N-isopropylacrylamide (NIPAM) was copolymerized in water with a binding monomer and a hydrophobic comonomer using a living radical polymerization (RAFT). The resulting copolymers were water-soluble and displayed narrow polydispersities. They formed tight complexes with basic proteins depending on the nature and amount of the binding monomer as well as on the choice of the added hydrophobic comonomer.A new family of linear polymers with pronounced affinity for arginine- and lysine-rich proteins has been created. To this end,  The ability of biological receptors to bind strongly and specifically to a particular molecular target is an essential part of biological machinery. The best example is the immune system where antibodies are generated in response to minute amounts of foreign antigens. A continual challenge in nanoscale chemistry is to mimic the biological molecular recognition functions by synthetic chemistry with the aim of producing systems of lower complexity. When successful, this will enable the manufacturing of robust and specific synthetic receptors for a given protein target . ProteinMultivalency represents the key to generate high-affinity materials for biomacromolecules with a sufficient number of binding sites for Coulomb attraction and hydrophobic interactions . A statiWith this principle in mind, several groups have designed relatively simple linear polymeric structures with branched ionic comonomers and thus achieved remarkable affinities and biological properties. In their elegant work, Kulkarni et al. reported the use of NIPAM-based copolymers for lysozyme recovery by affinity thermoprecipitation. These polymers contained multiple acetamido groups in a hydrophilic environment for maximum interaction with the catalytic cleft and achieved high affinities . RotelloProtein recognition by multifunctional polymeric hosts features two prominent advantages. On one hand, it simplifies the complex recognition interface to isolated 1:1 complexes between monomeric binding sites and single complementary amino acid residues, while simultaneously allowing for an extensive induced-fit process of the linear polymer on the protein surface – in other words they encourage polymer/protein self-assembly in order to maximize attractive noncovalent interactions.A second major advantage of multivalent polymeric hosts is their rapid and efficient synthesis at low cost as well as the high proteolytic stabilities of most polymer backbones. They also pose fewer racemization problems which often accompany proteinogenic amino acids in peptidic environments.In recent years, our group has developed water-soluble linear polymeric protein binders which contained one or more different binding monomers and displayed micromolar protein affinities , accompaReversible addition–fragmentation chain transfer (RAFT) polymerization  and atom8 /[CTA] was 3, and the concentration of CTA and V-50 were adjusted to target polymers with a molecular weight of 3000, 7000 or 17000 g mol −1 at full conversion. This is possible since the degree of polymerization under RAFT conditions is equal to the ratio between monomer and chain transfer reagent concentration. Conversion was almost 100%, and copolymers were isolated by precipitation in hexane. The absence of low-molecular weight impurities such as monomers was ascertained by 1H NMR spectroscopy. Molecular weights were determined by gel-permeation chromatography (GPC) analysis of the copolymers. Narrow polydispersities (≤1.3) were observed for the shorter copolymers, although the highest molecular weights (targeted at 17000 g mol−1) reached only experimental values of 11,000–12,000 g mol−1 and also produced slightly higher polydispersities (1.56). For comparison, some copolymers such as S20CH15 were also prepared with a molecular weight of ~3000 g mol−1.RAFT polymerizations were carried out in methanol at 60 °C for 48 hours in the presence of CTA 3 M−1 could be fitted to the binding isotherm when the second derivative values of the protein at 415 nm were plotted against the RAFT polymer concentration (6–108 g mol−1), cytochrome C possesses a relatively small molecular weight similar to the RAFT copolymers. As a result, the RAFT copolymers and cytochrome C favor 1:1 binding. The incorporation of a hydrophobic comonomer further improved binding. The maximum binding strength was observed for polymers containing 15 mol % of N-cyclohexylacrylamide and 20 mol % of sodium methacrylate ; obviously, the methacrylate anion is a weak binder for lysines and arginines on these protein surfaces. Moderate binding (3 × 104 M−1) was only detected with S20CH15, which carries twice the amount of carboxylate groups. Association constants were initially calculated for each 1:1 complexation event of a single protein by the copolymer [For an independent comparison, the same protein–polymer pairs were subsequently subjected to microcalorimetric titrations , which ctrations –1600 M−1Ka values (>106 M−1) which were about two orders of magnitude higher than those achieved with sodium methacrylate copolymer S20CH15 (~104 M−1). This is not surprising for the bisphosphonate, which carries twice the amount of negative charges. However, the monoanionic tetrazolate anion is very similar in acidity and hydrogen bond pattern to a carboxylate, so that similar affinities would have been expected. Most likely, the difference is explained by interactions with the π-face of the tetrazolate anion, which are not possible with a carboxylate.By contrast, tetrazolate copolymer T20CH15 and bisphosphonate copolymer B20CH15 showed large enthalpy changes and hence much higher In all cases, protein complexation by RAFT polymers was endothermic, i.e., entropy-driven. Hence, unspecific electrostatic attraction in combination with solvophobic forces contributed the most towards protein binding.Ka values dropped substantially by 1–2 orders of magnitude  and an average-size polymer (MW 12000 g mol−1). Direct comparison produced a drastic difference: No binding could be detected for the short version, indicating that size matters and promotes multivalent or cooperative binding.For biological applications, it is desirable to keep the polymer size close to the size of the protein, so that specific 1:1 complexation is favored . In ordeKd values even with BSA, T20CH15 hardly ever reached the micromolar regime. Obviously, the bisphosphonate’s high negative charge density is especially effective for protein surfaces with a high density of basic amino acids such as the DNA-binding histones or for those offering distinct clusters of cationic amino acid residues (e.g. BSA). Interestingly, although in most cases nonlinear regression converges with an assumed 1:1 complex stoichiometry, curve fitting is greatly improved with a sequential binding or 2-sites model [Kd value is 16 nM, followed by very weak binding at a second site with a Kd of 1 mM. With respect to varying pI values, both RAFT polymers display little selectivity: From lysozyme (pI > 9) down to BSA (pI < 6) protein affinities vary by less than one order of magnitude.Finally, the protein series was extended to lysine-rich histone (pI 10), lysozyme (pI 9), proteinase K (pI 8) and bovine serum albumin or BSA (pI 6). Again, the strong binders B20CH15 and T20CH15 were examined concerning their affinities towards proteins of varying pI . In direes model . In all Kd values. Copolymerization with N-cyclohexylacrylamide introduced additional nonpolar groups beneficial for protein binding, leading to a substantial entropy gain and significantly improving protein affinities. The best pair was a bisphosphonate-containing RAFT copolymer and lysine–rich histone (Kd = 16 nM). In the future, we intend to investigate if it is possible to interrupt the nucleosome complex formation by noncovalent detachment of ds-DNA from its “own” histone proteins using histone-binding RAFT copolymers.In summary, RAFT copolymerization of NIPAM with monomers containing anionic binding sites for basic amino acids led to polymers of low polydispersities which were effective protein binders in buffered aqueous solution, with tunable stoichiometries close to the ideal 1:1 ratio. Although molecular recognition is based on unspecific electrostatic attraction and hydrophobic forces, those proteins which feature a high density of positive charges on their surfaces are bound especially well by the bisphosphonate site, in some cases reaching micromolar or sub-micromolar File 1Full experimental procedures, characterization details, microcalorimetry measurements, UV titration procedures and potentiometric titrations.


TGF-β1 is overexpressed in wound repair and in most proliferative disorders including benign prostatic hyperplasia and prostate cancer. The stromal microenvironment at these sites is reactive and typified by altered phenotype, matrix deposition, inflammatory responses, and alterations in nerve density and biology. TGF-β1 is known to modulate several stromal responses; however there are few transgenic models to study its integrated biology. To address the actions of TGF-β1 in prostate disorders, we targeted expression of an epitope tagged and constitutively active TGF-β1 via the enhanced probasin promoter to the murine prostate gland epithelium. Transgenic mice developed age-dependent lesions leading to severe, yet focal attenuation of epithelium, and a discontinuous basal lamina. These changes were associated with elevated fibroplasia and frequency of collagenous micronodules in collapsed acini, along with an induced inflammation in nerve ganglia and small vessels. Elevated recruitment of CD115+ myeloid cells but not mature macrophages was observed in nerve ganglia, also in an age-dependent manner. Similar phenotypic changes were observed using a human prostate epithelium tissue recombination xenograft model, where epithelial cells engineered to overexpress TGF-β1 induced fibrosis and altered matrix deposition concurrent with inflammation in the stromal compartment. Together, these data suggest that elevated TGF-β1 expression induces a fibroplasia stromal response associated with breach of epithelial wall structure and inflammatory involvement of nerve ganglia and vessels. The novel findings of ganglia and vessel inflammation associated with formation of collagenous micronodules in collapsed acini is important as each of these are observed in human prostate carcinoma and may play a role in disease progression. In vivo modeling studies have shown that reactive stroma promotes angiogenesis and prostate cancer progression as well as proliferative responses in normal human prostate epithelial cells, although little is understood about the specific mechanisms of reactive stroma formation A reactive stroma microenvironment is observed in wound repair, fibrosis, and in most proliferative diseases, including prostate cancer and benign prostatic hyperplasia. The activation of stromal cell proliferation and biology in wound repair facilitates granulation tissue formation and tissue remodeling through ECM deposition and growth factor production in vivo using expression of active TGF-β1 in transgenic mice. Targeted overexpression in the liver in a murine transgenic model led to the development of severe cirrhosis and glomerulonephritis in vitro studies have demonstrated that TGF-β1 modulates differentiation of normal prostate stromal cells into myofibroblasts, a cell type that is unique to tissues undergoing fibrosis, remodeling, or reactive stroma formation in several disorders TGF-β1 is a key factor released by platelets at sites of wound repair and modulates angiogenesis and inflammatory responses. The expression of TGF-β1 is elevated in most carcinomas and many proliferative diseases including benign prostatic hyperplasia, prostate cancer and prostatitis Accordingly, TGF-β1 is emerging as a potential target of therapeutics in modulating fibrosis, inflammation, and tumor progression. It is clear that the pleiotropic nature of TGF-β action is due in part to the local cell and tissue-specific milieu. Therefore, we sought to address the potential etiological role of aberrant TGF-β1 action in inducing stromal microenvironment responses in normal intact murine prostate gland tissue. We report here a transgenic mouse model constructed with epitope tagged and constitutively active TGF-β1 expressed in the prostate gland epithelium. In addition, we have evaluated a tissue recombination model generated with human prostate epithelial cells engineered to overexpress active TGF-β1 and stromal cells in xenografts. Results reported here demonstrate that expression of constitutively active TGF-β1 results in age-dependent phenotypic alterations characterized by attenuation of secretory acini walls, induction of fibroplasia, and inflammation in vessels and nerve ganglia. We also report the formation of unique fibrotic collagenous micronodules, shown previously to be associated with human prostate cancer.All animals were handled in strict accordance with good animal practice as defined by the relevant national and institutional animal welfare bodies, and all animal work was approved by IACUC protocol AN-1867.The composite probasin promoter ARR2PB (provided by Dr. Robert J. Matusik) was used to target transgene expression specifically to murine prostate epithelium as reported previously 5′ CAGTGTGGTGGAATTGCCCTTATCTGGTACC and 5′CAGAGATGCTAGTCTGGCACGTCGTATGGGTAGCT. Amplification of MbCx7 served as an internal positive control using the following primers: 5′ GATGTGCTCCAGGCTAAAGTT and 5′ AGAAACGGAATGTTGTGGAGT. PCR reactions used Platinum Taq DNA polymerase (Invitrogen) and 2 mM MgCl2 for 32 cycles at a 55°C annealing temperature for 1 min. Five founder mice were transgenic and bred for examination of F1 generation. Screening of F1 mice revealed that four founders exhibited germline transmission.PCR amplification of tail-derived genomic DNA was used to identify transgenic mice. Primers designed to amplify a 1.2 kb region of transgene encompassing a region upstream of the start codon to the internal HA tag were as follows: 5′ATACCAACAGCTACCCATACGACG and 5′ CACTTCCAGCCCAGGTCC. GAPDH primers designed to anneal to endogenous genetic material were as follows: 5′ CCTACCCCCAATGTGTCCG and 5′ CCTTCTTGATGTCATCATACTTGGC. Reverse transcription was performed at 50°C for 30 min followed by PCR amplification for 28 cycles at a 60°C annealing temperature for 1 min.All housing and manipulations of mice followed an approved IACUC protocol (AN-1867). Male and female mice from the F1 generation were bred with wild-type mates and resulting F2 generation males were screened by PCR of genomic tail DNA. Wildtype and transgenic littermates from each line were examined for RNA production by RT-PCR. Primers designed to amplify a region encompassing the internal HA tag were as follows: LNCaP human prostate carcinoma cells were acquired from ATCC  and maintained in RPMI 1640 media  supplemented with 10% fetal bovine serum (FBS) , 100 units/ml penicillin, and 100 μg/ml streptomycin . The Phoenix A packaging cell line was purchased from ATCC and maintained in DMEM with high glucose (Invitrogen) supplemented with 10% heat inactivated FBS (Hyclone), 2 mM glutamine (Invitrogen), 100 units/ml penicillin, and 100 μg/ml streptomycin (Sigma). The C57BL/6 mouse prostate stromal cell line was generated as reported previously To engineer cell lines, primers were designed to amplify cDNA from the pEF6-HA-pTGFβ1(a) vector for cloning into the multicloning site of the pBMN-Ires-eGFP retroviral vector (courtesy of Dr. Gary Nolan). Sequences were verified to confirm construct fidelity and pBMN-HA-TGF-β1(a)-IRES-eGFP was then transfected into Phoenix A cells with a Calcium Phosphate Transfection kit (Invitrogen) for retroviral engineering of the NHPrE1.2 human prostate epithelial 5 NHPrE1.2 cells (engineered for TGF-β1 expression or vector control) in 50 μl collagen. Collagen plugs were then grafted under the kidney capsule of SCID mice for 8 or 12 weeks and removed for formaldehyde fixation and paraffin embedding. Sections were cut at 5-micron thickness for immunostaining.Tissue recombination was performed as described previously The intact urogenital tract including the bladder, seminal vesicle and prostate gland was removed en mass, fixed in 4% paraformaldehyde and processed for histology and immunohistochemistry as we have reported previously Ventral prostate and surrounding regions were evaluated as the initial focus of this analysis due to the well-characterized simple epithelial acini histology and stroma of this prostate lobe. Tissues from each respective transgenic line were evaluated separately and data grouped together for comparison with wild-type littermates. Phenotypic changes in inflammatory foci, epithelial wall attenuation (thinning), and stromal fibroplasia were each evaluated as separate histopathologic parameters using a semi-quantitative protocol similar to what we have reported previously t-tests were used to assess significant differences for each measured phenotype. Significance of the frequency of collagenous micronodules was evaluated using Fisher's exact test. For quantitative evaluation of CD115 and F4/80 staining, positive cells were counted manually in the nerve ganglia in a blinded study (reviewer blinded to the mouse genotype). Values were averaged for each mouse and Kruskal-Wallis non-parametric ANOVA and Mann-Whitney tests were used to determine significance. For all tests, data generating a P value less than 0.05 was considered statistically significant. All analyses were performed using the Prism statistical software package.Mann-Whitney and non-parametric Student's in vitro were engineered to express HA-TGF-β1(a) construct. Expression and secretion of full-length protein was confirmed by Western blot (HA epitope) and ELISA for TGF-β1 protein (data not shown). Biological activity of secreted protein was confirmed via induction of phenotypic changes in C57BL/6 murine prostate stromal cells as we have reported previously 2PB) was used to drive HA-TGF-β1(a) expression in the prostate gland Expression of constitutively active TGF-β transgene was targeted to epithelial cells of the male urogenital tract in order to assess TGF-β1 actions on the stromal environment. An HA-TGF-β1(a) construct was used containing mutations in Cys223 and Cys225 to provide constitutive biological activity and an internal HA epitope tag as previously reported 2PB induced expression in this region of murine prostate gland. All transgenic lines exhibited similar phenotypic changes in epithelium and stroma.Age-matched male reproductive tract was examined in all four transgenic lines and control littermate mice from 12 weeks to 144 weeks of age (n = 38 control and n = 41 transgenic mice) as outlined in Initial analyses suggested an alteration in the epithelial wall integrity in transgenic mice as compared with control mice . Some acConcomitant with alterations in epithelial wall integrity, transgenic mice also exhibited focal regions of inflammation in the vasculature and local parasympathetic ganglia. Vascular fibroplasia typified by a thickened tunica media in intermediate vessels was noted in some transgenic mice and was usually associated with inflammation in the vessel wall . Foci ofFibroplasia was observed in inter-acinar regions in transgenic mice. Similar to other phenotypic observations, the appearance of inter-acinar stromal fibroplasia was evident in transgenic mice over a year of age and consisted of more focal rather than widespread involvement. In regions of focal fibroplasia  the stroConsistent with age-associated inflammatory changes, transgenic mice exhibited differential immunoreactivity of CD115+ myeloid cells in the neurovascular bundles and ganglia compared to wild-type littermates . QuantitTGFβ cell lines were recombined with rat urogenital mesenchyme (rUGM) and placed under the kidney capsule of SCID mice for 8 or 12 weeks. Tissue recombinants constructed with HA-TGF-β1(a) expressing cells demonstrated a decrease in overall graft size over both time periods (To address HA-TGF-β1(a) in a human epithelium/rodent stroma recombinant model and to further address induced stromal fibroplasia, normal human prostate NHPrE1.2 epithelial cells were engineered to express HA-TGF-β1(a). Control NHPrE1.2 and HA-TGF-β1(a) expressing NHPrE1.2 periods . Both co periods . Compare periods  and elev periods . Immunoh periods , whereas periods . Compare periods , 12-week periods . AccordiData presented here shows that targeted expression of constitutively active TGF-β1 in prostate gland epithelium resulted in focal expression of the transgene that was associated with attenuation and breakdown of the glandular acini wall epithelium, degeneration of secretory acini, inflammation of vessels and nerve ganglia, and an induced stromal fibroplasia. Of interest, these phenotypes evolved as an age-dependent process with significant differences observed in mice over a year of age. The earliest focal phenotype was an attenuation of epithelial cells in acini walls, concomitant with a degeneration of basal lamina integrity in some but not all acini wall regions. Inflammatory and fibroplasia alterations appeared to be secondary to this focal epithelial attenuation. The appearance of pyknotic nuclei in conjunction with focal regions of severe epithelial wall thinning suggested cell damage. Over time, this process of epithelial disruption would be expected to generate a physical breach in the basement membrane thereby providing a gateway of entry for constitutively active TGF-β1 to enter the stromal compartment. Indeed, collagen IV immunohistochemistry showed focal regions of discontinuity in the basal lamina surrounding several ventral prostatic acini in transgenic mice. This is a key alteration in homeostasis since the stroma is normally restricted from access to proteins secreted by epithelium into the lumen within glandular tissues. Such an alteration would also be expected to generate host responses in the stromal compartment. As TGF-β1 is a potent inducer of reactive stroma, it is likely that a repair response was induced by active HA-TGF-β1(a) subsequent to release from damaged acini and loss of the epithelium integrity. This defect is conceivably more pronounced in mice over one year of age owing to both the natural aging processes and TGF-β1-induced accumulated defects in epithelial wall integrity. Our previous reports have shown that reactive stroma initiates adjacent to preneoplastic prostatic intraepithelial neoplasia (PIN) lesions in a heterogeneous pattern where epithelial cells had lost polarity, suggesting defects in acini wall integrity A novel finding in this study was inflammation associated primarily with the neurovascular bundles and local ganglia observed in nearly all transgenic mice over 1 year of age. Both prostate cancer and benign prostatic hyperplasia are associated with elevated inflammation and both exhibit overexpression of TGF-β1 in epithelial cells The function of the collagenous micronodules reported here has not been elucidated. One interpretation is that these micronodules evolve as a component of wound repair to induce a collapse of the epithelial acini in damaged, non-functional glands in order to effectively take these acini off-line as a compensatory response. Closure of damaged acini might prevent retrograde introduction of pathogens from the downstream urogenital tract into the prostate gland stroma that houses vessels. Accordingly, in conjunction with the other induced inflammatory responses in vessels and nerves, these nodules may form to ostensibly sequester or limit a potential nidus of infection from spreading in a prostate gland with damaged prostatic acini and epithelial walls with breached integrity. Indeed, these lesions are nearly identical in histopathology to collagenous micronodules associated with human prostate cancer The generation of focal fibroplasia, inflammation and collagenous micronodules in the stromal microenvironment of TGF-β1 overexpressing mice is consistent with the role of reactive in wound repair biology


Lycaon pictus) in Tanzania, killing 49 of 52 animals within 2 months. The causative agent was identified as Canine distemper virus (CDV) by means of histologic examination, virus isolation, reverse transcriptase-polymerase chain reaction analysis, and nucleotide sequencing. This report emphasizes the importance of adequate protection against infectious diseases for the successful outcome of captive breeding programs of endangered species.In December 2000, an infectious disease spread through a captive breeding group of African wild dogs ( Lycaon pictus) is a highly endangered carnivore found in Africa south of the Sahara. Its population, estimated at <5,500, has declined dramatically in recent decades. Suggested causes for this decline include habitat loss, killing by humans, reduced prey availability, competition with other carnivores, and infectious diseases, including rabies and canine distemper The African wild dog , determined by methods similar to those used in a study of large felids Neutralizing antibody levels to Morbillivirus-specific primers P1: 5´ATGTTTATGATCACAGCGGT 3´ and P2: 5´ATTGGGTTGCACCACTTGTC 3´, which have been used before for phylogenetic analysis of morbilliviruses Morbillivirus P-gene fragment. Phylogenetic analysis of the nucleotide sequences from the resulting PCR fragments demonstrated that the causative virus was most closely related to CDV (Canis familiaris), lions (Panthera leo), and bat-eared foxes  from East Africa in the 1990s , virus isolation (n=2), and reverse transcriptase-polymerase chain reaction (RT-PCR) with d to CDV  and clushe 1990s . P-gene These results show that the primary cause of death of these African wild dogs was CDV infection. Canine distemper is highly infectious for many species of carnivores and causes high death rates in immunologically naïve populations Potential routes of transmission of this virus to the captive breeding groups are by direct contact with infected domestic dogs or wild carnivores or indirectly by contact with humans or their equipment. Domestic dog populations in some parts of Tanzania are endemically infected with CDV and were considered to be the source of infection for canine distemper in Serengeti lions in 1994 Phoca vitulina) and dogs against phocine distemper virus infection Because vaccination with live attenuated virus had been suspected of causing past deaths of African wild dogs (Conservation of endangered species, both free-living and captive, has been jeopardized by infectious disease outbreaks in the past


ProNPY processing of less than 50% was correlated with poor clinical outcome (P = 0.004). MYCN oncogene amplification was also correlated to a low degree of proNPY processing (P = 0.025). In summary, a low degree of proNPY processing was correlated to clinical advanced stage and poor outcome in neuroblastomas. ProNPY/NPY processing generated molecular forms of NPY with known differences in NPY-receptor selectivity, implicating a potential for in vivo modulation of NPY-like effects in tumour tissue. © 2000 Cancer Research CampaignNeuropeptide Y (NPY) is found at high concentrations in neural crest-derived tumours and has been implicated as a regulatory peptide in tumour growth and differentiation. Neuroblastomas, ganglioneuromas and phaeochromocytomas with significant concentrations of NPY-like immunoreactivity were investigated for different molecular forms of NPY and for significance of proNPY processing. Gel-permeation chromatography identified intact NPY (1–36) in all tumours, whereas proNPY (69 amino acids) was detected only in control adrenal tissue and malignant neuroblastomas. Purification of NPY-like immunoreactivity in tumour extracts and structural characterization revealed that both NPY (1–36) and the truncated form NPY (3–36) was present. The degree of processing of proNPY to NPY in tumour tissue was lower in advanced neuroblastomas with regional or metastatic spread (stage 3 and 4) (


Cervical smears prepared around the time of menses have been linked to unsatisfactory specimens and false negative results; however, it is unclear whether liquid-based cytology is similarly affected and data relating date of last menstrual period (LMP) to human papillomavirus (HPV) DNA testing are conflicting. Accordingly, we evaluated liquid-based cytology and HPV test results using Hybrid Capture 2 and PCR by LMP . We studied 5060 participants in ALTS, the Atypical Squamous Cells of Undetermined Significance (ASCUS) Low Grade Squamous Intraepithelial Lesion (LSIL) Triage Study. On average, women had 3.4 examinations  during a 2-year period of observation permitting an examination of intra-individual variation in cytology and HPV by LMP. Although uncommon, unsatisfactory cytology specimens were most likely on days 0–10. For satisfactory specimens, the frequency with which cytologic categories were reported varied by time since LMP, although differences were modest and did not affect the chance of abnormal cytology or its severity among women diagnosed with CIN2+. The frequency of positive HC2 tests did not vary with date of LMP. Among HPV infected women, independent of eventual diagnosis and the number of viral genotypes present, mid-cycle specimens yielded the highest frequency of LSIL cytologic interpretations and the highest HPV load; however, the magnitude of these effects were small. Intraindividual correlations of cytology or HPV by LMP were generally weak. We conclude that mid-cycle specimens yield slightly higher HPV DNA loads and slightly increased LSIL interpretations, but the clinical impact is marginal. Standardizing collection times would slightly improve interpretation of trends in HPV load. Finally, these data are consistent with the view that the biological properties of the HPV-infected cervix vary with the date of the LMP. Important goals of cervical cancer research include improving detection of precancerous lesions and reducing equivocal results by employing better collection, preparation, and testing methods  DNA and cytologic testing in some settings highlights the need to clarify inconsistencies in reported analyses assessing the performance of HPV testing in relation to LMP (n=3488) or LSIL (n=1572) at four clinical centres in the US , was placed in PreservCyt  and used to prepare a ThinPrep (Cytyc) cytology slide and to perform the Hybrid Capture 2  test, which targets 13 oncogenic HPV types. The second sample, collected with a Dacron swab, was placed in Specimen Transport Medium (Digene), frozen, and later used for HPV typing by a polymerase chain reaction (PCR)-based assay.Subjects were randomized to one of three management arms: (1) Conservative Management consisting of colposcopy referral for repeat cytology of a high-grade squamous intraepithelial lesion (HSIL); (2) HPV triage, with colposcopy referral for a positive HC2 test for oncogenic types ; and (3) Immediate Colposcopy. Subjects who received histologic diagnoses at the Clinical Centers of cervical intraepithelial neoplasia 2 or worse (CIN2+) were treated with loop electrical excision procedure (LEEP). The follow-up protocol was identical for all women: repeat cytology every 6 months for 2 years, with colposcopy referral for Clinical Center cytology of HSIL. At the 24-month (exit) visit, all data were unmasked and reviewed for every subject, colposcopy was performed, and women with CIN2+ or persistent ASCUS or LSIL diagnosed by the Clinical Center were offered treatment with LEEP, permitting detection of histologic CIN2+ that was undetected by cytology and colposcopy. All referral community smears and enrollment ThinPreps, most follow-up ThinPreps, and all histology specimens were reviewed by a Pathology Quality Control (QC) Group to provide standardized interpretations and added subject safety .−1 of HPV 16 DNA (approximately 5000 copies) were considered positive. The content of HPV DNA in specimens that tested positive (‘HPV load’) was determined as relative light units (RLU), calculated as the ratio of the signal of the specimen to that of the standard, which is linearly related to load  using a PCR-based assay employing L1 consensus primers and PGMY09/11 amplification followed by reverse line blot hybridization as described elsewhere  and Pathology QC histology results (the reference standard for outcomes) were used for all analyses. For most analyses, we stratified these data in three periods, reflecting serum hormone fluctuations during a normal 28-day cycle: (1) days 1–10 ; (2) days 11–21 ; and (3) days 22–28 . For ease of interpretation of odds ratios (ORs), we selected the late period as the referent group, which generally yielded ORs ⩾1.0 for most comparisons.n=4098), failure to collect cytology specimens (n=16) and LMPs greater than day 28 day (n=5797) accounted for the exclusions. HC2 data were available for 14 490 of these visits.Of 25 300 potential cytologic examinations that could have been performed in ALTS (five examinations of 5060 women), cytology results with associated LMP data in the range of 0–28 days were available for 15 389 interpretations. Missed visits  to take into account possible intra-individual ‘dependencies’ or ‘auto-correlation’  because many women contributed multiple data points to the analysis . This approach provides an estimate of whether data from individual women are correlated; strong auto-correlation reduces the precision gains achieved for larger sample sizes, resulting in reduced statistical power. We present ORs with 95% confidence intervals (CI) using GEE models, although auto-correlation was generally weak and adjustment widened CI only slightly.We assessed specimen quality by LMP by tabulating the frequency of reports of Bethesda System categories of ‘satisfactory for interpretation’ or ‘unsatisfactory’ . Next, fTo consider how cytologic interpretations related to the most severe histologic diagnosis per woman during the entire trial, we stratified the cytologic results by whether or not a woman ever received a histologic diagnosis of CIN2+. Previous work within ALTS showed that subsequent risk of CIN2+ or CIN3+ was similar for women with a colposcopically-directed biopsy result of CIN1, a negative biopsy, or a normal colposcopic appearance, which did not prompt a biopsy . We only−1  for which the corresponding PCR test demonstrated only one of the 13 oncogenic types included in the HC2 kit. The specimens could contain other types as well, not targeted by the assay. Of 3739 specimens that tested positive for only one of the 13 types by PCR and which were associated with suitable LMP dates, there were 3004 specimens that tested positive by HC2 for which we assessed viral load. To confirm the conclusions from this approach, we analyzed associations between LMP data and viral load <1.0 , and also analyzed LMP associations for specimens containing any single HPV type, including types that are not targeted by the HC2 kit. These analyses were confirmatory and are not presented.Subsequent analyses regarding HPV results were restricted to specimens with positive HC2 tests  specimens collected early in the cycle, five of 6792 (0.07%) collected during mid-cycle, and eight of 3213 (0.25%) collected late in the cycle. Considering specimens collected late as the referent, the association with unsatisfactory specimens yielded an OR=1.87 (95% CI=0.88–4.43) for the early period and OR=0.30 (95% CI=0.09–0.89) for mid-cycle collections. Unsatisfactory reports were most common on days 0–4 (data not shown). Intra-individual correlation was minimal (−0.0019).Among the 15 351 satisfactory specimens that comprised the rest of the analysis, the reported frequency of cytologic interpretations in ALTS varied significantly by LMP, although the magnitudes of absolute differences were slight . For exaTo explore the possible clinical relevance of the variation in cytology by time since LMP, we restricted the study population to women who received a histologic diagnosis of CIN2+ in ALTS . Among tWe compared the frequency of HPV DNA positive test results by HC2 for the three intervals of the menstrual cycle, without restrictions. The auto-correlation estimate was 0.39, showing a slight tendency of women that were HPV positive once to be positive repeatedly. This strength of correlation was typical of all HPV analyses, whether assessed as negative vs positive or as viral load.n=6468 associated with one or multiple types by PCR), we obtained slightly significant associations. Compared to specimens collected late in the cycle, specimens obtained early in the cycle had viral loads that were only 83% (95% CI=0.71–0.98) as high, whereas mid-cycle specimens yielded average loads that were 22% higher (95% CI=1.05–1.43).Overall, 44.6% of specimens were HC2 positive. Frequencies of HC2 positive results were unrelated to the date of LMP. When we assessed viral load among all HC2-positive tests .To study the associations between HPV viral load per type and time of LMP, independent of number of HPV types present, we assessed the frequency of positive HC2 results among specimens associated with PCR detection of one of the 13 carcinogenic types targeted by HC2. Positive HC 2 results were obtained for 1094 of 1377 (79.4%) specimens collected early in the cycle, 1315 of 1614 (81.5%) obtained at mid-cycle, and 595 of 748 (79.6%) late cycle specimens . Compare−1=403.1, median 93.6) peaked at mid-cycle  containing only one of the 13 types, HPV load  and HPV18 (n=192) separately. Among specimens collected early in the cycle that were associated with concurrent single infections with HPV 16 (determined by PCR), 77.7% of HC2 tests were positive, which was less sensitive than HC2 tests performed on specimens collected late in the menstrual cycle . Viral load was nonsignificantly lower (21.9%) for the specimens collected early as opposed to late in the cycle. For mid-cycle specimens associated with HPV 16, the frequency of HC2 positive results and load determinations did not differ significantly from those obtained for specimens late in the cycle. Among specimens associated with concurrent detection of single infections with HPV 18 (detected by PCR), positive HC2 results were more frequent at mid-cycle  and load was 55% higher (though non-significantly) compared to specimens collected late in the cycle.Finally, we analysed data for single infections with HPV16  are probably optimal for detection of HPV DNA by HC2 testing and for identification of LSIL by thin-layer cytology. However, the effects of the date of LMP on the performance of cytology and HC2 testing among women diagnosed with histologic CIN2+ was minor, suggesting that it is unnecessary to defer testing for women who present outside this interval.Ideally, optimizing the timing of specimen collection would minimize unsatisfactory specimens and cytologic reporting of ASCUS  overall and maximize clear-cut results of SIL among women with underlying CIN2+. Similar to results obtained with conventional smears, specimens obtained early in the menstrual cycle were associated with the highest frequency of unsatisfactory samples  as compared to mid-cycle (4.4%) and late in the cycle (2.9%), although these differences were not significantly significant based on limited data. To account for these findings, we speculate that misclassification of endometrial cells as HSIL contributes to higher reporting of HSIL during the early part of the cycle. In support of this hypothesis: (1) endometrial cells are generally identified only during the first 10 days of the cycle; (2) endometrial cells may mimic HSIL because of their similar small size and high nuclear to cytoplasmic ratio, especially in liquid-based cytology; and (3) the inconspicuousness of blood in most thin-layer slides disguises the occurrence of menses. Previous studies have emphasized that interpretation of HSIL in thin-layer cytology may pose difficulties, especially with regard to false negative results  rather than analytical sensitivity (detection of the lowest number of copies possible). Finally, some collections that yielded positive PCR and negative HC2 tests could reflect errors in HPV typing, differences in HPV content between samples tested by HC2 and PCR or other factors.Although this analysis was based on a clinical trial of over 5000 women who were followed with repeated examinations for 2 years, we recognize some limitations. The majority of women in this study were young and eligibility criteria included a recent cytology report of ASCUS or LSIL, therefore, these results may not apply to all women. In addition, the measurement of HPV load in cervical cellular collections using HC2 does not necessarily reflect the concentration of the virus in cervical tissue. In fact, it is recognized that HC2 pg mlIn conclusion, the LMP date is not a critical clinical consideration if women with cytology of ASCUS+ are closely monitored and HPV test results are categorized as negative or positive for management. However, it is prudent when possible to schedule screening tests at mid-cycle to optimize sensitivity and limit the occurrence of unsatisfactory cytology. Standardized timing of specimen collections may also minimize the inherent variability of test results for individual patients, thereby facilitating interpretation of serial results. Improved understanding of the interactions between hormones and HPV may eventually provide clues that are useful for cancer prevention.


Small molecules have played an important role in delineating molecular pathways involved in embryonic development and disease pathology. The need for novel small molecule modulators of biological processes has driven a number of targeted screens on large diverse libraries. However, due to the specific focus of such screens, the majority of the bioactive potential of these libraries remains unharnessed. In order to identify a higher proportion of compounds with interesting biological activities, we screened a diverse synthetic library for compounds that perturb the development of any of the multiple organs in zebrafish embryos. We identified small molecules that affect the development of a variety of structures such as heart, vasculature, brain, and body-axis. We utilized the previously known role of retinoic acid in anterior-posterior (A–P) patterning to identify the target of DTAB, a compound that caused A–P axis shortening in the zebrafish embryo. We show that DTAB is a retinoid with selective activity towards retinoic acid receptors γ and β. Thus, conducting zebrafish developmental screens using small molecules will not only enable the identification of compounds with diverse biological activities in a large chemical library but may also facilitate the identification of the target pathways of these biologically active molecules. Small molecules have proved to be powerful tools for understanding the importance of molecular pathways in development and disease. When compared to traditional genetic mutations, they have the advantage that they can be used to reversibly perturb the functions of molecules at different points of embryonic development or disease progression. Due to the concentration-dependent effects of small molecules on their targets, it is also possible to uncover phenotypes that result from partial inactivation of a pathway. Their intracellular permeability permits their use in cultured cells as well as whole organisms and makes them candidates for use in therapeutics. This has made the identification of novel small molecule modulators of important genetic networks an area of intense interest.in vitro systems has been utilized successfully in many targeted small molecule screens, resulting in the identification of novel biological tools and therapeutic candidates ex utero, and permeability to small molecules In an effort to discover novel disease modulators, a number of synthetic and natural chemical libraries have been assembled in recent years www.zfin.org). Therefore, in many cases, the developmental defects caused by a small molecule can be linked with high resolution to a specific genetic pathway known to cause the same defect.A major challenge of employing phenotypic screens for identifying small molecules is delineating their molecular targets. Zebrafish embryogenesis progresses through a strictly stereotypical sequence of events choreographed by numerous signaling pathways. Perturbation of these molecular pathways leads to distinct and predictable defects in the embryos that are easily detectable and have been extensively characterized for thousands of genetic mutants  axis-patterning defect in the embryos. Guided by the known role of retinoic acid (RA) in A–P patterning in embryos we were able to show that DTAB exhibits a retinoid-like activity and is able to phenocopy the effects of RA treatment on zebrafish embryos. Using receptor activity assays, we demonstrate that DTAB is also able to activate the retinoic acid receptors (RARs and RXRs) with selectivity for RAR-γ.To identify small molecule modulators of development, 5760 compounds from a diverse synthetic chemical library  were screened. Embryos at 1–4 hours post fertilization (hpf) were treated with the compounds and scored for obvious developmental defects in the body-axis, brain, heart, blood and vasculature at 24, 48 and 72 hours. 5540 compounds out of the 5760 tested (96%) caused no discernible phenotype at 72hpf and 45 compounds (0.8%) resulted in toxicity leading to death. 175 (3%) compounds affected normal development of the zebrafish embryos. Of these, 61 caused general developmental delay or embryos failed to hatch out of the chorion and 8 compounds caused severe disruption of multiple structures during development. The remaining 106 compounds caused phenotypes that included effects on the brain (21), heart morphology and function (43), blood and vasculature (28), gut (3), trunk and tail (20), pigment formation (3), and touch responsiveness (3) . The smaSince 96% of the compounds in the library did not appear to have any effect on embryogenesis we compared the chemical properties of the active and inactive compounds to identify properties that might determine biological activity. The ability of a compound to produce a phenotype in embryos depends on its physico-chemical properties as well as its interaction with developmentally relevant molecules. In terms of their size distribution the active compounds exhibited no bias and included the full size-range of the library, from 200 daltons (Da) to 700 Da . The parTreatment of embryos with DTAB (12.5 µM) at 1hpf caused a severe patterning defect resulting in embryos with almost no identifiable morphological features except scattered melanocytes across the skin surface . At 3 µMIn order to determine if the molecular target of DTAB was functional early or late in development, the embryos were exposed to 3 µM DTAB at different developmental stages from 1hpf to 24hpf . DTAB apIn situ hybridization for early developmental markers was performed in order to elucidate the molecular nature of the short-axis phenotype. Dorso-ventral patterning defects result in short anterior or posterior structures krox20, a marker for dorsally derived hindbrain segments rhombomere 3 and 5. Krox20 expression was completely absent in embryos treated with 12.5 µM of DTAB at 4hpf onwards  axis in the embryos  and goosecoid (gsc) are expressed in overlapping domains in the dorsal region of the shield stage embryos. As gastrulation progresses, the two domains separate with gsc moving anteriorly and ntl moving posteriorly In situ hybridization revealed that expression of ntl as well as gsc was reduced upon exposure to DTAB, thus indicating there is a reduction in both the anterior and posterior derived structures  it is expressed in the dorsal side of the embryo in the region flanking the presumptive notochord . otochord . Treatmeng brain . Taken tall trans-RA, and DEAB, a small molecule inhibitor of Raldh2 activity. Embryos treated with DTAB or RA exhibited remarkably similar patterning defects leading to severely shortened anterior-posterior axis  and the myoD (somites) staining domains while the Raldh2 mutant necklesskrox20 and myoD domains in embryos. Consistent with previous reports, treatment with RA increased the distance between krox20 and myoD markers when compared to vehicle treated embryos  metabolism, is a very important regulator of development and differentiation. In the vertebrate embryo, RA plays a crucial role in a variety of processes such as patterning of the anterior-posterior axis Interestingly, we found that DTAB exhibits selective activity towards RARγ and to a lesser extent towards RARβ, compared to the broad activity of RA. RARγ is predominantly expressed in the skin in the adult organism ex vivo can induce HSC self-renewal Decreased RARγ activity has been implicated in ovarian cancer, and AHPN (CD437), an RARγ agonist, has been shown to induce apoptosis and inhibit growth of ovarian tumor cell lines as well as transplanted tumors in nude mice For all experiments TUAB zebrafish lines were used. Fish embryos were incubated in non-buffered E3.Zebrafish embryos at ∼1hpf were arrayed in round-bottomed 96-well plates in 100 µl of E3. Each 384-well plate of Chembridge diverse set E compounds (in DMSO) was transferred to four 96-well plates. The compounds from the 96-well plates were pin-transferred (approx. 100 nl) into wells with arrayed embryos. Embryos were incubated with the compounds in a humidified box at 28°C and were screened for phenotypes at 24hpf, 48hpf and 72hpf under a dissecting microscope. Compounds of interest were re-tested in 24-well plates.Log P values of the whole tested library were calculated from values provided by Chembridge.trans-retinoic acid (RA) and 4-Diethylaminobenzaldehyde DEAB) were purchased from Sigma. Stock solutions of RA (1 mM) and DEAB (10 mM) were prepared in DMSO. DTAB (5133623) was purchased from Chembridge Corporation and a 12.5 mM stock was made in DMSO.All-in situ hybridization was performed essentially as previously described krox20 and myoDcyp26a1 and raldh2goosecoid (gsc) and no tail (ntl) pax2aWhole-mount Hek293T cells were grown in DMEM containing 10% de-lipidated fetal calf serum for 24 hours and were transfected with either [pCMX-Gal-L-hRAR +tk-px3-luc+tk-βgal] or [pCMX-mRXR+tk-apoA1-luc+tk-βgal]. ‘No-receptor’ controls were transfected with [tk-luc+tk-βgal] and treated with various concentrations of RA. 24 hours after transfection the cells were treated with RA, DTAB or left untreated. Cell lysates collected at 48 hours post-transfection were assayed for luciferase activity using the Promega luciferase reporter kit. The assay results were corrected against β-gal activity assayed using Promega β-gal reporter kit. All the reported results are from assays done in triplicates and repeated at least once. All the plasmids were kind gifts from Ronald M. Evans


Aluminum (Al) toxicity is a major worldwide constraint to crop productivity on acidic soils. Al becomes soluble at low pH, inhibiting root growth and severely reducing yields. Maize is an important staple food and commodity crop in acidic soil regions, especially in South America and Africa where these soils are very common. Al exclusion and intracellular tolerance have been suggested as two important mechanisms for Al tolerance in maize, but little is known about the underlying genetics.2 linkage populations with approximately 200 individuals each were used to study genetic variation in this complex trait. Al tolerance was measured as net root growth in nutrient solution under Al stress, which exhibited a wide range of variation between lines. Comparative and physiological genomics-based approaches were used to select 21 candidate genes for evaluation by association analysis.An association panel of 282 diverse maize inbred lines and three FSB likeZea mays Alt (ZmASL), Zea mays aluminum-activated malate transporter2 (ALMT2), S-adenosyl-L-homocysteinase (SAHH), and Malic Enzyme (ME). These four candidate genes are high priority subjects for follow-up biochemical and physiological studies on the mechanisms of Al tolerance in maize. Immediately, elite haplotype-specific molecular markers can be developed for these four genes and used for efficient marker-assisted selection of superior alleles in Al tolerance maize breeding programs.Six candidate genes had significant results from association analysis, but only four were confirmed by linkage analysis as putatively contributing to Al tolerance:  This form of Al is highly toxic to plant roots. Approximately 30% of the worlds ice-free soils are acidic, 17% of which are considered arable Aluminum (Al) toxicity from acidic soil is a major constraint to worldwide crop production. Al, one of the most abundant elements in the soil, is solubilized as Al3+ ion becomes the dominant species in an acidic environment 3+ disrupts many physiological processes in plants through both apoplastic and symplastic interactions, but exact mechanisms remain elusive 3+ intoxication is the cell wall of the root apex 3+ exposure, callose formation seals off the cell walls, increasing rigidity, decreasing extensibility and preventing further transport into the cell 3+ displaces Mg2+ and Ca2+, which are required for ATPases, cell signaling, and altering or inactivating the function of many proteins 2+ channels The toxic effects of acid soil result from an interaction between pH and elements in the soil. Several metals, including Al and Mn, become soluble at and below pH 5.5, which causes stress in the plant. In a neutral or basic environment, Al is found in insoluble divalent and monovalent forms of Al-oxides or Al-hydroxides, but the soluble trivalent AlPlants have developed several mechanisms for dealing with Al toxicity, which can be classified as either external or internal tolerance mechanisms Maize has considerable genetic variation in levels of Al tolerance, but clear physiological bases and molecular mechanisms for this tolerance remain elusive. Physiological studies found that OA exudation contributes to maize Al tolerance, but is not the only mechanism, as some Al sensitive varieties have been shown to exude high amounts of OA from the roots The genetic variation for Al tolerance in maize indicates it is a complex trait, involving many genes and physiological processes Biparental crosses used in linkage mapping, in which one or a few loci controlling Al tolerance may segregate, provide limited insight into the analysis of complex traits in general In this study, we used an integrated approach combining association mapping with linkage mapping to identify and evaluate candidate Al tolerance genes in maize. Without positively identified mechanisms or biochemical pathways involved in Al tolerance, selection of candidate genes requires knowledge based on previous studies and proposed mechanisms. We tested 21 candidate genes for association with Al tolerance, in a maize diversity panel of 282 inbred lines, using the MLM approach discussed earlier 3+3+} at pH 4.0. A control treatment was carried out over the same time period in an identical hydroponic solution, containing no Al3+. A wide range of tolerance levels is seen in this panel for both control NRG and Al treated NRG (−27). Mean correlation between replications was 42.5% in Al stress treatments and 37.3% in control treatment replications.Phenotypic data for Al tolerance in the maize association panel was collected as net root growth (NRG) in a hydroponic nutrient solution with or without a toxic level of Alated NRG . Mean NR2) for NRG in the Diversity Panel, calculated using the relatedness (K) matrix, was between 30 and 32% in the Al stress environment and 22% without Al stress. Broad sense heritability (H2) for NRG under Al stress and without Al stress was 41% and 37%, respectively. These heritability estimates for net root growth in seedlings are similar to those observed in maize breeding programs for enhanced tolerance to low pH Narrow sense heritability  is the major Al tolerance gene in wheat (Triticum aestivum) and is the first true Al tolerance gene identified in any plant TaALMT1 were examined and are referred to as ZmALMTx. One gene homologous to SBAlt, the major Al tolerance gene in Sorghum bicolor, is referred to as ZmASL (Zea mays AltSB – like) TaALMT1 from wheat have been demonstrated as Al tolerance genes in Arabidopsis and rye SBAlt from sorghum have been demonstrated as Al tolerance genes in Arabidopsis and barley Genotypic data for association mapping came from polymorphisms identified in candidate gene sequences. Genes were chosen based one of two factors: as responsive to Al-stress treatments according to gene expression analysis or by sequence similarity to Al tolerance genes found in other species . Genes tInformation regarding the genes used in association mapping is shown in The mixed linear model (MLM) was used for association mapping 2 linkage populations: malic enzyme (ME); isocitrate lyase (ISL); SAH hydrolase (SAHH); ZmALMT2 (ALMT2); ZmASL (ASL); pectin methylesterase (PME)  for Al stress. In order to estimate the number of expected false positives due to multiple testing of sites, a false discovery rate (FDR) was calculated for each model using 1,095 random SNPs throughout the genome. FDR allows for the comparison of significant sites in our candidate genes to those we would expect to see by random chance alone. Based on the FDR values for the MLMs, about 24% of the sites under the Q+K model and 34% under the Q+K+C model under Al stress could be accounted for by false positives. Given this high rate for false discovery, it is crucial to test the connection between the six genes with putative association to Al stress tolerance using an independent line of reasoning.Six genes had statistically significant associations (p≤0.01) with NRG under Al stress and were selected for further study in Fse (PME) . Complet2 populations. F2 populations were phenotyped in the same manner as the association panel and genotyped for the sites of interest  or additive gene action , S-adenosyl-L-homocysteinase (SAHH), Malic Enzyme (ME), and ZmALMT2 (ALMT2), are described in further detail below. All genes possessed more than one statistically significant polymorphism associated with Al tolerance differences. The complete coding sequences for the ZmASL, SAHH and ME genes were characterized in the 27 DL to look for other regions of interest such as non synonymous sites, alternative splicing, and protein structure modifications. ALMT2 was not sequenced in the 27 DL subset due to constraints caused by abundant paralogs within the ZmALMT family. Individual sites in these genes explain only about 2% of the phenotypic variance in the association panel, but confer 13%–20% increase in NRG.The four genes with significant association and linkage to Al tolerance, ZmASL, which is highly similar to the Al-activated citrate transporter from sorghum, is described in ZmASL, including exons, introns and nonsynonymous sites, based on the genomic sequence of the 27 DL. Total length of ZmASL sequenced in the 27 DL was about 6 kb, including 11 exons, both 5′ and 3′ UTRs and an upstream region containing a 300 bp MITE insertion. The common polymorphisms (frequencies ≥10%), which are responsible for 12 amino acid substitutions, are shown. The 43 rare amino acid substitutions, insertions or deletions (<10% frequency) are not shown. Many of the rare polymorphisms are found in only one of the 27 DL (CML247 was responsible for 21 sites).SBAlt gene is associated with the number of MITE insertions in the regulatory region of that gene ZmASL was not associated with NRG.Two minimally overlapping regions of this gene were sequenced in the association panel, covering a total of ∼1.7 kb . These r2<0.2) were significantly associated with NRG in the region sequenced , were significant for NRG under the Q+K+C model  gave a significant result (ZmALMT family. Two SNPs in this gene (#2 and #12) were associated with NRG under the Q+K model (2<0.2), although LD was moderate to extensive between most of the SNPs found at this gene  in the linkage population than expected from the association population (20.2%).Site #11, an indel that was not associated with Al tolerance, was used for the linkage analysis in the B73×NC350 FZmALMT2 and ZmASL, are very similar to Al-activated organic transporters that play crucial roles in determining Al tolerance in other species ME and SAHH are involved in several central metabolism reactions and speculations on their contribution to Al tolerance can be made based on previous studies 2 populations (13–20% increases in NRG). While none of these new Al tolerance genes represent major effect QTL, combining multiple small QTL can make a significant impact to enhance the desired trait. In the B73×CML247 F2 population, combining the elite alleles of ZmASL and SAHH enhanced net root growth by 30%  is a maize gene homologous to SBAlt, the major Al tolerance gene from sorghum and is a member of the Multidrug And Toxic Compound Extrusion (MATE) family of transporters ZmASL gene mediates Al-activated root citrate efflux, as is the role of SBAlt in sorghum. Although many sites in ZmASL were associated with NRG under Al stress, none were amino acid substitutions. The significant sites we detected may be in LD with regulatory elements of the gene, as is the case in SBAlt, where polymorphisms in the promoter help to determine the level of gene expression ZmASL contained the most significant independent sites of any gene tested, but also contained extensive LD among many of the other significant sites. Fortunately, future experiments to evaluate the relationship of ZmASL with Al tolerance will be relatively straightforward given the presumed gene function.SAHH, S-adenosyl-L-homocysteine hydrolase, is an enzyme that removes the feedback inhibitor of SAM (S-adenosylmethionine) mediated methylation in any organism SAHH has a high degree of sequence conservation among eukaryotes SAHH is a cytokinin binding protein in plants, induced by auxin and cytokinin, and has been associated with salt-stress response in spinach and sugar beets and viral resistance in Arabidopsis SAHH we examined was previously found to be highly expressed in root tips under Al stress SAHH contributes to Al tolerance differences via cell wall modification NADP-ME (ME) catalyzes the conversion of malate to pyruvate. The maize ME examined in this study was the cytosolic rather than plastidic isoform of the enzyme. Maize Cyt-ME is highly similar to Cyt-ME found other in C3 and C4 plant species. This isoform was found to be expressed in the embryo and emerging roots, with expression responsive to hypoxia and drought ME was unusual among the genes we examined as the results from linkage and association studies were opposite in direction, while still both highly significant. In the association mapping, three significant sites were identified – a site in the first exon (SNP #4), which was in linkage equilibrium with all other sites, and two sites in the second intron (SNP #7 and SNP #11), which were in high linkage disequilibrium with all of nearby the SNPs . Non-B73 parents were selected on the basis of genotype information for the candidate Al tolerance genes.The maize association population has been previously described 3+ at pH 4.0, for 1 d, for acclimation to hydroponic conditions 3+} at pH 4.0 (Al stress treatments) or Magnavaca nutrient solution containing no Al3+ at pH 4.0 (control treatments), for 2 d. After 2 d of Al stress final root growth (FRG) measurements were taken. Net root growth (NRG) was calculated as FRG – IRG.Maize seeds were germinated in either autoclaved sand or on filter paper, moistened with deionized water, for 3–5 d at 28°C in continuous darkness. Seedlings were rinsed and placed into sample cups suspended in 8L vessels containing a nutrient solution without AlFive replicate experiments were performed for Al stress treatments, while three replicates were performed for the control treatment. In each experiment, 3–4 individuals for each of the 282 inbred varieties in the association panel were phenotyped in each replicate experiment. Least squares means (LSmean) for both traits were calculated in SAS version 9.1 for Windows  and used as the phenotypic values in all models .2 linkage populations were phenotyped in a similar manner, with {27 µM Al3+} at pH 4.0, with the modification that 200 F2 individuals were evaluated for each cross plus parental checks (n = 10). Leaf tissue was collected for DNA extraction and genotypic analysis after FRG measurements. Measurements of NRG under control and stress treatments are found in FAll DNA was isolated using a standard CTAB extraction method 2 populations were genotyped only for the loci that were expected to segregate in each cross. Molecular markers were developed from sequence analysis of each locus and evaluated using standard PCR methods on agarose gels for indels or by fluorescently labeled primers for SNPs (Ffor SNPs . Marker Primers were designed based on reference sequences obtained from the Maize Assembled Genomic Island (MAGI) Database TASSEL 1.9.6 was used to evaluate linkage disequilibrium (LD), extract polymorphic sites, calculate narrow sense heritability, and perform General and Mixed Linear Models  with incorporation of trait data, population structure (Q) and kinship matrix (K) The MLM approach and estimation of the kinship matrix (K) has been previously described The mixed model used, for vector of phenotypes, y, is:2 = 1) due to an excessive (73) number of sites in LD. Lines with quality scores less than 60% were discarded.Polymorphic sites tested, SNPs and indels, that occurred ≥10% were extracted from aligned sequence data. A total of 331 sites were across 21 genes were used . Sites fIn order to account for expected false positives present due to multiple testing, a False Discovery Rate (FDR) was calculated using 1095 SNPs that occur randomly across the maize genome 2) was calculated in TASSEL using the kinship matrix (K) as a parent-offspring regression. Broad sense heritability  for the association panel in the Al-stress condition  and control condition .(0.03 MB PDF)Click here for additional data file.Table S2Sequence polymorphisms utilized for association analysis. Polymorphic sites (SNPs and indels) were identified in each of the 21 candidate Al tolerance genes across the 282 member association panel. SNPs are coded as nucleotides (ACGT), indels are coded as numbers , while missing data appear as N.(0.61 MB PDF)Click here for additional data file.Table S3Mixed Linear Model (MLM) based association analysis. MLM analysis was used to evaluate the importance for each polymorphic site in every candidate Al tolerance gene for NRG. Al-stress and control growth conditions were evaluated separately. All results are reported here.(0.18 MB PDF)Click here for additional data file.Table S42 populations segregating for the candidate Al tolerance genes. This table reports phenotypic and genotypic information for the linkage experiments.Validation of association mapping via linkage mapping. Association mapping results were validated using linkage mapping of F(0.05 MB PDF)Click here for additional data file.Table S5PCR primers utilized for linkage mapping.(0.02 MB PDF)Click here for additional data file.


Deep sequencing of PCR amplicon libraries facilitates the detection of low-abundance populations in environmental DNA surveys of complex microbial communities. At the same time, deep sequencing can lead to overestimates of microbial diversity through the generation of low-frequency, error-prone reads. Even with sequencing error rates below 0.005 per nucleotide position, the common method of generating operational taxonomic units (OTUs) by multiple sequence alignment and complete-linkage clustering significantly increases the number of predicted OTUs and inflates richness estimates. We show that a 2% single-linkage preclustering methodology followed by an average-linkage clustering based on pairwise alignments more accurately predicts expected OTUs in both single and pooled template preparations of known taxonomic composition. This new clustering method can reduce the OTU richness in environmental samples by as much as 30–60% but does not reduce the fraction of OTUs in long-tailed rank abundance curves that defines the rare biosphere. Massively parallel pyrosequencing of ribosomal RNA (rRNA) coding regions that evolve rapidly allows the detection of very low abundance populations in complex microbial communities. Each processed sequence (pyrotag) more or less serves as a proxy for the occurrence of a microbial genome in an environmental DNA sample. Matching pyrotags to a reference rRNA database or clustering tags in a taxon-independent manner to identify operational taxonomic units (OTUs) suggests that taxonomic richness in marine, terrestrial and microbiome communities exceeds all prior estimates of microbial diversity. Many of the novel OTUs correspond to low-abundance organisms of the ‘rare biosphere’ ; 2007;. Quality-controlled sequence reads of 16S rRNA V6 hypervariable region amplicon libraries produced by the Roche Genome Sequencer 20 System (GS 20) have a per-base error rate of 0.25%  cultures of a single clone of Staphylococcus epidermidis, which contains six 16S rRNA operons with two distinct V6 sequences; (iii) a plasmid clone containing a nearly full-length 16S rRNA coding region from E. coli 16S rRNA operon A, (iv) a plasmid clone containing a nearly full-length 16S rRNA coding region from S. epidermidis 16S rRNA operon 9; and (v) a mixture of 43 plasmid clones (Clone-43) containing partial 16S rRNA coding regions that span the V6 hypervariable region of distinct 16S rRNA genes amplified from a deep-sea hydrothermal vent community  cultures of a single clone (colony forming unit) of E. coli and S. epidermidis genomic template data sets led to the formation of several obvious within-species chimeras that we identified and removed based upon our knowledge of the sequences.Prior to calculating the sequencing error rate, we eliminated the 11–19% of all reads that did not pass previously described quality filtering  eliminatDifferences between a pyrotag sequence and its template reflect a combination of PCR and pyrosequencing error. We calculated the combined error rate, understanding that any unfiltered contamination, chimeras or non-rRNA amplification will increase our error rate estimates. We define the error rate for a given sequence as the number of insertions, deletions and substitutions in a sequence as compared with its template, divided by the length of the template sequence. The error rate varied between data sets, ranging from 0.0021 to 0.0042 . This fin errors would follow the binomial distribution. If pyrosequencing errors occurred at random, the number of expected sequences with exactly E. coli OTUs, 89 rather than the two expected S. epidermidis OTUs and 694 Clone-43 OTUs rather than the expected 43  from their template . For each species, we inoculated three 250 ml flasks, each containing 50 ml nutrient broth, with 100 µl of the overnight culture. We incubated the flasks at 37°C with vigorous shaking and harvested the cultures at late stationary phase (OD600 of ∼1.25). We then divided each culture into three aliquots, extracted genomic DNA and used each DNA extraction in a separate amplification of the V6 region. We split one amplicon library from each species into two emPCR amplification prior to pyrosequencing for a total of 10 subsamples of single clones of E. coli and of S. epidermidis. We also generated an amplicon library from a previously prepared pool of plasmid DNA from 43 different cloned 16S rRNA genes from deep-sea vent organisms .We generated amplicon libraries using primer pools designed to span the V6 or V4-V5 hypervariable regions of as many known bacteria or archaea as possible . We usedData from environmental samples were collected as previously described , that did not have an exact match to the expected bar-coded forward primers, or that had an average quality score less than 30    to any of the sequences already in the cluster, the new sequence will be added to the cluster and not tested against subsequent clusters. If the sequence is not within a distance of 0.02 from any read in any of the existing clusters, it will establish a new cluster. Once all sequences have been assigned to clusters, sequences in the low abundance clusters (< 10 tags) are tested against the larger clusters and added to those clusters if possible. Ordering sequences by frequency ensures that those sequences that accurately represent the template pool will preferentially seed the establishment of a new cluster rather than those errant sequences that occur at lower frequency. This also helps avoid linking very different template sequences into a single cluster through a chain of low frequency errant sequences. The second round of preclustering incorporates small clusters into larger clusters in cases where the smaller cluster formed before the larger cluster included sequences that differ from the smaller cluster by less than 2%. For each final precluster, SLP assigns the sequence with the highest frequency to represent the cluster in downstream OTU clustering, with the count of all tags in the precluster assigned to it. The perl script that performs these operations, SLP, can be downloaded from We randomly selected 10 000 tags having an average quality score ≥ 30 and no ambiguous bases. We ran PyroNoise with default values (including −s 15.0 −c 0.05 for PCluster) and removed denoised sequences that did not map to Bacteria by GAST. The remaining sequences were clustered using pairwise alignments and average-linkage clustering as above.


Previous studies on primary biliary cirrhosis (PBC) have focused on             the role of T lymphocytes as potential effectors of tissue injury. We hypothesized              that single nucleotide polymorphisms (SNPs) of genes involved in lymphocyte              proliferation would be responsible for uncontrolled expansion of T cells and              autoreactivity. To address this, we genotyped DNA from 154 patients with PBC              and 166 ethnically matched healthy controls for SNPs of five candidate genes               using a TaqMan assay.		p = 0.0411).               Moreover, we found a significant association of the same allele and of the LYP⋆T               allele with anti-mitochondrial antibody (AMA) serum negativity . No association between any of the other studied SNPs and               PBC susceptibility, progression, or AMA status was observed. In conclusion, given               the high prevalence of SNPs in CTLA-4 detected in numerous autoimmune               diseases, we encourage a more detailed genetic analysis of this candidate gene.               Further, although obtained from a limited number of AMA-negative subjects, our               data suggest a potential genetic              heterogeneity for this specific subgroup of patients with PBC.We report herein a statistically significant decrease in homozygosity rate              for the 60A⋆CTLA-4 allele in patients with PBC compared to controls (