ApcMin (Min) mouse model of CRC is a classic quantitative trait that is subject to complex genetic and environmental factors, and therefore serves as an ideal platform to study modifiers of disease. While disparate inbred genetic backgrounds have well-characterized modifying effects on tumor multiplicity, it is unclear whether more closely related backgrounds such as C57BL/6J and C57BL6/N differentially modify the phenotype. Furthermore, it is unknown whether the complex gut microbiota (GM) influences the effects of these background strains. We assessed tumor multiplicity in F1 mice generated from the original Min colony from the McArdle Laboratory at the University of Wisconsin (C57BL/6JMlcr-ApcMin) crossed with either C57BL/6J or C57BL/6N wild-type mice. We also used complex microbiota targeted rederivation to rederive B6NB6JMF1-ApcMin embryos using surrogate dams harboring complex GMs from two different sources to determine the effects of complex GM. Both B6/J and B6/N backgrounds significantly repressed tumor multiplicity. However, the B6/N background conferred a stronger dominant suppressive effect than B6/J. Moreover, we observed that complex GM likely modulated B6/N-mediated adenoma repression such that two distinct communities conferred differential tumor multiplicity in isogenic B6NB6JMF1-ApcMin mice. Although we cannot rule out possible maternal effects of embryo transfer, we show that B6/J and B6/N have modifier effects on Min, and these effects are further altered by the complex GM. Foremost, strict attention to genetic background and environmental variables influencing the GM is critical to enhance reproducibility in models of complex disease traits.Tumor multiplicity in the The online version of this article (10.1007/s00335-019-09814-3) contains supplementary material, which is available to authorized users. ApcMin (Min) mouse model of CRC, which harbors an autosomal dominant mutation in the Apc tumor suppressor gene causing the development of intestinal adenomas, provides an extensively studied and quantitative platform to investigate factors contributing to disease initiation  loci  necessitate the use of animal models that enable the resolution of complex traits. The il10−/− mice, which develop intestinal inflammation due to loss of immunoregulatory IL-10, were colonized with three distinct GM communities. The isogenic mice subsequently developed differing severity of disease based on GM colonization, corroborating the important role for intestinal microbial communities  has also emerged as an important factor in complex disease traits such as inflammatory bowel disease (IBD) and CRC , and either B6/J or B6/N wild-type females. F1 offspring from the B6/N lineage (B6NB6JMF1) were rederived using surrogates harboring two different complex GMs, such that isogenic mice were born with two distinct GMs. We describe differential adenoma suppression mediated by the B6/J and B6/N strains across GM-controlled groups, and illustrate potential maternal effects that may contribute to phenotype variability. We further demonstrate GM-mediated phenotype modulation in isogenic mice. Through these approaches, we develop a platform for identifying genetic and bacterial modifiers of the Min phenotype, and concurrently illustrate how complex variables shape complex quantitative traits in a classic cancer model.In this study, we addressed two questions in an effort to further our understanding of different factors contributing to phenotypic variability across Min colonies. First, we aimed to determine how the B6/N genetic background modifies adenoma multiplicity in comparison to the B6/J strain. Furthermore, we asked whether colonization with distinct complex GM communities further modulates adenoma multiplicity. We established F1 Min cohorts using Min males from the original colony at the McArdle Laboratory of the University of Wisconsin  compared to the B6/JM line  , as they developed significantly fewer adenomas than the B6/JM line and significantly more adenomas than the B6/J parents (p < 0.001)  and a high-richness community (GM4) using CD-1 surrogate dams with GMs that originally represented The Jackson Laboratory (GM1) or Envigo (GM4)  to establish B6NB6JMF1-M4) Fig. a. Of notentclass1pt{minimaApcMin mice, we characterized GM1 and GM4 through 16S rDNA sequencing. Fecal samples were collected from B6NB6JMF1GM1 and B6NB6JMF1GM4 mice at 1 month of age and subject to sequencing of the 16S rRNA microbial gene to determine relative abundances of all detected bacterial phyla and operational taxonomic units (OTUs). Fecal samples were also collected from a subset of the B6/J and B6/JM parental colonies and B6JB6JMF1 mice. To visualize overall GM community similarity of parental colonies and F1 mice, a Principal Component Analysis (PCA) was used  males from the original Min colony from the McArdle Laboratory at the University of Wisconsin with C57BL/6J (B6/J) females to generate a B6JB6JMF1 Min cohort  gene in B6/J  accredited facility based on the guidelines provided by the Guide for the Care and Use of Laboratory Animals. All animal studies were approved by the University of Missouri Institutional Animal Care and Use Committee.ApcMin/Mmmh (B6/JM) embryos from the original Min colony at McArdle Laboratory, University of Wisconsin were acquired and rederived in our facility at the University of Missouri using CD-1 surrogate dams with a GM representing The Jackson Laboratory (Crl:CD1GM1), previously generated in our laboratory  was acquired from the Jackson Laboratory. Both B6/JM and B6/J colonies were maintained at the University of Missouri as separate colonies. To generate B6JB6JMF1-ApcMin mice, 6–8-week-old B6/J-Apc/++ females were mated with 6–8-week-old B6/JM-ApcMin+/ males. Four- to five-week-old female C57BL/6NHsd (B6/N) were purchased from Envigo . To generate B6NB6JMF1-Apc/Min+ mice, in vitro fertilization (IVF) was performed as described previously using 5- to 8-week-old B6/N females and B6/JM males   were previously acclimated and maintained as a colony at the University of Missouri prior to their use as surrogates. The aforementioned Crl:CD1GM1 females harboring a GM representing The Jackson Laboratory were used as the second cohort, such that half of the B6NB6JMF1-ApcMin embryos were transferred into CD-1GM1 surrogates, and half into CD-1GM4 surrogates. All female CD-1 embryo recipients were mated with vasectomized CD-1GM1 or CD-1GM4 males, respectively, and those that were copulatory plug-positive were used for embryo transfer. CD-1 females were anesthetized via IM injection of ketamine/xylazine cocktail at 5.5 mg and 1 mg per 100 g body weight, respectively. A dorsal midline incision was made and the uterine oviducts located by dissecting through the retroperitoneal muscle. Embryos in 3 to 5 µL of media were injected into the oviducts using a glass hand-pipette. Skin incisions were closed with sterile surgical staples and mice received a subcutaneous injection of 2.5 mg/kg of body weight flunixin meglumine (Banamine®) prior to recovery on a warming pad.Frozen C57BL/6JMlcr-es Takeo . Presumees Takeo . In ordeApcMin would acquire the GM of their respective surrogate dams. Thus, these mice are denoted as Surrogate dams were allowed to naturally deliver pups such that resulting B6NB6JMF1-2 asphyxiation. Following exposure through the abdominal cavity, whole small and large intestines were incised longitudinally, flushed with saline and placed on bibulous paper with the luminal side facing up for formalin fixation. Grossly visible adenomas were counted manually using a Leica M165FC microscope at ×12.5 magnification.At 100 days of age, mice requiring adenoma counts were euthanized via CO® (Qiagen) as previously described  for further processing. An amplicon library of the hypervariable V4 region of the bacterial 16S rRNA gene was generated using the U515F/806R primer set as described previously . Quality control of DNA, assembly of contiguous sequences, sequence removal after trimming for base quality, and chimera removal were completed as described  with the Student Newman–Keul post hoc test in Sigma Plot 14.0 . For statistical comparisons of bacterial phyla and α-diversity indices, the student’s Supplementary material 1 (DOCX 79 kb)Below is the link to the electronic supplementary material.


The distribution of m6A peaks within HIV transcripts in HIV Tg rats largely corresponded to the ones observed for HIV transcripts in cell lines and T cells. Host transcripts were found to be differentially m6A methylated in HIV Tg rats. The functional roles of the differentially m6A methylated pathways in HIV Tg rats is consistent with a key role of RNA methylation in the regulation of the brain transcriptome in chronic HIV disease. In particular, host transcripts show significant differential m6A methylation of genes involved in several pathways related to neural function, suggestive of synaptodendritic injury and neurodegeneration, inflammation and immune response, as well as RNA processing and metabolism, such as splicing. Changes in m6A methylation were usually positively correlated with differential expression, while differential m6A methylation of pathways involved in RNA processing were more likely to be negatively correlated with gene expression changes. Thus, sets of differentially m6A methylated, functionally-related transcripts appear to be involved in coordinated transcriptional responses in the context of chronic HIV. Altogether, our results support that m6A methylation represents an additional layer of regulation of HIV and host gene expression in vivo that contributes significantly to the transcriptional effects of chronic HIV.Internal RNA modifications have been known for decades, however their roles in mRNA regulation have only recently started to be elucidated. Here we investigated the most abundant mRNA modification, N It has long been known that transfer RNA (tRNA), ribosomal RNA (rRNA) and both mRNA and noncoding RNA (ncRNA) contain multiple modifications –5. At leg others . Of thesne (m6A) , 2. The  in 1974 , 12. Whis of m6A . mRNAs a6A-modified RNA sites has been suggested [6A methylated, while highly regulated mRNAs often contain m6A residues [6A methylated within the consensus motifs G(m6A)C and A(m6A)C [6A appears to contribute to the regulation of gene expression and is increasingly being implicated in disease. Evidence suggests that RNA m6A methylation contributes to the regulation of splicing, nuclear RNA export, mRNA stability, and translation [6A methylation is at least in part dynamically regulated and potentially reversible [6A RNA methylation and the fate of m6A methylated RNA [6A demethylase, has been linked to a variety of biological processes including dopaminergic neuron regulation [6A contents in the RNAs of T2DM patients and diabetic rats are significantly lower than controls, while the level of FTO in T2DM patients was significantly higher [6A content in mRNA and impaired fertility [Reminiscent of DNA epigenetics, the occurrence of both stable and dynamically regulated muggested , 14–16. residues , 10. RNA A(m6A)C . RNA modnslation –23. RNA versible . Multiplated RNA . The fatgulation  and a vagulation –31. The ertility .6A modified. Classic studies by Shatkin and associates in the 70s showed specific patterns of m6A modification in transcripts from both DNA and RNA viruses such as influenza virus, adenovirus, papovavirus, and flavivirus, among others [6A modified [6A modification in HIV-1 transcripts contributes to regulate HIV-1 mRNA in vitro [6A modification of viral RNAs appears to be antiviral innate immune evasion, since the presence of m6A on viral RNA has been shown to reduce its activation of toll-like receptor 3 signaling [Multiple viral RNAs have long been known to be mg others –35. Retrmodified , 37. Themodified –41. Recein vitro –44. Anotignaling .6A methylation of RNA in the hippocampus of HIV Tg rats. HIV-1 Tg rats harbor a non-replicating HIV-1 transgene and express multiple HIV-1 proteins under the viral LTR in macrophages, lymphocytes and disease-relevant glial cells, such as microglia and astrocytes, but not in neurons [Here we investigated m neurons , 47. HIV neurons  and reve neurons , 50, bef neurons . This is neurons .6A RNA methylation in HIV transcripts in HIV Tg rats largely corresponds to the ones reported for HIV transcripts in cell lines and T cells in vitro [6A methylation of genes involved in several pathways related to neural function, suggestive of synaptodendritic injury and neurodegeneration, consistent with our previous observations in both HIV Tg rats and humans with HIV [6A methylation and expression are also involved in immune response and inflammation, also consistently with gene expression changes in HIV Tg rats and humans with HIV [6A methylation in transcripts of pathways involved in RNA splicing and processing, which are processes previously implicated in the role of m6A RNA methylation [We observed that distribution of peaks of min vitro –44. Furtwith HIV , 53. Trawith HIV , 53. We hylation .6A. As shown in 6A methylation was similar, although a slightly larger number of genes with down-regulation of m6A methylation was observed than genes with up-regulated methylation in the hippocampus of HIV Tg rats  in the distal CDS and 3’UTR of the mRNAs and especially in the immediate vicinity of the stop codon , which partially overlaps with the 3’ LTR  algorithm  to ident Tg rats . A total Tg rats  of whichhylation . The topc injury –60, Fig  Tg rats , 53, Fig6A pathways related to neural function include “Reactome Neuronal Systems”, “KEGG Axon Guidance”, and “Reactome Trafficking of Glur2 containing AMPA Receptors”  vivarium on a 12h/12h light/dark cycle with ad libitum access to food and water. Rats were sacrificed by decapitation under deep CO2 anesthesia. All procedures adhered to the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of The Scripps Research Institute.Adult male HIV Tg rats in Sprague Dawley background (Harlan Sprague-Dawley) 5–6 months of age were housed in groups of two per cage in a temperature-controlled (20–26 6A (Synaptic Systems), which was pre-immobilized to M-270 Epoxy using the Dynabeads Antibody Coupling kit (Thermo Fisher Scientific) for easy capture of the m6A-modified RNA in subsequent elution with a Proteinase K-containing buffer. Eluate with the anti-m6A captured RNA was extracted by phenol/CHCl3 followed by ethanol precipitation.Total RNA was extracted from dissected hippocampi of the wildtype and HIV Tg rats with RNEasy kit (Qiagen) and poly-A selected with Dynabeads Oligo(dT)25 (Thermo Fisher Scientific) following manufacturer’s instructions. Poly-A enriched RNA was recovered by ethanol precipitation overnight, fragmented using RNA Fragmentation Reagents (Thermo Fisher Scientific) and ethanol precipitation overnight. An aliquot of fragmented RNA was set aside serving as input or control for RNA sequencing. Twenty μg of fragmented RNAs were denatured at 75°C for 5 min, cooled on ice for 3 min, and then incubated for 2h at 4°C with 3 μg of pre-equilibrated anti-mLibraries for the anti-m6A-captured and control  RNAs were generated for next gen sequencing, following the protocol recommended in the ScriptSeq v2 RNA-Seq Library Preparation Kit (Illumina), and uniquely barcoded with the ScriptSeq Index PCR Primers (Illumina) to run a 16-library sample multiplex in a single flowcell on a NextSeq500 platform (Illumina) to generate ~30M 1x75 bp reads/sample.Sequences were first trimmed using Trimmomatic with default setting (version 0.33). All the samples passed the quality control by fastQC (version 0.11.3). Fastq files were aligned to a combined rat genome (Rnor_6.0) and HIV-1 genome (NC_001802) using STAR (v2.4) with default setting. The overall mapping rate range from 79.78% to 85.79% 6A peaks within host mRNAs, per base coverage was generated for all the m6A treated samples using R package “RiboProfiling” for a fixed interval of 400 bases around the start codon and stop codon.RNA methylation sites were detected using exomePeak  in HIV infected rat samples and control samples separately. Aligned bam files of m6A treated samples and non-treated samples were directly passed to exomePeak to test for statistically significant enrichment. A total of 9696 and 9051 consist RNA methylation sites with at least 100 bp were identified in HIV infected samples and control samples respectively . 201 genes were identified with significant changes of methylation   . Differe < 0.05) . A set o < 0.05) .A total of 1,452 well-known gene sets from the MSigDB collection was tested with the GSEA algorithm. 166 gene sets were significantly changed in expression   and 74 gS1 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.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.


The high genetic diversity of HIV-1 has been shown to influence the global distribution, disease progression, treatment success, and the development of an effective vaccine. Despite the low HIV prevalence in Cameroon, all the major HIV subtypes alongside several circulating recombinant forms (CRFs) and unique recombinant forms (URFs) have been reported in Cameroon. To date, HIV-1 diversity in some parts of Cameroon has been largely studied however, information on circulating HIV-1 subtypes in the Northwest region (NWR) of Cameroon is dearth. Therefore the aim of this study was to determine the current circulating HIV-1 subtypes among adults in the NWR of Cameroon.pol gene was performed on 81 samples. The samples were collected from drug naïve patients aged between 18 and 61 years residing within the rural and urban towns in the NWR during the period between February and April 2016. Viral RNA was extracted from plasma, reverse-transcribed, further amplified by nested-PCR before sequencing using an in-house protocol. Generated sequences were then phylogenetically analyzed together with references using MEGA 7.The genetic analysis of the reverse transcriptase region of the Phylogenetic analysis revealed a broad viral diversity including CRF02 _AG (74.1%), F2 (7.4%), D (7.4%), G (3.7%), A1 (1.2%), CRF22_01A1 (2.5%), CRF06_cpx (1.2%), CRF09_cpx (1.2%), CRF11_cpx (1.2%). Three close epidemic clusters were found among F2 (1) and CRF02_AG (2) variants. For the first time we are reporting the CRF22_01A1 subtype in this region.Our findings update HIV-1 subtypes information in Cameroon and uphold previous studies that CRF02_AG is the most prevalent subtype. This CRF02_AG subtype may have important public health, research, and clinical consequences. Studies involving other parts of the country like the NWR have not been adequately evaluated. The few studies undertaken in this region, even though sample sizes have been small (< 30 samples), have revealed a number of new strains , 12. DueA cross sectional study was conducted and five ART clinics systematically sampled; Bafut, Bali, Ndop and Santa District Hospitals and Bamenda Regional hospital of the NWR of Cameroon. Study participants were enrolled on the day of antiretroviral therapy (ART) initiation from February 2016 to April 2016. A standardized questionnaire and patient record file were used to collect demographic.The National Ethics Committee of Cameroon approved the study protocols (NO2016/01/685/CE/CNERSH/SP). The purpose of the study and research procedures were fully explained to participants in the language  best understood by the clients. Signed informed consent was obtained from each of the participants prior to the interviews and blood collection.(HAART) naïve patients, male or female aged ≥18 years while the exclusion criteria were pre-exposure to HAART, and patients looking severely ill . Of the 100 patients enrolled, 81(81%) samples were sequenced in required reverse transcriptase coding regions giving a successfully sequencing performance of 81%.A total of 100 HIV-1 patients were enrolled from February 2016 to April 2016. The eligibility criteria were HIV-1 Highly active anti-retroviral therapy + T cell counts was performed using a FACS Calibur flow cytometer  equipped with automated acquisition and analysis software according to the manufactures instructions [The CD4ructions .pol-RT gene corresponding to 2075–3703 bp (the position is given referring to theHXB2 strain) was amplified by nested PCR using in-house protocol. Of the 100 samples, 81(81%) were successful amplified, purified, and directly sequenced using ABI 3130 genetic analyzer  according to previously published in-house protocol with the following primers as shown on Table Five milliliters of whole blood was drawn from each subject into an EDTA (Ethylene Diamine Tetra acetic Acid) tube and centrifuged at 3000 rpm for 5 min. Two plasma aliquots of 1 ml each were collected from each patient and stored at − 80 °C until transported in an ice box to the Molecular Biology Laboratory of CIRCB-Yaoundé for analysis at CIRCB-Yaoundé where the samples were stored at − 20 °C. Plasma Viral RNA was extracted from 1 ml plasma using the QIAamp Viral RNA Mini kit  according to the manufacturer’s instructions. Extracted RNA was transcribed to cDNA using in-house RT –PCR protocol using a one-step kit . Partial HIV-1 http://www.hiv.lanl.gov/content/sequence/RIP/RIP.html).Generated sequences were aligned using CLUSTAlW version 1.8.3 with subsequent inspection and manual modification with pair wise evolutionary distances estimated by Kimura’s two-parameter method with bootstrap analysis of 1000 replicates. Resulting trees were visualized using Fig tree software. Bootstrap resampling (1000 data sets) of multiple alignments was performed to test the statistical robustness of the trees . Baseline characteristics of study patients were described using frequencies and percentages.+ T cell count was 194.3 (8–498) cells/mm3 and a greater part of the participants had CD4+ T cells count of < 200 cells/mm3 59.3% (48). The majority of the patients were residents in an Urban setting 54.3% (44)  were female. The mean (range) CD4From the 81 samples that were successfully amplified and sequenced, analysis of these sequences revealed; four pure subtypes and five CRF; F2 (6: 7.4%), G (4: 4.9%), D (3: 3.7%),A1 (1: 1.2%) pure subtypes and CRF02_AG (60: 74.1%), CRF22_01A1 (2: 2.4%), CRF06_cpx (1: 1.2%), CRF09_cpx (1: 1.2%) and CRF11_cpx (1: 1.2%) CRFs  patients were infected with HIV variants that carried resistance associated mutations associated with Nucleoside reverse transcriptase inhibitor (NRTI); 8.6% (7/81), Non nucleoside reverse transcriptase inhibitor (NNRTI); 4.9% (4/81) and protease inhibitors (PI); 1.2% (1/81). In this study, the most common mutations were K219Q  and E138A  which confers resistance to NRTIs and NNRTIs respectively. Singleton mutations that were associated with NRTI included: D67N, K70R, T215F, T215TA, M184MV, M184 V, D67N, K70R, K70 T, and M41ML; while that of, NNRTI was K103 N, V108I, V179E, and Y181C and for PI was I54IFV. Dual-class drug resistance mutation involving both NRTI and NNRTI was observed in three patients (3.7%).Previous studies have shown that CRF02_AG is the dominant virus circulating in Cameroon. This study showed that CRF02_AG was the dominant HIV-1 subtypes in circulation involving 74.1% of the entire samples analyzed  in this study falls within the 48.6–80% range of previous studies conducted in Cameroon and other countries in West Africa, Central Africa, and Europe , 19–21. Compared with previous studies from different urban towns in Cameroon, the prevalent subtypes after CRF02_AG were F2, G, and D contrary to detected G, F2, and D  and D, FThe CRF22_01A1 variant seen in this study with a very low prevalence has not been reported in the NWR , 12.The Phylogenetic analysis of these sequences showed clusters with reference sequences from Cameroon and surrounding countries indicate possible local origins. This implies that transmissions of these viral strains are within the country.Transmitted drug resistance (TDR) was however determined in this study. Overall prevalence of TDR was 11.1%. This prevalence was found to be higher compared to those detected (7.2%) in West and Central Africa in 2016 . The mod, [The high prevalence of NRTI over NNRTI mutations detected was contrary to previous finding in other urban centres in Cameroon , 26. How, . This hi, , 30. The, . Detecti, , 32. Nev, , 33. Bas, . The detOur data show an increasing diversity of HIV-1 in the NWR. This study confirmed previous findings that CRF02_AG subtype is still the most predominant subtype. In addition, the overall prevalence of TDR among recently diagnosed individuals in Northern region of Cameroun was moderate. We suggest that there is need for implementation of effective measures that could strengthen monitoring and guide ART usage and surveillance of HIV genetic diversity.


Group A rotaviruses (RVA) are one of the major causes of acute gastroenteritis (AGE) in young children worldwide. Owing to lack of proper surveillance programs and health facilities, developing countries of Asia and Africa carry a disproportionately heavy share of the RVA disease burden. The aim of this hospital-based study was to investigate the circulation of RVA genotypes in Rawalpindi and Islamabad, Pakistan in 2015 and 2016, prior to the implementation of RVA vaccine. 639 faecal samples collected from children under 10 years of age hospitalized with AGE were tested for RVA antigen by ELISA. Among 171 ELISA positive samples, 143 were successfully screened for RT-PCR and sequencing. The prevalence of RVA was found to be 26.8% with the highest frequency (34.9%) found among children of age group 6–11 months. The most predominant circulating genotypes were G3P[8] (22.4%) followed by G12P[6] (20.3%), G2P[4] (12.6%), G1P[8] (11.9%), G9P[6] (11.9%), G3P[4] (9.1%), G1P[6] (4.2%), G9P[8] (4.2%), and G3P[6] (0.7%). A single mixed genotype G1G3P[8] was also detected. The findings of this study provide baseline data, that will help to assess if future vaccination campaigns using currently available RVA vaccine will reduce RVA disease burden and instigate evolutionary changes in the overall RVA biology. The high prevalence of RVA infections in Pakistan require to improve and strengthen the surveillance and monitoring system for RVA. This will provide useful information for health authorities in planning public health care strategies to mitigate the disease burden caused by RVA. Reoviridae are the leading cause of fatal dehydrating diarrhea in infants and young children worldwide particularly in the developing countries of Africa and Asia  MH109741-MH109754 and MH236884-MH236897, P[6] MH109777-MH109806 and MH255676-MH255696, P[8] MH109755-MH109776 and MH255709-MH255740.The VP7 and VP4 nucleotide sequences were submitted to the GenBank with following accession numbers: t-test was performed to ascertain the equality of means of RVA positive and negative for continuous variables; age, weight, height, temperature, vomiting (duration and episodes), diarrhea (duration and episodes) and dehydration. Statistical significance was defined as p < 0.05.Statistical analyses were performed with Stata version 13 (StataCorp. 2013) . A chi-sp>0.05) (p<0.05). Whereas, no significant difference (p>0.05) was found between different age groups positive for RVA through ELISA . The RVA prevalence detected in BBH, Rawalpindi was 27.4% (96/350) and in PIMS, Islamabad was 26.0% (75/289). RT-PCR conducted on all ELISA positive samples showed that 83.6% (143/171) of these samples were positive by RT-PCR. There was no statistically significant gender difference in the distribution of RVA genotypes (p>0.05) . There wgh ELISA . Howevergh ELISA .From the 171 RVA antigen-positive samples, 143(83.6%) were confirmed positive by RT-PCR and the remaining 16.4% were RT-PCR negative. All 143 RT-PCR positive samples were successfully genotyped for VP7 and 140 samples were genotyped for VP4. The most common G type was G3 followed by G12, G9, G2 and G1. A single mixed G-type (G1G3) was detected in one sample (0.7%). The most common P-type was P[8] followed by P[6], P[4] and P[Non-typeable]. The most prevalent genotype combination was G3P[8] followed by G12P[6], G2P[4], G1P[8], G9P[6] and G3P[4], whereas the least prevalent genotypes were G9P[8], G3P[6], G1G3P[8], and G9P[NT] .Annual genotypic distribution of RVA strains in 2015 to 2016 is shown in RVA infections were present throughout the year over the period of the two years of surveillance (2015–2016), with highest prevalence in dry winter season. During 2015, the highest number of RVA positive cases (37%) were observed in dry winter months  of the year followed by . Similarly, RVA infections peaked with 53% positive cases in dry winter months (October-December) of the year 2016.Maximum likelihood trees were constructed for selected RVA strains based on partial genome sequences of VP7 gene segment. The total number of nucleotide sequences obtained for VP7 genotypes G1, G2, G3, G9 and G12 are 20, 15, 44, 24 and 30 respectively.G1: The G1-VP7 tree was constructed based on 20 Pakistani G1 strains sequenced in this study and representative members of the G1 genotype  and Russia (NN464-16/). Finally, a single strain PAK613 formed a separate cluster, most closely related (97.3–97.5% nt similarity) with strains circulating in India, Russia, USA, China, Japan and Brazil and other Pakistani strains isolated in this study. All P4 strains i.e reference strains and strains isolated in this study included in the tree diverge considerably from USA G1 strain (Wa/1974/G1P[8]) used as an outgroup.tic tree , showingP[6]: A total of 50 Pakistani P[6] strains sequenced in this study and representative members of the P[6] genotype were selected for the construction of P[6] phylogenetic tree  reported from Pakistan during 2008–2014 ,27,30,31–34% repoThe RT-PCR assay failed to detect RVA in 28 faecal samples positive by ELISA. This could be due to one or more of the following reasons; i) false positive ELISA result, ii) PCR inhibitors that cause positive samples to be negative by PCR, iii) drop of the viral titer below PCR detection limit due to additional shipment and freeze-thaw cycles.There are seasonal and geographical variations in RVA based infections globally. In temperate climates, RVA epidemics commonly appear during the dry, winter months of the year. However, in most tropical areas, RVA gastroenteritis is prevalent throughout the year without seasonal fluctuation . In the In the present study, no gender differences were found in RVA infections, which is in accordance with the previous reported cases from Pakistan ,27. A laThe rotavirus molecular epidemiology varies from country to country, depending upon the socio-economic status and weather conditions . The mosThe detection rate of the G1 genotype in the current study is 16% which is in accordance with two previous hospital based studies (11.6% and 14.5%) conducted during the years 2008 and 2014, respectively ,27. HoweIn this study the G2 genotype is reported in 13% of the cases, which is less than the previous studies 19–24%) reported during 2010 and 2014 ,24. Phyl% reporteThe G3 genotype is recognized as the third most predominant RVA genotype in humans mostly found in combination with P[8] . RotaTeqThe human G9 RVA genotype has gained epidemiological significance since mid-1990s and is now acknowledged as the fifth main human RVA genotype . AccordiThe first G12 genotype (G12P[4]) was discovered in 1987 from the Philippines, in children less than 2 years old, affected with acute diarrhea . A decadThe dominance of G3P[8] genotype in 2014, G12P[6] genotype in 2015 and further re-emergence of G3P[8] as a dominant genotype in 2016 in Pakistan in the current study suggested that the G3 and G12 strains might be competing and excluding one another in the same vulnerable population. Moreover, the increasing implementation of RVA mass vaccination worldwide might be the reason for genotypic fluctuations and emergence of new genotypes that lead to their global spread including Pakistan.The P[4] genotype of human RVAs is mostly identified with VP7 genotypes G2 and G8 . HoweverThe RVA P[6] genotype is detected all over the world with many G genotypes  ,108. In A distinct subtype of the P[8] genotype, P[8]-4 or OP354-like P[8] has been detected in many countries of Europe, Africa and Asia . AccordiRVA mixed infection rate has increased globally from 7.9–11.7% from 1997 to 2007 . The RVAThe unusual genotypes  accounted for 25% of total RVA genotypes in this study, which is lower than previous data reported by Masab and colleagues . The unuPakistan is one of the countries with the highest disease burden of RVA infections in children of less than five years of age. The introduction of two RVA vaccines has reduced the global mortality rate and number of hospitalizations in many countries ,14,115. However, we believe that with the inclusion of more samples for sustained time period and recruiting multiple sentinel sites throughout the country will be required for better understanding the scale of problem. Furthermore, whole genome characterization of human RVA strains and inclusion of samples from animal and environmental sources in future studies is recommended to monitor the ongoing interspecies transmission and reassortment events.This study concludes the high prevalence of G12 and G3 RVA strains in Pakistan in 2015 and 2016 in Rawalpindi and Islamabad, Pakistan. Although it provides an incomplete epidemiological picture of gastroenteritis caused by RVA throughout the country, the results of this study will provide comprehensive knowledge to the researchers and public health authorities to calculate the RVA disease burden in the country. The observed large diversity of RVA genotypes and their yearly fluctuations demand an intensive, large scale surveillance system in the country. This will help to monitor evolving of unusual RVA genotypes and to assess the impact of vaccine introduction on these recently emerged RVA strains in Pakistani population.S1 Table(DOCX)Click here for additional data file.S1 FigLane 1: positive control, Lane 2: DNA ladder 50 bp, lane 3, 4, 5, 6, 8, 9, 10, 11, 13: Amplified product of gene segment VP7(1062 bp size) of samples no 1, 2, 3, 4, 6, 7, 9, 12 and 15, Lane7, 12: Samples number 5, 13 negative for VP7 gene, Lane 14, 15: Negative control and non-target control(NT-C), respectively.(TIF)Click here for additional data file.S2 FigLane 1: DNA ladder 50 bp, lane 2, 3, 4, 5, 6, 8: Amplified product of gene segment VP4 (876 bp size) of samples no 1, 2, 3, 4, 6 and 16, Lane7: Samples number 13 negative for VP4 gene, Lane 9, 10 and 11: Positive control, Negative control and non-target control (NT-C), respectively.(TIF)Click here for additional data file.


CYP3A28 gene promoter were cloned and sequenced, and putative transcription factor binding sites were predicted. The CYP3A28 proximal promoter  contained DNA elements conserved among species. Co-transfection of bovine nuclear receptors (NRs) pregnane X and constitutive androstane receptor (bPXR and bCAR) with various CYP3A28 promoter constructs into hepatoma cell lines identified two main regions, the PP and the distal fragment F3 (-6899/-4937 bp), that were responsive to bPXR (both) and bCAR (F3 fragment only). Site-directed mutagenesis and deletion of NR motif ER6, hepatocyte nuclear factor 1 (HNF-1) and HNF-4 binding sites in the PP suggested either the involvement of ER6 element in bPXR-mediated activation or the cooperation between bPXR and liver-enriched transcription factors (LETFs) in PP transactivation. A putative DR5 element within the F3 fragment was involved in bCAR-mediated PP+F3 transactivation. Although DNA enrichment by anti-human NR antibodies was quite low, ChIP investigations in control and RU486-treated BFH12 cells, suggested that retinoid X receptor α (RXRα) bound to ER6 and DR5 motifs and its recruitment was enhanced by RU486 treatment. The DR5 element seemed to be recognized mainly by bCAR, while no clear-cut results were obtained for bPXR. Present results point to species-differences in CYP3A regulation and the complexity of bovine CYP3A28 regulatory elements, but further confirmatory studies are needed.The regulation of cytochrome P450 3A (CYP3A) enzymes is established in humans, but molecular mechanisms of its basal and xenobiotic-mediated regulation in cattle are still unknown. Here, ~10 kbp of the bovine  CYP3A genes: the xenosensors pregnane X-receptor , the constitutive androstane receptor , the hepatocyte nuclear factor-4 (HNF-4), HNF-3, the vitamin D receptor , and the CCAAT/enhancer binding protein-α (C/EBPα) thiazole-5-carbaldehyde O-oxime (CITCO) and 5--3-phenyl-4,5-dihydroisoxazole (FL81) have been documented [The established activators of human and mouse CAR and PXR have been reviewed –31. DEX,cumented –34. OligCYP3A28 promoter is described in detail in The cloning of ~10 kbp Putative binding sites for CAR and PXR heterodimers with RXR (DR3-5 and ER6-9 motifs) were identified with NUBIscan and NHR scan algorithms ,36. MatICYP3A28 promoter and containing the putative NR and transcription factor (TF) binding sites, were amplified using TaKaRa LA Taq Hot Start DNA polymerase  and specific primers  and full-length PXR (XREM-3A4-luc) have been previously described ,39 and uIn brief, site-directed mutagenesis of the ER6 and HNF-4 (DR1) sites was performed using mutagenic primers , amplifihCAR and hPXR expression [2) and cultured overnight to have 50–70% confluence. HepG2 cells were transfected using PEI25 and a reverse transfection method [CYP3A28 reporter constructs, or with positive control reporters hPBREM-tk-luc or hCYP3A4-XREM-luc, or with negative control reporter pGL4.10-luc (500 ng/well) and expression vector pCI-neo, bCAR or bPXR (100 ng/well). C3A cells were transfected using the calcium phosphate precipitation method [CYP3A28 reporter constructs or positive/negative control reporters (50 ng/well) and expression vectors (25 ng/well) as with HepG2. DMEM complemented with HyClone 5% delipidated serum  was used for the treatment with the PXR agonist SR12813 (10 μM) or its vehicle . After 24 hours, cells were lysed and assayed for luciferase and β-galactosidase activities with the VICTOR2 multiplate reader  as described before [HepG2 (ATCC HB-8065) and C3A (ATCC CRL-10741) cells, characterized by very low constitutive pression ,41, weren method , with con method  with cond before .CYP3A28 expression. BFH12 cells (passages 16–21) were cultured in Williams’ E medium containing 5% heat-inactivated fetal bovine serum (FBS), 1% penicillin/streptomycin, 2 mM L-alanyl-L-glutamine, 100 nM DEX and 0.2 U/mL insulin, at 37°C and 5% CO2 in a humidified atmosphere as described [4 cells/well). At day 4, the medium was changed with complete medium but without DEX. At day 7, stock solutions of PXR ligands, solubilized in DMSO and diluted in culture medium, were added into the wells. Cells treated with 0.1% DMSO  were used as control.The novel bovine SV40 large T-antigen-transduced fetal hepatocyte-derived cell line BFH12  was usedPreliminary induction studies were performed using DEX, PCN, RIF, RU486 and SR12813 at a fixed concentration (10 μM), according to , and difRXRα, the gene-specific primers forward (F): 5’-GCGTACTGCAAACACAAGTACC- 3’ and reverse (R): 5’-AGGCACTTGAGGCCAATG-3’ were selected, while for CYP2B22, CYP3A28, CAR, PXR and RPLP0 , previously published bovine primers were used [CAR assay, 300 nM/300 nM for CYP2B22, CYP3A28, RXRα and RPLP0, 600 nM/600 nM for PXR. For each qPCR assay, negative controls  were run. PCR amplification was performed in duplicate. The ΔΔCt method [Total RNA was extracted with the RNeasy Mini kit (Qiagen) following manufacturer’s instructions and quantified by NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific). Complementary DNA (1 μg) was synthetized using the High Capacity cDNA Reverse Transcription kit . The quantitative real-time PCR (qPCR) amplification was carried out in a final volume of 10 μL, using 12.5 ng of cDNA, the Power SYBR Green PCR Master Mix (Applied Biosystems) and Stratagene Mx3000P thermal cycler . Standard qPCR conditions were used. For the amplification of ere used ,17,25. Dt method  was used2 flasks at a density of 5 x 103 cells/cm2. At day 4, the medium was replaced with fresh medium without DEX. At day 7, cells were incubated for 6 hours with 100 μM RU486, 30 μM FL81 or 0.1% DMSO. Then, the cells, after trypsinization, were subjected to ChIP assay. The process for ChIP assay is detailed in BFH12 cells were seeded in 75 cmCYP3A28 fragment activity in HepG2 cells, were performed at least in triplicates. Data were normalized to the pGL4.10 empty vector and pCI-neo when no bPXR or bCAR were co-transfected ; pGL4.10 co-transfected with bPXR and SR12183 activities were used for experiments involving bPXR; pGL4.10 co-transfected with bCAR were used to normalize luciferase activity in bCAR transfections. An arbitrary value of 100 was set for pGL4.10 + pCI-neo, pGL4.10 + bPXR + SR12183, pGL4.10 + bCAR.All transfections, apart from the initial screening of P value < 0.05 was considered as statistically significant.For the statistical analysis of data from gene reporter assays and induction studies, the Student’s t-test or the analysis of variance (ANOVA), followed by the Tukey’s post-test was used . A CYP3A28 transcription start site , one potential binding site for CAR at position -4338/-4323 bp was detected by MatInspector. This element was flanked by an HNF-3β site (5’) and two HNF-4 sites (3’). A putative binding site for C/EBPα was also predicted in F2 at -3288/-3273 bp. NUBIscan identified a DR5 motif at -5541/-5525 bp within the fragment F3 (-6899/-4937 bp)  . Additio4937 bp) .CYP3A28 PP (-283/+71 bp) fused upstream of the luciferase reporter and appended by different upstream sequences  to the PP appeared to decrease NR-dependent activation. This result was particularly evident for the fragment F2 (-4998/-3133 bp), where the basal activity and the NR-mediated activation of CYP3A28 PP were completely abolished. This suggests the presence of silencing elements in F2. In contrast, bCAR and bPXR increased PP+F3 reporter activity of 70% and 150%, respectively, compared to empty pCI-neo. The responsiveness of F3 (-6899/-4937 bp) to both bPXR and bCAR indicated that the NR elements within this fragment should be analyzed further.The relevance of these putative NR sites was examined by reporter gene assays using the CYP3A28 PP (-284/+71 bp) and F3 (-6899/-4937bp) trans-activation by bPXR, the constructs PP and PP+F3 were extensively tested in C3A cells by co-transfecting the relevant expression vectors . The C3A cells were chosen over HepG2 because of their better functionality in reporter gene-based assays [P < 0.05; see To verify the consistency of d assays . The traP < 0.01; Next, the ER6, HNF-1 and HNF-4 elements located in the PP construct were deleted. This shorter PP  displayed slightly decreased activity (-25%) in bPXR co-transfected cells, and significantly reduced activation (-57%) by SR12813  present in F3 (-6899/-4937 bp) was performed to clarify its role in the observed response to bPXR and SR12813. The PP_del and the mutated PP_mER6 were used in combination to eliminate or reduce, respectively, the contribution by the intact PP. Both basal (empty pCI-neo) and bPXR-mediated activity of PP_del+F3mDR5 and PP_mER6+F3 were reduced by about 50%, indicating the presence of some important regulatory elements in F3. The mutation of DR5 (mDR5) reduced the reporter activity by 18% only in the context of PP_mER6 that contained a HNF-4 site but not in the context of PP_del that lacked both the HNF-4 and the proximal ER6 sites. This suggested that DR5 may have only a marginal role in bPXR-mediated activation that also depended on the intact CYP3A28 5’-flanking regions was examined by transient transfection of C3A cells. As shown in P < 0.05) as compared to the empty expression vector pCI-neo.The ability of bCAR to transactivate the The same set of deleted and mutated constructs used to assess bPXR-dependency was employed to investigate bCAR-dependent activity . HoweverCYP3A28 inducibility in the recently available BFH12 cell line, cells were exposed to prototypical CYP3A inducers and their time- and dose-responses were determined.To assess CYP3A28 mRNA expression was modestly but significantly induced by 10 μM RU486 after 6, 12 and 24 hours of exposure (P < 0.001)  were non-toxic after 6 hours of incubation (Alamar blue cytotoxicity test). A dose-dependent increase of CYP3A28 mRNA expression was observed only for RU486 (P < 0.01) and at 100 μM . SR12813 and RIF did not affect the expression of CYP3A28 mRNA  CYP2B22 mRNA levels after 12 hours of incubation; however, no transcriptional effects on CYP3A28 mRNA were ever noticed after 6 or 12 hours of incubation (Among the selected bCAR activators (CITCO and FL81), only the highest tested concentration of FL81 (30 μM) significantly increased , RXRα was bound to both ER6 and DR5 within the PP and the distal F3 fragment, respectively , probably due either to the low constitutive expression of NRs in BFH12 cells or the weak responsiveness of this fetal cell line to prototypical CYPs inducers, as described above.CYP3A28 transcription was investigated through DNA re-sequencing, in silico prediction of NR/TF binding sites, gene reporter assays of wild-type and mutated promoter fragments, induction studies in a bovine-derived hepatic cell line and ChIP assays.In the present study, NR-mediated regulation of bovine CYP3A28 expression via an ER6-type motif at -156 bp within the PP. This ER6 element is conserved among most of the CYP3A promoters in primates and other placental mammals [CYP3A4 reporters [CYP3A4 activation is also dependent upon distal enhancer modules XREM and CLEM [in silico analysis did not identify XREM and CLEM modules within the 10 kbp CYP3A28 promoter, although CAR- and PXR-responsive upstream regions were present (fragment F3). In a bioinformatic study using human CYP3A4 sequences as a query [CYP3A. In similar analyses, we found a CLEM-like sequence located ~34 kb upstream of the CYP3A24 sequence or ~77 kb upstream of the CYP3A38 gene, but not in the vicinity of the CYP3A28 promoter. Therefore, the lack of CLEM that could synergize with the proximal ER6 element might explain its modest responsiveness to bCAR or to its moderate induction in vivo [We showed that the bPXR is involved in the transcriptional regulation of  mammals . It conf mammals  and bindeporters –50. CYP3and CLEM ,38,51. O a query , the pro in vivo .CYP3A28 PP (bases -284 to +71) was activated by the ectopically expressed bPXR, the reporter gene expression increased about 2- to 3-fold. The extent of activation was about the same magnitude as with the human CYP3A4 ER6 element [CYP3A28 PP, in accordance with the recent profiling of bPXR ligand-binding domain [When the  element . RIF faig domain .CYP3A28 PP construct. A putative DR5 element (-5525 bp) and several HNF-3, HNF-4 and C/EBPα binding sites could contribute to F3 bPXR and bCAR responsiveness, because also DR5 elements can serve as functional sites for CAR and PXR [The distal fragment F3 -6899/-4937 bp) conferred a higher basal activity and modest 2-fold responsiveness to bPXR to the 37 bp conlement -55 bp and  and PXR .The additional NR-like elements elsewhere in the promoter  were also surrounded by LETF sites, but we did not detect any responsiveness to either bCAR or bPXR in co-transfection analyses. They cannot be completely ruled out, because reporter plasmids are not organized in the same fashion as in the natural chromatin context .CYP3A4 gene, and at least in case of CLEM, the proximal CYP3A4 promoter activity was decreased [CYP3A28 promoter context.The upstream region F2 (-4998/-3133 bp) was found to suppress the basal and bPXR- and bCAR-dependent reporter activity, even though it contained HNF-3, HNF-4 and CAR binding sites. Similar clusters of HNF-4 sites are present in the human ecreased . Althougecreased , they caecreased ,58. TherCYP3A28 reporter gene activity (fragments PP and F3). The deletion of the ER6, HNF-1 and HNF-4 binding sites in the CYP3A28 PP eliminated the bPXR-mediated activation by SR12813. Upon mutagenesis, the ER6 motif but not the HNF-4 site appeared to be critical. This suggested that the ligand-activated bPXR is able to recognize ER6 and that HNF-1 could participate in bPXR-mediated activation. Recently, an HNF-1 binding site located at -32 kbp upstream of the rat CYP3A1 was proved to act synergistically with DR4 and DR2 motifs to mediate the transactivation by rat CAR [Next, we focused on two NR site-containing regions that increased the  rat CAR .CYP3A28 mRNA and a strong activator of bPXR [CYP3A28 mRNA, which was consistent with their poor ability to activate bPXR [in vivo and in liver slices [To confirm the relevance of the ER6 motif, ChIP assays were performed in BFH12 cells treated with RU486, the most potent inducer of  of bPXR . Among oate bPXR  or weak r slices ,25,60.ChIP assays in DMSO- and RU486-treated cells showed that RU486 weakly increased the recruitment of RXRα, the obligate heterodimer partner for CAR and PXR, for the binding to ER6. It was not possible to reliably detect binding of PXR, probably due to its low expression in BFH12 cells and its modest cross-reactivity with the anti-human PXR antibody. The contribution of HNF-4 in the PP activation was not evaluated by ChIP, because of its slight mRNA expression in BFH12 cells . ConversCYP3A28 and CYP2B22 mRNA expression in BFH12 cells, comparatively lower than that elicited by FL81 in human hepatocytes [Concerning DR5, the binding site located at -5525 bp in F3, luciferase assays did not clearly identify it as the element responsible for the bPXR- or bCAR-dependent activation. It is thus possible that many other LETF sites in this 1.5-kbp fragment cooperate with either DR5 or the proximal ER6 to confer NR responsiveness. Therefore, a deeper characterization of the fragment F3 is required. Nevertheless, ChIP experiments suggested that DR5 motif could recruit both CAR and PXR. DR5 seemed the main DNA binding site recognized by CAR, and the immunoprecipitation of both PXR and RXRα was qualitatively and slightly increased by RU486 treatment. Unfortunately, FL81 did not affect CAR-mediated DR5 immunoprecipitation, probably for two reasons. First, its rather modest increase (1.4-fold) of atocytes ; second,atocytes , as demoatocytes .in vitro model because it does not express the whole set of LETFs and CYPs and does not fully respond to prototypical CYP inducers. As an example, the CYPs induction by PB, a hallmark of CAR-mediated regulation in vivo in calves [CYP3A28 mRNA expression. Secondly, as negative controls, we used no-antibody control (beads only) and a negative locus (CYP3A28 exon 13) as already reported in previous publications [The limitations of the present study are mainly attributable to ChIP assay results. First, a low specific antibody enrichment of ER6 and DR5 regions was generally obtained. This result could be justified by the fact that antibodies specific to bovine NRS were not available; moreover, the present bovine liver cell line (the only available at present) probably is not the optimal n calves ,15, in rn calves ) could nn calves  did not ications –65 insteCYP3A28 gene promoter, the proximal ER6 motif and a distal element F3. We hypothesized after protein/DNA interaction investigations that bCAR, despite the weak response in reporter assays, is recruited to the DR5 element in the distal F3 fragment. However, further molecular studies are needed to confirm ChIP results here obtained.In summary, we have localized two regions potentially responsive to bPXR in the CYP3A regulation and it opens fresh avenues for more detailed analysis.Overall, this work represents the first mechanistic study on bovine S1 File(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.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.S7 Table(PDF)Click here for additional data file.S8 Table(PDF)Click here for additional data file.S1 FigCYP3A coding genes are known: CYP3A28, CYP3A38, CYP3A48 and the predicted CYP3A24. The GenBank IDs and nomenclature are displayed together with the nomenclature proposed by [Four posed by .(PDF)Click here for additional data file.S2 FigCYP3A28 promoter region was able to solve the first gap starting at –1492 bp revealing a sequencing artefact (A), as well as the second gap at base –2988 with a new genomic sequence (B). Two of the five single nucleotides polymorphisms (-2899T>G and -2981_-2979insA) are here shown circled.The re-sequencing of (PDF)Click here for additional data file.S3 FigCYP3A28 proximal promoter (PP) and the contribution of the binding motif DR5 identified in F3. The parental PP was deleted of the whole putative region containing several TF binding-sites resulting in the PP_del; through site-direct direct mutagenesis the ER6 (PP_mER6) and DR1 (PP_mDR1) motifs were inactivated. The parental PP+F3 was deleted of the whole putative region containing several TF binding-sites resulting in the PP_del+F3; through site-direct direct mutagenesis the ER6 (PP_mER6+F3), the DR5 motif (PP+F3_mDR5 and PP_del+F3_mDR5) or both (PP_mER6+F3_mDR5) were inactivated. Details are reported in Several constructs were produced to study the binding elements identified in the (PDF)Click here for additional data file.S4 FigCYP3A28 was detected by qPCR in control (0.1% DMSO) and treated cells, using RPLP0 as internal control gene. The relative expression of DMSO-treated cells was set to 1 and its value was used for the normalization of the other groups. Data are expressed as the mean ± SD of three independent experiments . Statistical analysis: ANOVA + Tukey’s post test. Significance was defined as P < 0.05: *; P < 0.01: **; P < 0.001: ***.BFH12 cells were treated with different CYP3A inducers  at the fixed concentration 10 μM for 0 (A), 1 (B), 3 (C), 6 (D), 12 (E) and 24 (F) hours. The expression of (PDF)Click here for additional data file.S5 FigCAR (A), PXR (B) and RXRα (C) was detected by qPCR in control (0.1% DMSO) and treated cells, using RPLP0 as internal control gene. The relative expression of DMSO-treated cells was set to 1 and its value was used for the normalization of the other groups. Data are expressed as the mean ± SD of three independent experiments . Statistical analysis: ANOVA + Tukey’s post test.BFH12 cells were treated with different CYP3A inducers  at the fixed concentration 10 μM for 0, 1, 3, 6, 12 and 24 hours. The expression of (PDF)Click here for additional data file.S6 FigCYP3A28 was detected by qPCR in control (0.1% DMSO) and treated cells, using RPLP0 as internal control gene. The relative expression of DMSO-treated cells was set to 1 and its value was used for the normalization of the other groups. Data are expressed as the mean ± SD of two independent experiments . Statistical analysis: ANOVA + Tukey’s post-test.BFH12 cells were treated with different concentrations of SR12813  and RIF  for 6 hours, as described in Materials and Methods. The expression of (PDF)Click here for additional data file.S7 FigCYP3A28  and CYP2B22  mRNA was detected by qPCR in control (0.1% DMSO) and treated cells, using RPLP0 as internal control gene. The relative expression of DMSO-treated cells was set to 1 and its value was used for the normalization of the other groups. Data are expressed as the mean ± SD of three independent experiments . Statistical analysis: ANOVA + Tukey’s post-test. Significance was defined as P < 0.01: **; P < 0.001: ***. A, C: 6 hours of incubation; B, D: 12 hours of incubation.BFH12 cells were exposed to different concentrations  of the bCAR activator, FL81, for 6 and 12 hours. The expression of (PDF)Click here for additional data file.S8 FigBovine liver cytosolic and nuclear extracts were isolated according to Renisalo et al. (2012) with minor modifications. Proteins (30 μg) were subjected to immunoblotting analysis following the protocol previously published by . Membran(PDF)Click here for additional data file.S9 FigCYP3A28 DNA region without NR binding sites, is reported in the graph. In all experiments, negative and positive controls behaved as expected.BFH12 cells were exposed to 0.1% DMSO and 100 μM RU486 for 6 hours. Chromatin was isolated, subjected to ChIP using anti-human RXRα antibody and quantified by qPCR as described in (PDF)Click here for additional data file.S10 FigCYP3A28 DNA region without NR binding sites, is reported in the graph. In all experiments, negative and positive controls behaved as expected.BFH12 cells were exposed to 0.1% DMSO for 6 hours. Chromatin was then isolated, subjected to ChIP using anti-human CAR antibody and quantified by qPCR as described in (PDF)Click here for additional data file.S11 FigCYP3A28 DNA region without NR binding sites, is reported in the graph. In all experiments, negative and positive controls behave as expected.BFH12 cells were exposed to 0.1% DMSO and 100 μM RU486 for 6 hours. Chromatin was isolated, subjected to ChIP using anti-human PXR antibody and quantified by qPCR as described in Material and Methods. Results for ER6 and DR5 DNA regions are reported in panel A and B, respectively. Data are normalized to input DNA and expressed as % ChIP/input. The experiment was performed four times independently, and similar results were obtained. The data shown derived from a representative experiment. Chromatin samples from control cells immunoprecipitated with or without Histone H3 antibody are shown as Histone H3 and beads, respectively. A further negative control (exon 13), representing a (PDF)Click here for additional data file.


Icariside D2 is a plant-derived natural glycoside with pharmacological activities of inhibiting angiotensin-converting enzyme and killing leukemia cancer cells. Production of icariside D2 by plant extraction and chemical synthesis is inefficient and environmentally unfriendly. Microbial cell factory offers an attractive route for economical production of icariside D2 from renewable and sustainable bioresources.Escherichia coli. We screened the uridine diphosphate glycosyltransferases (UGTs) and obtained an active RrUGT3 that regio-specifically glycosylated tyrosol at phenolic position to exclusively synthesize icariside D2. We put heterologous genes in E. coli cell for the de novo biosynthesis of icariside D2. By fine-tuning promoter and copy number as well as balancing gene expression pattern to decrease metabolic burden, the BMD10 monoculture was constructed. Parallelly, for balancing pathway strength, we established the BMT23–BMD12 coculture by distributing the icariside D2 biosynthetic genes to two E. coli strains BMT23 and BMD12, responsible for biosynthesis of tyrosol from preferential xylose and icariside D2 from glucose, respectively. Under the optimal conditions in fed-batch shake-flask fermentation, the BMD10 monoculture produced 3.80 g/L of icariside D2 using glucose as sole carbon source, and the BMT23–BMD12 coculture produced 2.92 g/L of icariside D2 using glucose–xylose mixture.We metabolically constructed the biosynthetic pathway of icariside D2 in engineered E. coli for the de novo efficient production of icariside D2 with gram titer. It would be potent and sustainable approach for microbial production of icariside D2 from renewable carbon sources. E. coli–E. coli coculture approach is not limited to glycoside production, but could also be applied to other bioproducts.We for the first time reported the engineered  O-β-d-glucoside of tyrosol), an active natural product, was first isolated from traditional medicinal herb Epimedium diphyllum  under positive-ion mode. All of the HPLC analysis were quantified using a five-point calibration curve and the Additional file 1: Fig. S1. Production of tyrosol glycosides by heterologous expression of regio-promiscuous UGTs of RsUGT73B6, RrUGT17 and Yjic in E. coli BL21 (DE3).Additional file 2: Fig. S2. The LC–MS of the (A) icariside D2 standard and (B) fermentation supernatant sample of strain BMD2. Strain BMD2 produced a new product that has identical retention time with standard icariside D2 and the primary ion fragment at m/z 318 ([M+NH4]+) corresponds to icariside D2 with molecular weight of 300.Additional file 3: Fig. S3. The high resolution LC–MS/MS of the (A) icariside D2 standard and (B) fermentation supernatant sample of strain BMD2. The main fragments of new compound were identical with icariside D2 standard.Additional file 4: Table S1. The main primers used in this study.Additional file 5: Table S2. Nucleotide sequences of codon optimized genes.Additional file 6: Fig. S4. Calibration curve of BMT23 obtained by counting colony forming units at different OD600 without the IPTG.


Aspergillus fumigatus has developed worldwide resistance to azoles largely through mutations in cytochromeP450 enzyme Cyp51. In this study, we indicated that in vitro azole situation results in emergence of azole-resistant mutations. There are previously identified azole-resistant cyp51A mutations  and we successfully identified in this study two new mutations  conferring azole resistance among 18 independent stable azole-resistant isolates. The Galleria mellonella model of A. fumigatus infection experiment verified that Cyp51A mutations N248K/V436A and Y433N reduce efficacy of azole therapy. In addition, a predicted Cyp51A 3D structural model suggested that Y433N mutation causes the reduced affinities between drug target Cyp51A and azole antifungals. This study suggests that drug selection pressure make it possible to isolate unidentified cyp51A mutations conferring azole resistance in A. fumigatus.The opportunistic pathogen  Aspergillus fumigatus, the most common Aspergillus species, is a major opportunistic fungal pathogen and may lead to a variety of allergic reactions and life-threatening systemic infections in humans, including invasive pulmonary aspergillosis, pulmonary aspergilloma, and allergic bronchopulmonary aspergillosis  and mutant isolates are stored in CMCC . A. fumigatus A1160 strain (ku80 pyrG) was purchased from Fungal Genetics Stock Center. The strain A1160C (ku80 A1160:pyrG) belonged to a laboratory strain generated from reference strain A1160 transformed with pyrG selectable marker  and uridine (1.2 g/L). RPMI 1640 medium is composed of 10.4 g/l RPMI 1640  and 34.53 g/l MOPS [3-(N-morpholino) propanesulfonic acid]. All strains were cultured at 37 or 35°C (according to the M38-A2 guidelines) for the times indicated. All the strains used in this study are listed in The clinical e marker . All A. C strains was cultured onto YAG medium for 3 days at 37°C. Then 5 million freshly harvested spores from one parental strain were spread onto medium  supplemented with 4 μg/ml itraconazole and cultured them for 5 days. The surviving isolates on plates were serially streaked and cultured on itraconazole-containing plates (16 μg/ml) continuously four generations with an incubation period of 5 days at 37°C. The surviving strains on the final-step plate amended with 16 μg/ml itraconazole were cultured on drug-free medium continuously ten generations and then were spotted back onto medium with 16 μg/ml itraconazole. Isolates with stable drug resistance finally were named and kept for further studies.First, parental strain NO.3 or NO.7 or A1160A. fumigatus ATCC MYA-3626 and AF293 were included in related tests as quality control and reference strains. For the test of antifungal susceptibility in the constructed point mutant strains PY01 and PY02, RPMI1640 minus uracil and uridine liquid medium was used in order to maintain the growth of control stain A1160.All of the antifungal drugs were purchased from Sigma. Testing for susceptibility to the drug was carried out by the broth-based microdilution methods according to the CLSI document M38-A2 guidelines . Brieflycyp51A gene coding region and its promoter were amplified by PCR with two pairs of primers cyp51A-F1/cyp51A-R1 and cyp51A-F2/cyp51A-R2. Gene cyp51A nucleotide sequence (AFUB_063960) of A. fumigatus 1163 reference strains from Ensembl Fungi database1 was used as reference sequence. Primers used in this study are listed in The The Cyp51A 3D structural model used in this study was constructed as described previously . BrieflyA. fumigatus was the result of the mutations of Cyp51A N248K/V436A or Y433N, the strains containing cyp51A gene mutation sites were created. To construct strain PY01 (N248K/V436Acyp51A), the 5′-flanking region and ORF of N248K/V436Acyp51A from isolated strain NO 3-339 were amplified with primers cyp51A-F1 and P3. The 3′-flanking region of the N248K/V436Acyp51A gene was amplified from strain NO 3-339 with primer pair P4/P6, and the Neurospora crassa pyr4 gene (a selectable marker) was amplified from plasmid pLA5 with primer pair Pyr4-up/Pyr4-down. The three PCR products were fused with the nested primer pair P2/P5 and then transformed into recipient strain A1160 (a standard A. fumigatus strain susceptible to azoles) to generate mutant strain PY01 (N248K/V436Acyp51A). Using the same method, to construct PY02 (Y433Ncyp51A) stain, the 5′-flanking region and ORF of Y433Ncyp51A from isolated strain NO 1160-73 were amplified with primers cyp51A-F1 and P3. The 3′-flanking region of the Y433Ncyp51A gene was amplified from strain NO 1160-73 with primer pair P4/P6, and the Neurospora crassa pyr4 gene was amplified from plasmid pLA5 with primer pair Pyr4-up/Pyr4-down. The three PCR products were fused with the nested primer pair P2/P5 and then transformed into recipient strain A1160 to generate mutant strain PY02 (Y433Ncyp51A). The transformants were confirmed by diagnostic PCR with corresponding primers pairs  were used and 15 caterpillars per group were employed. Larvae were stored in the dark at 37°C prior to use. The conidial suspensions were collected in water. Preliminary experiments were performed to define an appropriate range of inoculum concentrations that causes > 80% mortality after 72–96 h post-infection (data not shown). A hamilton syringe  was used to infect the larvae with 10 μl of the inoculum suspension (8 × 107/ml) into the hemocoel of each G. mellonella via the last left proleg. Within 2 h of infection, 10 μl of antifungal solution was injected in a different proleg using the same technique. Following antifungal treatment, caterpillars were maintained in plastic containers at 37°C and larval survival was monitored daily. The larvae were considered dead when they did not respond to touch. Infected larvae injected with water were used as an untreated control group. Other control groups were also included: untouched and injected with PBS. Each experiment was performed at least three times and the results are reported as mean values. In order to simplify some of the figures, untouched control groups may have been omitted from the graphs. Stock solutions of antifungals were first prepared in dimethyl sulfoxide (DMSO) and dilutions were then made to obtain the selected concentrations in water for injection. Therapeutic dose of VRC (40 mg/kg), POS (10 mg/kg), ITC (25 mg/kg) were selected. The log-rank (Mantel-Cox) test was performed for survival analysis. A p-value < 0.05 was considered significant.A. fumigatus isolates  as the parent strains. Strains NO.3 and NO.7 were isolated from the patient sputum, and the strain A1160C belonged to the laboratory strain generated from the clinical-derived reference strain A1160  for a 5-day period, and then growing viable progenies were re-transfer to next round of 16 μg/ml ITC media for a 5-day period, and so on , and two strains were resistant to VRC (MIC = 4 μg/ml), suggesting usage of high-dose ITC may lead to cross-resistance phenomenon.Their colony phenotypes on both drug-free and drug-containing (16 μg/ml ITC) media were further observed. As shown in –8  in this cyp51A mutations is common reason for emergence of A. fumigatus azole resistance, we amplified entire cyp51A gene including its promoter region and sequenced using the primers . It is reported that M220K, M220I, M220R, G54E and G54W confer azole resistance and in our study these mutants displayed indeed high MIC value for ITC and POS (A. fumigatus azole-resistant isolates. We found that in vitro the isolate (3-339) carrying the N248K/V436A substitution shows resistant to ITC and POS but VRC-intermediate resistance and the isolate (1160-73) harboring Y433N mutation displays cross resistance to ITC, VRC and POS , compared with the group infected with the control parental strain showing an improvement in survival .Because mers see  for the  and POS . Notably and POS . AdditioA. fumigatus strains, and it is believed that this site does not correlate with drug resistance  with the A. fumigatus Cyp51A protein (A. fumigatus Cyp51A (A. fumigatus Cyp51A protein (as shown in Both the strain NO.3 (parent strain) and mutant strain 3-339 harbor amino acid mutation N248K in Cyp51A . In factsistance . Therefo protein . In the s Cyp51A , a typicComparison of wild-type and mutant structures for the Cyp51A of V436A is shown in A comparison of the wild-type and mutant structures for Cyp51A of Y433N is shown in The strain 3-339 contains both mutations V436A and N248K. Therefore, the mutant structures for Cyp51A of V436A in combination with N248K was analyzed in protein modeling . MutatioA. fumigatus. Using a PEG-mediated gene transformation system of protoplasts, the cyp51A gene in strain A1160 (a standard wild-type A. fumigatus strain susceptible to azoles) was replaced with the one from mutant strain 3-339 (N248K/V436Acyp51A). Similarly, the cyp51A gene in strain A1160 was replaced with the one from mutant strain 1160-73 (Y433Ncyp51A). The diagnostic PCR and sequencing analysis for cyp51A gene in the transformants showed that the mutated cyp51A gene (N248K/V436Acyp51A or Y433Ncyp51A) had been integrated into at the original cyp51A gene locus in the genome of strain A1160 (N248K/V436Acyp51A) and PY02 (Y433Ncyp51A) showed obviously reduced susceptibility to azoles  and T1368A (Y433N) contribute the azole resistance in in A1160 , S2. As o azoles . CompareN248K/V436Acyp51A and Y433Ncyp51A in an in vivo Galleria mellonella model, we examined virulence and the efficacy of azole therapy for the two reconstituted mutant strains PY01 and PY02. The group infected by the mutant PY01 or PY02 showed a mortality rate similar to the group infected with control parental wild-type strain  was given via injection administration 2 h after fungal infection. As shown in p = 0.0002; from 26% up to 60% for ITC, p = 0.0056; from 40% up to 93% for VRC, p < 0.0001), indicating that using the selected azoles therapeutic dose, azoles therapy in our fungal-infection Galleria mellonella model was successful. In contrast, with similar therapeutic dose, POS and ITC treatment fail to increase the survival of the larvae infected with mutant strain PY01  or PY02 . On the other hand, after VRC treatment, the group infected with strain PY01 showed an similar improvement in survival compared with the control parental strain group . Although the survival rate (33%) of group infected with the PY02 strain was from 13% up to 33% after VRC administration (p < 0.0001), it was significantly lower than survival rate (93%) of the group infected with the control parental wild-type strain after VRC administration (p = 0.0125). These results suggest that strains PY01 were not easily treatable with POS and ITC, and PY02 were not easily treatable with POS and ITC and VRC. These data support a conclusion that the point mutations T815A/T1378C (N248K/V436A) and T1368A (Y433N) in the A. fumigatus cyp51A gene are able to reduce in vivo the susceptibilities to azoles and decrease the efficacy of azole therapy.To evaluate any effects of e strain , suggestA. fumigatus including cyp51A mutation, overexpression of drug efflux, and modification of intracellular signaling pathways involved in stress response pathways  of drug resistant progenies generated G54E/W and M220I/K/R, which is consistent with that the G54 and M220 amino acid substitutions in the Cyp51A protein were reported as hot spot mutations  does not confer C. albicans azole resistance  resulted in meaningful changes in the fluconazole resistance but not in the itraconazole and voriconazole resistance  show high-level resistance to ITC in above in vitro and in vivo experiments. However, it is reported that N248K had been found in many azole-susceptible A. fumigatus strains .PC, QZ, ZZ, WL, and MS completed the experiments. PC, HS, and LL conducted the experiments and data analysis. PC and LL wrote the manuscript. All authors read and approved the manuscript.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.


Cyclophilin A (CypA) is a multifunctional protein that exhibits an isomeraseactivity and exists in the intracellular and secretory forms. Secretory CypApromotes regeneration of the hematopoietic and the immune systems of anorganism by stimulating stem cell migration from the bone marrow. Newapproaches based on CypA are currently being developed for the treatment oflimb ischemia, neutralization of the side effects of Cyclosporine A (CsA)therapy, etc. However, the role of CypA in the antitumor immune response isstill unexplored. In this work, we used the model experimental system oflymphoma EL-4 rejection in B10.D2(R101) mice and showed that recombinant humanCypA (rhCypA) stimulates the antitumor immune response via early recruitment ofgranulocytes to the tumor cell localization site and rapid accumulation ofeffector T-killers Cyclophilin A (CypA) is a member of the peptidyl-prolyl isomerase family andexists in the intracellular and secretory forms. Cytosolic CypA is detected inall tissues and has multiple functions . This prSecretory CypA is a pro-inflammatory factor that attracts innate immunity cells to the inflammation site andmediates the pathogenesis of various diseases . The proin vivoelimination of lymphoma cells was observed under rhCypA treatment.Moreover, it was shown using the model of antitumor immune response to lymphomaEL-4 in transgenic 1D1b mice [However, the role of CypA in the induction and development of the antitumorimmune response remains poorly understood to date. The aim of the present studywas to determine the functions of CypA in the early stages of the antitumorimmune response. Here, we studied the effect of recombinant human CypA (rhCypA)on the rejection of lymphoma EL-4 cells in B10.D2(R101) mice. Theimmunomodulatory effect of rhCypA was identified, aimed at stimulating both theinnate and the adaptive immune system. As a result, accelerated D1b mice  thatrhCMice C57BL/6 (KbI-AbDb) and B10.D2(R101) (KdI-AdI-EdDb) mice were obtained from thebreeding facility of the N.N. Blokhin National Medical Research Center ofOncology . The transgenic mouse line 1D1b was generated on thegenetic background of the B10.D2(R101) line in the Laboratory of RegulatoryMechanisms in Immunity of the N.N. Blokhin National Medical Research Center ofOncology [Production of rhCypA E.coli BL21(DE3)Gold transformed with the recombinant plasmid pETCYPoptithat contained the full-length gene of human CypA [The recombinant protein was isolated from the bacterial biomass of man CypA . RhCypA Immunization of mice 5 and 1.0 × 106, respectively, in 500μl PBS.B10.D2(R101) and 1D1b mice were i.p. immunized with lymphoma EL-4 (KbDb) cellsat doses of 3.0 × 10Mode of rhCypA administration B10.D2(R101) mice were i.p. injected with 5 mg rhCypA/kg (100 μg/mouse)during 3 days post-immunization. The first protein injection was made 3 hpost-implantation of EL-4 cells. 1D1b mice were subcutaneously dosed with 10 mgrhCypA/kg during 10 days post-immunization. Control mice received PBS as aplacebo in a similar manner.Cell isolation B10.D2(R101) mice were euthanized by cervical dislocation on days 6, 9, and 12post-immunization. The peritoneal cavities of mice were washed with 2 ml ofice-cold PBS to obtain the lavage. Splenocyte suspensions were prepared byisolating the murine spleens and homogenizing in a Potter tissue homogenizer in3 ml of PBS. The 1D1b transgenic mice were euthanized on day 12post-immunization; their splenocytes were isolated using a similar procedure.Erythrocytes were lysed in a lysis buffer . Cells were then washed inPBS and centrifuged . Viable cells were counted in a Goryaevchamber after Trypan Blue-Eosin staining.Antibodies The following monoclonal antibodies were used for the analyses: anti-CD3ε– eFluor450 (clone 17A2) ; anti-CD8 – Pacificblue (clone 53-6.7) ; anti-CD44 – APC (clone IM7)(eBioscience); anti-CD62L – APC-Cy7 (clone MEL-14) (eBioscience);anti-Vb6 – PE (clone RR4-7) (eBioscience); anti-Gr1 – APC (cloneRB6-8C5) (BD Pharmingen); and anti-CD11b-PE – Cy7 (clone M1/70) (BDPharmingen).Flow cytometry analysis 6 events were analyzed. The FlowJo 7.6. software  wasused for further processing of the results.The lavage and spleen cell samples (1.0–5.0 × 106) were incubatedwith Fc block  for 5 min at 4°C andstained with monoclonal antibodies for 40 min at 4°C. Cells were thenwashed with PBS by centrifugation , followed by analysis on aFACS CantoII flow cytometer  using the FACSDiva 6.0. software. Deadcells were excluded from the analyses comprising staining with propidium iodide. In order to characterize cell subpopulations, 0.5–1.0 ×10Statistical analysis p ≤ 0.05.Statistical data analysis was performed using the Student’s t-test inExcel . The differences were considered statisticallysignificant at Fig. 1).In this study, we used an allogenic system in which the EL-4 (KbDb) lymphomacells were rejected in B10. D2(R101) (KdI-AdI-EdDb) mice because of thedifference in a single MHC I class molecule (H2-Kb). It was shown that rhCypAadministration results in complete EL-4 elimination by day 9post-transplantation, whereas complete tumor rejection in the absence of rhCypAwas observed on day 12. On day 9post-immunization, rhCypA stimulated recruitment of immature granulocytes (GrhiCD11blo) and promyelocytes and myelocytes (Grint CD11bint) in the peritonealcavity of dosed mice. Cell counts in these subpopulations were increased underrhCypA administration 2.5- and 4.5- fold, respectively, compared to therespective cell counts in immunized control mice.The immune response to EL-4 was accompanied by granulocyte accumulation at thetumor cell localization site. The rhCypA induced intensive accumulation ofmature granulocytes in the peritoneal lavage of mice on day 6post-immunization: the relative count of these cells was threefold higher thanthat in the immunized control mice.These data correlated well with the tumorrejection dynamics under rhCypA treatment.Next, we evaluated the effects of rhCypA on quantitative and subpopulationchanges in CD8+ T cells in tumor-bearing mice. The protein under study did notinfluence the dynamics of CD8+ T cell accumulation neither in the tumorlocalization site  nor at the systemic level. However, analyses ofCD8+ T cell subsets of naive cells (CD62L+CD44-), central memory cells(CD62L+CD44+), and effectors (CD62L-CD44+) revealed that rhCypA inducedenhanced accumulation (by 65% as compared to the placebo control) of effectorcytotoxic T cells on day 9 post-transplantation of EL-4 tumor cells .Interestingly, administration of rhCypA significantly increased (2.0- fold ascompared to the placebo control) the count of effector CD8+ T cells with thetransgenic TCR β-chain.The According to these data, we have assumed that rhCypA can modulate an antitumorimmune response both in mice with the native TCR repertoire (B10. D2(R101)) andin transgenic 1D1b mice with the contracted TCR repertoire by inducing theaccumulation of effector CD8+ T cells.In this study, we evaluated the role of rhCypA in the development of theantitumor immune response to lymphoma EL-4 cells in B10.D2(R101) mice. It wasshown that rhCypA stimulates granulocyte accumulation at the tumor celllocalization site and systemic accumulation of effector cytotoxic T cells,which results in rapid tumor elimination. It is well-known that tissueinfiltration with neutrophils is the first phase of the immune response toinfections and inflammation. These cells can take up an antigen and migrate tothe draining lymph nodes and the spleen, where neutrophils come in contact withthe antigen-presenting cells (APCs) and lymphocytes  or direcIn 1D1b mice, the expression of the transgenic TCR β-chain in T cells bothcontracted the TCR repertoire and reduced the count of activated T cells . The immHence, we have demonstrated that rhCypA has an immunostimulating effect, as itfacilitates the development of the antitumor immune response by stimulatingboth innate and adaptive immunity.


H3F3A or H3F3B gene, respectively. These were, except for one uncommon G34L mutation in a GCT, reliably and specifically detected by mutation-specific H3.3 G34W or H3.3 K36M immunohistochemistry and DNA sequencing. In the diagnostic substantiation of CB, DOG1 staining was less sensitive compared to H3.3 K36M immunohistochemistry. 47% of ABC specifically showed translocations of the USP6 gene, while mutations in H3F3A/B were absent.The diagnosis of giant cell-rich lesions of bone can be challenging if radiological findings are ambiguous and tissue of the biologically deciding component is underrepresented in biopsy specimens. The frequent association of giant cell tumor of bone (GCT) and chondroblastoma (CB) with (secondary) aneurysmal bone cysts (ABC) may further impede correct classification. The present study evaluates the potentials and limitations of mutation-specific histone H3.3 and DOG1 immunohistochemistry, Sanger-/next generation sequencing (NGS) and FISH analysis in the differential diagnosis of 23 GCT, 14 CB and 19 ABC. All morphologically typical GCT and CB harbored mutations in the USP6 FISH analysis enable a reliable diagnostic distinction of GCT, CB and ABC of morphologically and radiologically difficult cases.Based on the results of this study, we conclude that mutation-specific H3.3 immunohistochemistry (selectively complemented with NGS-based DNA sequencing) and  Diagnosis of giant cell-rich lesions of bone is usually based on a combination of morphological and radiological features. This interdisciplinary approach has proven particularly helpful with regard to lesions that may display a significant overlap of radiographic and morphological features, as is the case for aneurysmal bone cysts (ABC), giant cell tumor of bone (GCT) and chondroblastoma (CB). However, cases in which specific primary bone tumors are disguised by a (dominant) ABC component may still elude correct diagnostic classification, particularly if tissue of the underlying biologically leading tumor is underrepresented in the biopsy specimen.ABC is a benign expansive lytic lesion of bone composed of multiple blood-filled cysts that are separated by fibrous septa containing variable numbers of lesional fibroblast-like cells as well as osteoclast-like giant cells. Traditionally, ABC is classified into primary ABC and secondary ABC . PrimaryGCT of bone is most frequently located in the epi-metaphyseal region of long bones , 7. In mCB is typically related to the growth plate of long bones with involvement of the epiphysis or apophysis , 14. HisThough the specific clinical, radiological and histological features of these three lesions are most often evident, the exact histological classification, even in open biopsy specimens, may be difficult due to a significant morphological overlap. From the clinical perspective, exact diagnostic typing is elementary: As ABC, GCT and CB of bone most frequently represent locally aggressive lesions, the therapeutic concept for all of them is mainly based on local removal e.g. by curettage. However, in contrast to ABC, GCT (and even CB) may rarely spread, particularly to the lung, making an adequate staging of the patient essential. Beyond that, GCT may therapeutically benefit from treatment with Denosumab, which is not an established treatment for CB or ABC. Eventually, rare cases of malignant GCT of bone have been described.USP6 translocations [H3F3A (G34W) or H3F3B (K36M) gene mutations, respectively [H3F3A and H3F3B has therefore been successfully introduced as specific tool in routine diagnostics [While a subset of primary ABC is molecularly characterized by ocations , GCT andectively . Mutatiognostics . Beyond gnostics , 19. In H3F3A/B mutational testing using next generation sequencing (NGS) and Sanger DNA sequencing were comparatively performed with immunohistochemical stainings applying mutation-specific histone H3.3 antibodies, complemented with immunostainings against DOG1 as a marker of CB [USP6-break apart fluorescent in situ hybridization (FISH) for the characterization of ABC. Based on these analyses, we were able to substantiate diagnoses even in morphologically challenging giant cell-rich lesions of bone.The present study was performed to comparatively evaluate the potential of different diagnostic approaches. To this end, er of CB  and USP6H3F3A (G34W or G34L) and H3F3B (K36M) genes, respectively. The type of mutation detected by DNA sequencing correlated with the immunohistochemical staining using the mutation-specific antibodies against H3.3 G34W and H3.3 K36M  radiographically interpreted as ABC and histologically showing a predominant membrane-like fibrohistiocytic pattern focally stained positive with the mutation-specific H3.3 K36M antibody Figure . In caseDOG1 staining was evaluated as positive in 11/14 cases of CB . There was no immunoreactivity with the antibody against DOG1 in any of the cases of GCT and ABC, respectively  ABC.H3F3A and H3F3B. Tumor-type specific mutations have been demonstrated in pediatric glioma, CB and GCT, qualifying them as driver mutations. While in pediatric glioma and glioblastoma K27M and G34R/V variants occur in H3F3A, GCT harbor mainly H3F3A G34W alterations whereas CB display predominantly K36M mutations in H3F3B [H3F3A mutation acts via a dominant negative gain of function by inhibition of the methyltransferase activity of EZH2, thus abolishing Polycomb-mediated repression of numerous genes [The DNA synthesis-independent replacement histone H3.3 is encoded by two different genes, in H3F3B , 21. Whius genes . Concernus genes . This suH3F3A mutations (G34W) in GCT ranges from 85–96% [H3F3A mutational status a reliable diagnostic tool. A recently developed monoclonal antibody targeting the typical H3.3 G34W mutation was described as a sensitive and specific marker for diagnosis [H3F3A. While the majority of the mutations was detectable employing Sanger sequencing, NGS sequencing  revealed one additional mutation-positive case (#21) with low mutation frequency, underlining the diagnostic value of NGS in samples with low content of lesional cells. The immunohistochemical approach led to a correct diagnosis in 36/37 cases of GCT and CB. In the single case (#15) of a H3.3 G34W IHC-negative GCT specimen, a rare G34L mutation could be detected by both DNA sequencing techniques  who screened 235 GCT and found nuclear immunostaining with the H3.3 G34W antibody in 213 cases. In 6 cases, IHC staining was negative due to underlying G34L, G34M or G34V H3F3A alterations. Therefore, since the immunohistochemical approach in GCT is limited to the G34W mutation, a small number of cases of GCT carry the risk to be misdiagnosed based on immunohistochemistry alone. By performing  DNA sequencing in case of suggestive morphology and negative H3.3 G34W immunohistochemistry, uncommon H3.3 alterations can also be detected, with NGS being the advantageous technique due to its higher sensitivity. It has to be emphasized that the differential diagnosis of a malignant tumor associated with a secondary ABC should always be considered in these patients, particularly since rare H3.3 hotspot mutations in malignant bone tumors, especially osteosarcomas, have been described previously [The reported frequency of m 85–96% , 24, makiagnosis , 25. In s Figure . This iseviously , and sinH3F3 mutations resulting in K36M alterations in 95% of cases. In most cases, mutations are found in H3F3B with only very few alterations detected in H3F3AH3F3B K36M mutation detected by both DNA sequencing methods. The immunohistochemical approach led to a correct diagnosis in 14/14 cases using the H3.3 K36M antibody and in 11/14 cases using DOG1 immunostaining. Discovered on gastrointestinal stromal tumours 1 (DOG1) was initially described as a highly expressed calcium-dependent chloride channel in gastrointestinal stroma tumors (GIST) [PDGFRA-mutated tumors (wild-type for KIT) [CB is described to harbor s (GIST)  and showfor KIT) . In a stfor KIT) . While ifor KIT) , its funIn one case (#27), it was not possible to detect the mutation by DNA sequencing due to underrepresented mutated lesional cells in the biopsy material. However, immunohistochemistry revealed a small area with distinct nuclear staining indicating a K36M mutation in at least few cells in that case Figure . DOG1 imUSP6 gene locus, indicating a true neoplastic nature of these lesions [USP6 chromosomal translocations, qualifying these cases as primary ABC. The somewhat minor frequency of USP6 translocations in our group of (primary) ABC compared to what was reported by Oliveira and colleagues [USP6 gene morphologically undetected components of GCT and CB might be present, H3F3A/B mutational status was assessed. DNA sequencing and immunohistochemistry  of all 19 ABC cases included in our study revealed no mutation in H3F3A/B and showed no positive IHC staining, ruling out secondary ABC on the basis of GCT or CB.More than 65% of primary ABC have been shown to carry clonal rearrangements of the  lesions . In the lleagues  is probaUSP6 FISH as diagnostic tools. If diagnosis is not possible with certainty, DNA sequencing should be performed to detect uncommon H3F3A/B mutations that have been described especially in GCT. Considering the sequencing approach, NGS represents the more sensitive technique to be employed, especially if a low mutational frequency needs to be detected.In the present study we correlated the results from mutation-specific immunohistochemical tissue typing with results from two different DNA sequencing techniques in giant cell-rich lesions of bone. We provide evidence that ambiguous cases with no clear-cut radiological and morphological appearance can reliably be diagnosed by the use of H3.3 mutation-specific immunohistochemistry and USP6 FISH analysis (step III) in cases negative with both mutation-specific antibodies. DNA sequencing (employing NGS as the more sensitive technique) of the H3F3A and H3F3B genes (step IV) is only necessary in cases negative in H3.3 G34W or K36M immunohistochemistry and lacking a USP6 rearrangement, which might point either to a GCT or CB with uncommon mutations or a USP6-negative ABC , followed by immunohistochemistry with mutation-specific H3.3 G34W, K36M) antibodies (step II) and W, K36M aIn total, 23 GCT, 14 CB and 19 ABC analyzed in the routine diagnostics of the Gerhard-Domagk-Institute of Pathology and of the Basel Bone Tumor Reference Centre, Switzerland, were included in the study. Diagnoses were based on the synopsis of radiographic features, morphological and molecular findings. In the GCT cohort, mean age was 30.6 (±8.6) years . A subset of 6/23 GCT was classified as recurrence following clinical data. Mean age in the CB group was 18.4 (±4.1) years . Two of the lesions presented as local recurrences. Within the ABC group, two cases were categorized as local recurrences. Mean age was 19.4 (±15.5) years . Approval of the study was obtained from the local Ethical Committees in Münster and Basel .H3F3A/B mutational status was performed by Sanger sequencing according to standard procedures using the BigDye Terminator v3.1 Cycle Sequencing Kit . Following primer sets were applied: (I) H3F3A: 5′GTC TCT GTA CCA TGG CTC GT-3′ (for) and 5′ACA AGA GAG ACT TTG TCC CAT TT-3′ (rev) and (II) H3F3B: 5′TCT TCG GGG CGT CTT TCT TA-3′ (for) and 5′GAG CAG GGG AGG AGT GAG-3′ (rev).The mutational status was analyzed by Sanger- and next generation amplicon based DNA sequencing (NGS). A customized GeneRead DNAseq Mix-n-Match V2 panel  was applied to simultaneously amplify the mutational hot spot regions of 19 cancer-related genes. Target enrichment was processed by means of the GeneRead DNAseq Panel PCR V2 Kit , following the manufacturer’s instructions. All purification and size selection steps were performed employing Agencourt AMPure XP magnetic beads . End repair, A-addition and ligation to NEXTflex-96 DNA barcodes  were conducted using the GeneRead DNA Library I Core Kit . Amplification of adapter-ligated DNA was performed using NEXTflex primers  and the HiFi PCR Master Mix . NGS was performed applying 12.5 pM library pools (2% PhiX V3 control) and the MiSeq Reagent v2 chemistry . NGS data analysis was performed by means of the CLC Biomedical Genomics Workbench software . Additional validation of the positive or negative. DOG1 was graded as absent, weak, moderate or strong cytoplasmatic/membranous staining (0–3) as published by Akpalo et al. [negative while moderate and strong stainings were evaluated as positive.IHC was performed on 3µm-thick sections from paraffin-embedded tissue using an automated immunohistochemistry staining system . Two monoclonal rabbit antibodies detecting the mutated H3.3 histones  were purchased from RevMab . Both staining protocols were established using formalin-fixed and paraffin-embedded tissue slides from GCT and CB cases with known mutational status. A monoclonal DOG1 antibody was purchased from Cell Marque . In brief, sections were deparaffinized and pre-treated with Cell Conditioning 1 or 2  for 32 minutes at 95° C  or 64 minutes at 100° C (DOG1). The primary antibodies G34W and K36M H3.3 were then incubated for 32 minutes at 36° C (dilution 1:500) and DOG1 for 16 minutes at 36° C. Immunoreactions were visualized using the Ventana Optiview DAB IHC detection kit . The sections were finally counterstained with haematoxylin  and bluing reagent . Nuclear stainings with the H3.3 monoclonal antibodies were evaluated as USP6 dual-colour break-apart probe  was used for analysis of the USP6 gene locus, employing the criteria published by Oliveira and colleagues [The lleagues . FISH prlleagues .


Following publication of , the folFigure 1A: E7 and GAPDH panels appear to contain background irregularities;Figure 1B: the bands in the total p38 and total JNK panels appear similar, cropped and adjusted for brightness and contrast;Figure 1C: total ERK1/2 panel contains vertical discontinuities;Figure 1C: GAPDH panel appears similar to the Figure 1B GAPDH panel 1B;Figure 2A: E7 panel has a vertical change in background between the nc and +tet lanes;Figure 2A: GAPDH panel background appears dissimilar between bands;Figure 4B: Phospho-p38 panel has a vertical change in background between the middle lanes;Figure 5A: Phospho RhoA and total RhoA panels have been heavily adjusted for brightness/contrast;Figure 6A: Active RhoA panel has vertical discontinuities between the 3 and 6 hr bands.The corresponding author does not agree with the concerns raised and provided images in relation to Figure 1B totp38 and totJNK panels, Figure 1C total ERK panel, Figure 2A GAPDH and E7 panels, Figure 4B Phospho-p38 panel, Figure 5A phospho RhoA panel and Figure 6A Active RhoA panel, but these do not satisfactorily resolve the concerns raised for these items. The primary data underlying other figure panels has not been provided, which was attributed to the time that has passed since publication.PLOS ONE Editors retract the article.In light of the unresolved concerns that question the validity of the study’s findings, the RAC, MRG, PC, VC, MT, and SJR did not agree with retraction. GB, AB, RA, AC, SM, and AP did not respond.