angiogenesis through upregulation of VEGF rather than a direct effect. In our system, we stimulate cord formation with VEGF, bFGF, or EGF but only the VEGF driven cords are dramatically altered by inhibition of VEGF with bevacizumab (a VEGF antibody). Even with growth factor combinations, only the VEGF driven phenotypes appeared to be blocked by the addition of bevacizumab. Similarly, inhibition of bFGF with an antibody only blocked bFGF driven cords. Likewise, an EGFR inhibitor only blocked EGF driven cords. This indicates that these angiogenic growth factors act independent of one another, but can work together to elicit particular angiogenic phenotypes. This also highlights the redundancy in proangiogenic factors and the ability of multiple growth factors to drive angiogenesis. This is particularly important in response to inhibition of a particular pathway, as what has been seen with bFGF driving angiogenesis in response to inhibition of the VEGF pathway In addition to the examination of phenotypic differences between cords driven by various growth factors and the study of dominant growth factor phenotypes in combination treatments, this ADSC/ECFC co-culture system can be used to screen for pro-angiogenic factors or drugs. To address whether this assay system can be used to identify factors that can promote cord formation and potentially be pro-angiogenic, we used a TGF-β inhibitor (LY2157299) in the cord formation assay. TGF-β is known to inhibit endothelial cell proliferation In normal epithelial and endothelial cells TGF-β is antiproliferative. However, in tumor cells, parts of the TGF-β signaling pathway are mutated and TGF-β no longer controls the cells. Both tumor and stromal cells have been shown to have increased TGF-β production which is associated with increased tumor invasiveness Despite the evidence that numerous growth factors drive angiogenesis, the majority of the in vitro studies examine a single factor or are performed in media or matrix with numerous growth factors. These studies limit one's ability to understand the different phenotypes driven by the growth factors and how combinations may lead to a particular phenotype versus another. In addition, without the use of a growth factor free medium, many proangiogenic factors may be missed. It would be interesting to relate these phenotypes with function in an in vivo setting to determine whether these phenotypes are also associated with other functional or physiological differences. This may lead to improvements in therapeutic angiogenesis efforts and or better rational for anti-angiogenic therapy in different pathological settings.Figure S1Association of SMA-positive cells to endothelial cords. Co-cultures of ADSCs and ECFCs were stained for cords (CD31) and smooth muscle actin (SMA) and imaged with the ArrayScan. High magnification images show a close association of the SMA-positive pericyte-like structures with the endothelial cords. Scale bars are 250 µm.(TIF)Click here for additional data file.Figure S2High throughput quantitative validation of the assay. (A) Co-cultures of ADSCs and ECFCs were stained for nuclei (Hoechst), cords (CD31), and smooth muscle actin (SMA) and whole wells were imaged with the ArrayScan. Grayscale images show the image of each marker and the black and white image shows what was analyzed. (B) Quantitative validation of Acumen eX3 images was performed in a 384 well assay format. Maximum, minimum and concentration response curves for total tube area and SMA area were used to calculate the overall Z′ and minimum significant ratio (MSR) for each parameter. Scale bars are 250 µm.(TIF)Click here for additional data file.Figure S3Induction of cords with HGF following heparin wash. (A) To address whether HGF can induce cord formation in the ADSC/ECFC co-culture assay, a heparin wash was performed prior to addition of HGF. After 3 days, the cords were fixed and stained for cords , smooth muscle actin , and nuclei . (B) ArrayScan quantification of heparin washed HGF induced total tube area and inhibition with an anti-HGF antibody. * p<0.05 vs basal. n = 3 per group. Scalebars are 250 µm.(TIF)Click here for additional data file.Table S1101 Image J descriptors.(TIF)Click here for additional data file.Table S2Statistical comparison from cluster analysis.(TIF)Click here for additional data file.


Vibrio cholerae encodes six resistance-nodulation-division (RND) efflux systems which function in antimicrobial resistance, virulence factor production, and intestinal colonization. Among the six RND efflux systems, VexAB exhibited broad substrate specificity and played a predominant role in intrinsic antimicrobial resistance. The VexAB system was encoded in an apparent three gene operon that included vexR; which encodes an uncharacterized TetR family regulator. In this work we examined the role of vexR in vexRAB expression. We found that VexR bound to the vexRAB promoter and vexR deletion resulted in decreased vexRAB expression and increased susceptibility to VexAB antimicrobial substrates. Substrate-dependent induction of vexRAB was dependent on vexR and episomal vexR expression provided a growth advantage in the presence of the VexAB substrate deoxycholate. The expression of vexRAB increased, in a vexR-dependent manner, in response to the loss of RND efflux activity. This suggested that VexAB may function to export intracellular metabolites. Support for this hypothesis was provided by data showing that vexRAB was upregulated in several metabolic mutants including tryptophan biosynthetic mutants that were predicted to accumulate indole. In addition, vexRAB was found to be upregulated in response to exogenous indole and to contribute to indole resistance. The collective results indicate that vexR is required for vexRAB expression in response to VexAB substrates and that the VexAB RND efflux system modulates the intracellular levels of metabolites that could otherwise accumulate to toxic levels. Vibrio cholerae is a noninvasive gram negative bacterial pathogen that causes the disease cholera. Cholera is a severe acute diarrheal disease that affects an estimated 3–5 million people per year . Thpression .vexRAB expression may be regulated in response to bile salts. To test this we cultured V. cholerae JB58 (ΔlacZ) under virulence gene inducing conditions in AKI broth containing sub-lethal concentrations of deoxycholate and measured vexRAB expression using a vexRAB-lacZ reporter (i.e. pXB233). AKI growth conditions were selected because previous studies had indicated a linkage between RND efflux and virulence gene expression [vexRAB expression in response to both erythromycin  into JB58 and its isogenic vexR deletion strain. The resulting strains were then cultured in LB broth in the presence or absence of 0.02% deoxycholate for two hours when β-galactosidase activity was quantified. The results showed that there was no significant difference in the expression of the five tested RND efflux systems in WT relative to the ΔvexR mutant under either growth condition  with pBAD18 (empty vector control) or pBAD18::vexR. The resulting strains were then grown in LB broth plus or minus 0.2% arabinose (to induce vexR expression from the arabinose inducible promoter in pBAD18) and luminescence production was measured at zero, one and two hours. The results showed that the empty vector control (i.e. pBAD18) did not affect vexRAB expression  and -59 to +21 (vexR2) relative to the ATG start site  further supported the conclusion that the vexRAB efflux system is expressed at a low basal level in the vexR mutant.The fact that sistance . Becausehenotype . TherefoD mutant . The intvexB mutant . We considered that this result could have been an artifact of the pBAD18 expression system, and therefore we cloned vexR into the low copy number IPTG-inducible pMMB66EH expression vector and repeated the complementation experiments and obtained identical results.We hypothesized that if vexR mutant is not clear. The inability to complement the vexR deletion likely did not result from the introduction of a secondary mutation during the construction of the vexR mutant since DNA sequencing confirmed the integrity of the vexR deletion construct (i.e. pWM91::ΔvexR) and the vexRAB locus in the vexR deletion strain. Further, the ΔvexR and ΔvexRΔvexD mutants were independently created from different parental strains, making it unlikely that the complementation defect was due to an unlinked spontaneous mutation. The lack of complementation was also not due to mutations introduced into the complementing plasmids as we confirmed the DNA sequence of the complementing plasmids. This latter conclusion is further supported by the observation that the presence of pBAD18::vexR activated vexRAB-lacZ expression in E. coli  and WT(pBAD18) in LB broth containing a sub-lethal concentration of deoxycholate. The results of this analysis showed that in the absence of deoxycholate, WT(pBAD18::vexR) grew equally well during logarithmic phase of growth as WT(pBAD18) through six hours  exhibited a growth advantage over the empty vector control regardless of the presence or absence of arabinose (vexR) relative to the same culture grown in the absence of arabinose  control strain was slightly attenuated relative to ΔvexB(pBAD18::vexR) in LB broth , but to a level that was less than observed in JB485 (vexB. The expression of vexRAB in a ΔvexDFHKM (vexB+) was not significantly different from WT. This latter finding was consistent with previous work showing that vexB, due to its broad substrate specificity, was able to complement for the loss of the other five RND efflux systems [vexRAB expression was responsive to the RND efflux status of the cell and that deletion of vexB resulted in vexRAB expression being elevated. The higher level of vexRAB expression in JB485 relative to the vexB mutant suggested that one or more of the other RND efflux systems were able to efflux the inducing factor(s). Based on these results we inferred that V. cholerae compensates for reductions in RND efflux activity by upregulating vexRAB expression. Exactly how V. cholerae senses efflux activity was unclear, but we speculated this resulted from the intracellular accumulation of natural substrates of the RND efflux systems in the absence of RND efflux activity.We next tested whether increased in JB485 . This su systems . Taken tV. cholerae upregulated vexRAB expression in response to the accumulation of metabolic byproducts. Our approach for these experiments was based on the assumption that the mutation of metabolic genes would disrupt biosynthetic pathways and result in the intracellular accumulation of chemical intermediates of the targeted biochemical pathway which would then activate vexRAB expression. To conduct these studies we obtained a number of metabolic mutants from a defined transposon mutant library that was constructed in V. cholerae El Tor strain C6706 [E. coli acrAB expression [We tested whether in C6706 . We selepression . This invexRAB-lux) and cultured in AKI broth for five hours when vexRAB expression was quantified. While we were most interested in the mutants that activated vexRAB expression, we noted several mutations reduced vexRAB expression. Based on the idea that vexRAB expression is modulated by metabolite accumulation, one explanation for reduced vexRAB expression is the loss of down-stream activators. However, we cannot exclude that the observed reduction of vexRAB expression was an artifact resulting from cellular byproducts inhibiting the luciferase reporter [vexRAB expression  resulted in the strongest induction of vexRAB expression suggesting that tryptophan biosynthesis intermediates may function as inducers of the vexRAB operon. To investigate this we examined vexRAB expression in six different tryptophan biosynthetic transposon mutants  is predicted to result in indole accumulation while mutation of trpA (VC1169) and trpD (VC1172) would result in indole-3-glycerol phosphate and anthranilate accumulation, respectively. This suggests that indole, indole-3-glycerol phosphate and anthranilate could contribute to vexRAB induction.Mutation of  mutants . The res mutants . MutatiovexRAB inducers, we hypothesized that adding these compounds to the growth media would activate vexRAB expression. As anthranilate and indole were available from commercial sources, we tested if the addition of these compounds affected vexRAB-lacZ expression in WT. The results showed that indole at 1.5 mM increased vexRAB expression by ~1.5 fold  on indole gradient agar plates. The results showed that the WT MIC was 2.1 mM while the JB485 MIC was 1.6 mM . Only the vexB mutant had a small but significant decrease in the indole MIC (1.8 mM), which was higher than the MIC exhibited by JB485 . In E. coli acrR functioned as a negative regulator of acrAB while global regulatory systems such as the Mar operon functioned to positively regulate acrAB expression in response to antimicrobial exposure  or the isogenic ΔvexR mutant bearing the transcriptional fusion reporters for the indicated RND efflux system were diluted 1:100 into LB broth and grown with shaking for 3.5 h at 37°C when deoxycholate (DOC) was added to 0.02%. The culture was incubated with shaking at 37°C for an additional 30 min when aliquots were collected in triplicate and β-galactosidase activity was quantified as described in the methods. (A.) vexCD; (B.) vexEF; (C.) vexGH; (D.) vexIJK; (E.) vexLM. The reported results are in Miller Units (MU) and are the mean ± SD of three independent experiments.Overnight cultures of (PDF)Click here for additional data file.S2 FigcpxP-lacZ transcriptional reporter were cultured overnight in LB broth before being diluted 1:100 into fresh LB broth. The cultures were incubated an additional hour and diluted 1,000-fold into PBS. Aliquots of the diluted cultures were then inoculated onto the surface of LB agar plates containing X-gal (160 μg/mL) plus and minus 500 μM CuCl2 (to induce expression of the Cpx system). The plates were then incubated overnight at 37°C before being photographed.The indicated strains containing a chromosomal (PDF)Click here for additional data file.S3 FigV. cholerae N16961 strains containing a vexRAB-lacZ transcriptional reporter were cultured to middle logarithmic phase in LB broth when vexRAB-lacZ expression was quantified by a β-galactosidase assay as described in the methods. The results are the average ±SD of five independent experiments.The indicated (PDF)Click here for additional data file.S4 FigV. cholerae N16961 ΔvexR containing pJB703 (pBAD18::vexR) or pBAD18 were grown in triplicate wells of microtiter plates containing (A) LB broth or (B) LB broth plus 0.015% deoxycholate. Arabinose (0.1%) was added to the media as indicated to induce expression of vexR from the arabinose regulated promoter in pBAD18. Cell growth was then monitored as the change in the optical density at 600 nm and plotted versus time as the mean ±SEM.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Arabidopsis, limited efforts have been made to address their possible role in crop like wheat. In the present work, 18 wheat ABCC-MRP proteins were identified that showed the uniform distribution with sub-families from rice and Arabidopsis. Organ-specific quantitative expression analysis of wheat ABCC genes indicated significantly higher accumulation in roots , stem (TaABCC1), leaves (TaABCC16 and TaABCC17), flag leaf (TaABCC14 and TaABCC15), and seeds  implicating their role in the respective tissues. Differential transcript expression patterns were observed for TaABCC genes during grain maturation speculating their role during seed development. Hormone treatment experiments indicated that some of the ABCC genes could be transcriptionally regulated during seed development. In the presence of Cd or hydrogen peroxide, distinct molecular expression of wheat ABCC genes was observed in the wheat seedlings, suggesting their possible role during heavy metal generated oxidative stress. Functional characterization of the wheat transporter, TaABCC13 a homolog of maize LPA1 confirms its role in glutathione-mediated detoxification pathway and is able to utilize adenine biosynthetic intermediates as a substrate. This is the first comprehensive inventory of wheat ABCC-MRP gene subfamily.The ABCC multidrug resistance associated proteins (ABCC-MRP), a subclass of ABC transporters are involved in multiple physiological processes that include cellular homeostasis, metal detoxification, and transport of glutathione-conjugates. Although they are well-studied in humans, yeast, and  Arabidopsis. Each of these MRP proteins contains at least one highly conserved ATPase domain as an energy provider (~200 aa residues long) also referred as nucleotide binding domains (NBD). This ATPase domain comprises of a Walker motif A and Walker motif B on either end of an ABC signature motif  proteins are found in all living organisms and constitute one of the largest known superfamily  are reported from model plant Arabidopsis     have been speculated for xenobiotic detoxification , is an important cereal crop, consumed as a staple food by a large population in the developing countries. Although, plant ABCC-MRP transporters were identified from model crops, limited attempts were made to characterize this subfamily from wheat. In this study, to address the importance of ABCC proteins for their diverse biological and physiological processes in wheat, systematic analysis and molecular function of ABCC genes was performed. Molecular gene structure and chromosomal locations of the putative wheat ABCC genes were predicted by both genome-wide analysis and ESTs searches. Eighteen wheat ABCC genes were identified and subjected to phylogenetic analyses along with Arabidopsis and rice counterparts. Their expression using qRT-PCR was studied to reveal their transcript accumulation in different tissues i.e., roots, stem, leaves, flag leaf, developing wheat grains, and in roots exposed to Cd stress. In addition, we demonstrated that TaABCC13 . Healthy plants with 14 days after anthesis (DAA) mature seeds were used to collect different vegetative tissues i.e., roots, stem, leaves, flag leaf, and seeds. Tissue samples were collected under random bulk up experiment method from different plants and snap frozen. Collected samples were stored at −80°C before RNA extraction. In total three biological replicates were used for the tissue samples. To study gene expression through seed maturation, the main individual spikes of the biological replicates were tagged at the first day after anthesis (DAA). The tagged spikes were harvested at subsequent developing days at 7, 14, 21, and 28 DAA and frozen in liquid nitrogen for RNA extraction.Bread wheat cv. C306, a good processing quality Indian variety  or 10 μM H2O2 for 24 H separately. After 24 h of stress, leaves and root samples were snapped frozen in liquid nitrogen and stored at −80°C for further use. Seedlings without stress treatment were used as the experimental control. The CdCl2 treated seedlings were subjected to estimate relative metal accumulation. Roots and leaves were collected and dried overnight at 60°C. The equal weight of treatment and control tissues (roots and leaves) was microwave digested in HNO3 . These digested samples were used for metal analysis by using inductively coupled plasma mass spectrometry .For treatments, surface sterilized seeds were soaked in half strength Hoagland's solution in Petri dishes for germination. Germinating seedlings were transferred to a hydroponic growing system with half-strength Hoagland's solution. Seedlings of 7 days old (with one fully expanded leaf) were treated with Hoagland's solution supplemented with the 50 μM Cd (CdCl3) were used as suggested earlier with minor modifications  containing 20 mM CaCl2. Control seeds imbibed in CaCl2buffer were used. Three replicates containing 22–25 seeds per plates with their respective hormonal treatments were used for the experiment. After 60 min of incubation seeds were collected and rapidly chilled before storage at −80°C. RNA was extracted from the respective treatments and was subjected for qRT-PCR. Five technical replicates from biological replicates were used for qRT-PCR analysis.To study the effect of hormones, abscisic acid (ABA) and gibberellic acid  and further these annotated ORFs were qualitatively checked with the gene structure database of wheat reported by Mayer et al.  of wheat plants. Total RNA was extracted using the RNeasy Plant MiniKit , following manufacturer's instructions. Genomic DNA contamination was removed by DNase-I treatment . Transcriptor First Strand cDNA Synthesis Kit RT-PCR  was used for cDNA preparation from two micrograms of RNA. Reverse transcription was performed using random hexamer primers following the manufacturer's guidelines. Gene specific primers Table  were useMATα, ura3–52; leu2- Δ 1; lys2–801; his3 Δ 200; trp1 Δ 63; ade2–101 was used as a positive control. Yeast mutant for ycf1 (ABC 470) with genotype of MATα, Dycf1::KanMX2; ura3–52; leu2-D1; lys2–801; his3D200; trp1D63; ade2–101 prepared in the same auxotrophic background was also used for the current study (37). For complementation experiment, TaABCC13 + pYES263 harboring in yeast Δycf1 mutant was used. Pre-culture of wild type, mutant and transformed yeast colonies were grown overnight in YPD broth and brought to the same OD600. The colonies were subsequently streaked on 0.5% YPD media for 4 days along with negative controls. The red pigmentation produced by the yeast colonies that includes ade2 mutants was quantified spectrophotometrically as described earlier  defective in adenine biosynthesis pathway, with a genotype of TaMRP3 gene  was also identified previously and characterized in wheat tissues  and ABCC-MRP (NBD) like domain those are conserved ubiquitously among members of ABCC-MRP transporter subclass. The predicted full length amino acid sequences from all TaABCC proteins were analyzed on CDD and Expasy PROSITE. The analysis revealed specific signatures of TM-NBD-TM-NBD with the possible location on membrane except for TaABCC members, without any mapped EST Figure . A ClustT. urartu, Ae. speltoides, and Ae. tauschii  . The CDS sequences for selective wheat ABCC genes were further confirmed by 5′ or 3′ RACE sequencing and subsequently refined by using IWGSC for their chromosomal locations  for wheat ABCC genes  was observed for TaABCC5, TaABCC7, TaABCC10, and TaABCC18; hence they were not selected for the expression study. The expression pattern of the remaining wheat ABCC genes was studied in roots, stem, leaves, and flag leaf that could help in speculating their site of molecular function. Analysis of the transcript abundance revealed that all the genes displayed differential expression patterns across the set of wheat tissue samples. Expression data, suggested the induction of wheat ABCC genes in leaves, root, stem, and flag leaf at different folds  followed by TaABCC2, TaABCC11 (~25-fold), and TaABCC3 (~12-fold) . TaABCC13 analysis was excluded since it was previously reported . In the current study, we chose GA3 and ABA, since these are the two major hormones that control the dynamics of seed maturation and grain filling  toxicity is one of the major abiotic stresses leading to hazardous effects in plants. Multiple plant ABCC-MRPs were speculated for their functional role for vacuolar sequestration, thus reducing the cellular metal toxicity  . Results showed down-regulation of TaCu/ZnSOD and TaCAT transcript when wheat seedlings were exposed to H2O2 , GA3 (GA-Myb) regulated domains and phosphate responsive domain (P1BS) (data not shown). This suggests that TaABCC3 can be a prime candidate for plant genetic engineering to enhance tolerance against heavy-metal accumulation.Studying the expression patterns in specific tissues and organs suggests the molecular clues for their role and help to address their functionality in plants. The preferential expression patterns suggested their specificities for the respective tissue/organs. For example, TaABCC14 and TaABCC15 are highly abundant in flag leaf and both are clustered together in the phylogenetic tree. Flag leaf is the important organ for the biosynthesis of transitory starches, micronutrients and other metabolites that contribute to the seed maturation during the senescence  and nutrients between the seed tissues  rescued Δycf1 sensitivity toward cadmium but its functional activity in utilizing glutathione-conjugates were not studied  mutant. These toxic metabolites are conjugated with glutathione and transported inside the vacuole primarily by YCF1  an MRP transporter has been classically utilized for confirming the functional role of ABCC-MRP transporters from plants (Tommasini et al., ABCC genes. Wheat ABCC genes showed differential expression in the presence of Cd or H2O2 suggesting their importance in abiotic stresses. Additionally, TaABCC13 was able to rescue the functional activity of yeast mutant by utilizing glutathione-conjugated substrates. The insight provided herein will be a much needed foundation regarding the wheat ABCC protein function that could be directed for cereal crop improvement.The current study provided the spatial-temporal characterization of 18 wheat The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Different grazing strategies impact grassland plant production and may also regulate the soil carbon formation. For a site in semiarid temperate steppe, we studied the effect of combinations of rest, high and moderate grazing pressure over three stages of the growing season, on the process involved in soil carbon sequestration. Results show that constant moderate grazing (MMM) exhibited the highest root production and turnover accumulating the most soil carbon. While deferred grazing (RHM and RMH) sequestered less soil carbon compared to MMM, they showed higher standing root mass, maintained a more desirable pasture composition, and had better ability to retain soil N. Constant high grazing pressure (HHH) caused diminished above- and belowground plant production, more soil N losses and an unfavorable microbial environment and had reduced carbon input. Reducing grazing pressure in the last grazing stage (HHM) still had a negative impact on soil carbon. Regression analyses show that adjusting stocking rate to ~5SE/ha with ~40% vegetation utilization rate can get the most carbon accrual. Overall, the soil carbon sequestration in the temperate grassland is affected by the grazing regime that is applied, and grazing can be altered to improve soil carbon sequestration in the temperate steppe. This is widely thought to be the major cause of grassland degradation and the subsequent decrease in soil carbon pools. Improved grazing management regimes have been considered as an important strategy to renew degraded grassland and to promote carbon storage in the soil3This study investigated livestock production systems to determine the effect of different seasonal grazing pressures on the process involved in soil carbon sequestration. Combinations of rest, high and moderate grazing pressure were implemented over three grazing stages. Our objective was to address: 1) how soil carbon responds to varying seasonal grazing pressures, 2) what major mechanisms mediate soil carbon input under these grazing regimes, and 3) what optimal grazing pressure is suitable for soil carbon sequestration in the temperate steppe.The standardized stocking rate [50 kg Sheep Equivalent (SE)] for each regime (treatment), across all grazing years and stages is shown in 4,10 = 46.8, p < 0.05; Plant aboveground production was significantly influenced by grazing regimes  were also detected for soil total nitrogen, and their interaction effects were significant  changes in aboveground production; and (ii) variation in vegetation composition in this temperate grassland. The relationship between the proportion of grasses and forbs with stocking rate illustrated that grazing can change aboveground biomass production indirectly through altering vegetation composition and directly with high utilisation reducing plant production. However, grazing induced changes to aboveground production might contribute little to the soil carbon change because much of the aboveground mass consumed and respired by animals would be respired anyway by microbes during degradation and would not be available for soil carbon formation anywayBelowground biomass grows in the soil and contributes higher levels of organic matter to soilsPrevious studies show that the effects of grazing on plant production is variable, but most have a negative influence on aboveground production243 volatilization. In addition, some nitrogen losses in HHH, HHM and MMM could partly be explained by a redistribution of soil nitrogen into the plant or litter nitrogen since the plant aboveground nitrogen contents were significantly elevated under these grazing regimes (293 volatilization (maintained high soil organic nitrogen level) and relatively lower root litter deposition, causing unchanged soil C:N ratio. Regression analysis between soil nitrogen change and stocking rate showed that nitrogen was retained best at a stocking rate of ~4 SE/ha  there was a limited sample size to determine the trend and the optimal position was somewhat ambiguous. However, despite this limitation, our analyses revealed informative patterns that reflect the importance of setting a grazing pressure that is appropriate to the productivity of the site. It also provides evidence that grazing management can be designed to mitigate greenhouse gases and store soil carbon.Leymus chinensis (Trin.) Tzvel., a C3 perennial rhizomatous grass that is widely distributed in the Eurasian steppe region.This study was conducted at a temperate steppe site in Guyuan County, Hebei Province, China , at an altitude of 1475 m. This area is a typical temperate zone characterized by a mean annual (1982–2009) precipitation of 430 mm and a mean annual temperature of 1.4 °C. Precipitation mainly falls in the growing season (June to August), which coincides with the highest temperatures. Minimum monthly mean air temperature is −18.6 °C in January and the maximum of 21.1 °C occurs in July. The site has a calcic-orthic Aridisol soil , with a loamy-sand texture. The plant community is dominated by Based on the phenology of dominant and common species, and long-term seasonal patterns of temperature and precipitation in this area, we divided the growth-season (from June to end of September) into three distinct stages. Stage 1 (S1): Starting growth period (beginning of June to mid-July) when plants begin to grow. Stage 2 (S2): Active growth period (mid-July to end of August), up to when plants have finished flowering and have set seed. Stage 3 (S3): Nil growth period (end of August to end of September) when plant growth terminates because of an abrupt decrease in temperature and precipitation. We selected the study area in 2009 for the study of different grazing options on livestock production and grassland sustainability, which had been fenced to exclude grazing since 2004 because of grassland degradation. In this paper we concentrate on the effects of different grazing regimes on soil carbon. Five grazing treatments were implemented: HHH: high grazing pressure at stages 1–3; HHM-high grazing pressure in stages 1 and 2, and moderate grazing pressure in stage 3; MMM: moderate grazing pressure in stages 1–3; RMH: deferred grazing regime, with rest (stage 1), and moderate (stage 2) and high (stage 3) grazing pressure; RHM: deferred grazing regime with rest (stage 1), high (stage 2) and moderate (stage 3) grazing pressure. The detailed information of the grazing practices was shown in Soil samples were collected in mid-May 2009 (just before the experiment began) and mid-August 2013 (when aboveground biomass is at its peak) at 0–10, 10–20, 20–30 cm soil depth increments. Cores were collected using a 4-cm diameter soil corer at nine locations, with three cores bulked at each location, within each of the 15 plots. Samples from each plot were then sieved at 2-mm to remove roots. To determined soil organic carbon, the soil inorganic C (including carbonates) was removed by treating the soil with dilute hydrochloric acid  and stirring until no further effervescence was observed. Then the soil samples were dried at 40℃ for 4 days, ground and analyzed for carbon using an autoanalyzer . The total nitrogen concentration was determined according to the Kjeldahl wet digestion procedure, using a 2300 Kjeltec Analyzer Unit .et al.The remainder of the fresh soil (depth 0–10 cm) was immediately stored at −80 °C and then analyzed for soil microbial communities. The microbial community in soil samples was assessed using phospholipid fatty acids (PLFAs). PLFAs were extracted from the soil as described by Bossio in situ soil core incubation method4+-N and NO3−-N) were determined using a Flow-Solution analyzer . Daily net nitrogen mineralization rates were calculated by subtracting initial core total inorganic nitrogen values from incubation tube total inorganic nitrogen values, and then dividing by the incubation period.Net nitrogen mineralization rates were determined using 2 movable exclosures following McNaughton et al.In mid-August 2013, nine sampling quadrats measuring 50 cm × 50 cm (three transect lines for each plot) were cut to ground level using scissors for determining aboveground biomass. The fresh samples were returned to the laboratory separated according to plant species, dried at 65 °C for 48 h and weighed. Plants were grouped into three functional types: grasses, sedges and forbs. The fresh root and foliage samples were analyzed at the China Agricultural University Laboratory for total carbon and nitrogen, and the percentage of lignin. Aboveground production was measured using 1-m4+ and NO3−) content and net nitrogen mineralization rate. The data for standing root biomass were run as a repeated-measures mixed model analysis. Treatment, sampling date and their interaction were considered fixed effects, and plot considered a random effect. Date was treated as a repeated measure variable. The soil organic carbon and total nitrogen data were also analyzed with the mixed procedure with treatment, soil depth and their interaction in the model as fixed effect, and plot as a random effect. Depth was treated as a repeated measure variable. Various types of variance–covariance matrices were fitted and the one with the lowest AIC value was used for the final analysis. Regress models employing the averaged stocking rate across years for each treatment as a independent variable were used to determine the relationship between stocking rate and vegetation UR , vegetation production and soil net nitrogen mineralization rate. The relationships between UR and grasses, forbs proportions were also examined. We calculated the soil organic carbon and total nitrogen changes (using g kg−1 as unit) in top 10 cm layer by subtracting the initial soil organic carbon and total nitrogen contents from the soils collected in 2013. Relationships between stocking rates (independent variable) and top soil carbon, nitrogen change or soil Fungi: Bacteria ratios (dependent variables) were then fitted using a regression model separately. No transformation was conducted if data meet the assumption of normality, otherwise data was log-transformed prior to analysis to ensure normality.Statistical analyses were performed using SAS Version 8.2 . ANOVA with Duncan’s multiple-range tests were used to compare the effect of grazing regimes on the plant above- and belowground production and quality data, PLFAS, soil inorganic nitrogen .