Streptococcus pneumoniae 19A ST320, a multidrug-resistant strain with high disease severity that notoriously spread before the use of expanded pneumococcal conjugate vaccines, was derived from a capsular switching event between an international strain Taiwan 19F-14 (ST236) and a serotype 19A strain. However, the molecular mechanisms underlying the adaptive evolution of 19F ST236 to 19A ST320 are unknown. In this study, we compared 19A ST320 to its ancestral clone, 19F ST236, in terms of adherence to respiratory epithelial cells, whole transcriptome, and ability to colonize a young mouse model. Serotype 19A ST320 showed five-fold higher adherence to A549 cells than serotype 19F ST236. High-throughput mRNA sequencing identified a prophage region located between dnaN and ychF in both strains; however, the genes in this region were expressed at significantly higher levels in 19A ST320 than in 19F ST236. Analysis by polymerase chain reaction (PCR) showed that the prophage is able to spontaneously excise from the chromosome and form a circular episome in 19A ST320, but not in 19F ST236. Deletion of the integrase in the prophage of 19A ST320 decreased spontaneous excision and cell adherence, which were restored by complementation. Competition experiments in mice showed that the integrase mutant was six-fold less competitive than the 19A ST320 parent . The 19A ST320 prophage-deleted strain did not change cell adherence capacity, whereas prophage integration strains (integrase mutant and 19F) had decreased expression of the down-stream ychF gene compared to that of 19A ST320. Further deletion of ychF significantly reduced cell adherence. In conclusions, these findings suggest that spontaneous prophage induction confers a competitive advantage to virulent pneumococci. Streptococcus pneumoniae serotype 19A ST320 was prevalent in many countries  is unclear. In this study, we used whole transcriptomic analysis via high-throughput mRNA-sequencing to study the mechanism responsible for the difference in colonization effectiveness between the 19A ST320 and 19F ST236 clones.Prior to implementation of the 13-valent pneumococcal conjugate vaccine , ountries . This clountries ; surged ountries ; and expountries . S. pneusyndrome . It was syndrome . Althougsyndrome , 2011. Isyndrome . This evad libitum.Our animal procedures of this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Chang Gung University , and the Committee recognizes that the proposed animal experiment follows the guideline as shown in the Guide for Laboratory Animal Facilities and Care as promulgated by the Council of Agriculture. Executive Yuan, ROC. BALB/cByJNarl mice were provided by National Laboratory Animal Center (NLAC), NARLabs, Taiwan. All animals were housed in an animal facility at 22°C, with a relative humidity of 55%, in a 12 h light/12 h dark cycle, with sterile tap water and food available 2. S. pneumoniae is a biosafety level-2 microorganism. The experiments handling the bacteria should follow all appropriate guidelines and regulations. Escherichia coli was grown in Luria broth. Antibiotics were added at the following concentrations when requited: spectinomycin at 250 mg/L for S. pneumoniae and 100 mg/L for E. coli and chloramphenicol at 4 mg/L for S. pneumoniae and 30 mg/L for E. coli.Pneumococcal isolates form Chang Gung Children’s Hospital (CGCH) were grown at 37°C in Todd–Hewitt broth supplemented with 0.5% yeast extract (THY) or on blood agar supplemented with 5% defibrinated sheep blood in the presence of 5% CO2 and subcultured twice a week. Adhesion assays using A549 were performed as described previously (5 cells/well) and maintained in culture medium at 37°C in a 5% CO2 incubator. Mid-log phase (OD600 = 0.4 to 0.6) S. pneumoniae in THY were washed once with serum-free DMEM medium and then added to each well at a multiplicity of infection (MOI) of 100 and co-incubated for 2 h at 37°C in a 5% CO2 incubator. To determine the total numbers of adherent bacteria, the wells were washed three times with phosphate-buffered saline (PBS) to remove non-adhering bacteria, and 1 mL of deionized distilled water was added and incubated for 5 min to lyse cells. The adherent bacteria were enumerated by plating serial dilutions of the lysate on blood agar and counting the number of viable bacterial colony-forming units (CFUs). The experiments were performed in triplicate and repeated independently four times.A549 (ATCC CCL-185) human lung epithelial carcinoma cells were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  at 37°C in a humidified incubator at 5% COeviously , with soCP000921.1 and CP001993.1, respectively). The expression level of each gene, as determined by RNA-Seq, was normalized to the number of reads per kilobase of exon region per million mapped reads (RPKM). The cut-off value for determining gene transcription was determined based on the 95% confidence interval for the RPKM values of each gene.For Illumina sequencing, total RNA was extracted from each sample (serotype 19A ST320 and Taiwan 19F-14 ST236) using the RNeasy mini kit (QIAGEN) and then treated with RNase-free DNase I (QIAGEN) for 15 min according to the manufacturer’s protocols. The integrity of the isolated total RNA was checked using an Agilent Technologies 2100 Bioanalyzer. Then, cDNA libraries were prepared according to the manufacturer’s instructions (Illumina), and an ABI StepOnePlus Real-Time PCR System was used for quantification and qualification of the sample library. The genome and gene information for serotype Taiwan 19F-14 ST236 and serotype 19A ST320 were downloaded from the NCBI database  and an ABI 7900 Real-Time PCR system. The cycling conditions for the quantitative real-time PCR (qPCR) were as follows: 50°C for 2 min and 95°C for 2 min, followed by 50 cycles of 95°C for 15 s and 60°C for 30 s, and after 50 cycles, melting curve analysis was performed from 60°C to 95°C, with a 2% ramp rate. Sequences of the primers used for RT–qPCR are listed in An aliquot (400 ng) of total RNA from each bacterial strain was subjected to cDNA synthesis using SuperScript IV Reverse Transcriptase (Thermo Fisher). The cDNAs of attB site on the chromosome; non-prophage-specific oligonucleotides designed to amplify a 72-bp fragment (RT-10621-F/RT-10621-R) of HMPREF0837_10621. First, 19A ST320 genomic DNA was used as a template to amplify target gene fragments by PCR using the primer pairs 10621-F/10621-R  and A/B , which were then cloned into pJET1.2/blunt (Thermo Fisher) to create plasmid standards for each target . Each amplification was carried out in a 20 μl reaction containing KAPA SYBR® FAST qPCR Master Mix on an ABI 7900 system. Serial 10-fold dilutions of the plasmid standard containing 0.1–1 × 106 copies were used to establish a standard curve to evaluate the Ct V.S. log10(copy number). To determine the changes in prophage excision over time, a culture of 19A ST320 was grown in THY at 37°C and 5% CO2 and genomic DNA was extracted at different time points. Then, the percentage of cells with excised prophage at each time point were analyzed by qPCR and normalized to the standard curve. To estimate the cell population with excised prophage at different time points, the number of copies of excised-chromosomal target region was divided by the number of copies of SP_10621 at each time point.The population with excised prophage were determined by qPCR. The following oligonucleotide pairs were designed for the experiment: prophage-excised-specific oligonucleotides to amplify a 151-bp fragment (RT-A/RT-B) near the integrase (int), dnaN and ychF gene-deletion strain  was generated by replacing the gene with a spectinomycin resistance gene cassette as follow and primer used are listed in int, dnaN and ychF form the serotype 19A ST320 were amplified by PCR using primer pairs: int-up-F/int-dn-R for int, dnaN-up-F/dnaN-dn-R for dnaN and ychF-up-F/ychF-dn-R for ychF, respectively. The resulting PCR products were cloned into a pGEM®-T easy (Promega) plasmid. The coding regions of the int, dnaN and ychF were then removed by inverse PCR (iPCR) with primer pairs: int-inverse-F/int-inverse-R for Δint, dnaN-inverse-F/dnaN-inverse-R for ΔdnaN and ychF-inverse-F/ychF-inverse-R for ΔychF, respectively and ligated to a PCR amplified spectinomycin antibiotic cassette (spec-F/spec-R) form pDL278 plasmid to create a deletion construct vector. This vector was transformed into S. pneumoniae serotype 19A ST320 by Competence Stimulating Peptide-1 , and transformants were selected on spectinomycin.The integrase deletion mutant strain (19A-Δint::int), a single copy of the integrase gene was inserted into the non-coding region between the HMPREF0837_12134 and HMPREF0837_12135 as follows. The integrase gene was PCR amplified from 19A ST320 by using the primer pair Pint-F/int-R, and then cloned into the pGEM®-T easy plasmid to generate Pint::pGEM-T. A chloramphenicol (cat) antibiotic cassette from pKO3-Km plasmid was PCR amplified by using the primer pair Pcat-F/cat-R, and then sub-cloned into the SacII site of Pint::pGEM-T to generate Pint-cat::pGEM-T. Then, the primer pair Pint-F/cat-R was used to produce a Pint-cat PCR fragment. Next, a 2,501-bp fragment of the insertion site was PCR amplified by using the primer pair 12133-F/12136-R and cloned into pGEM®-T easy to create p12133-12136::pGEM-T. A fragment generated by iPCR using the above plasmid and the primer pair 12134-5-inverse-F/12134-5-inverse-R was then ligated with the Pint-cat PCR fragment to form the complementation vector p12133-Pint-cat-12136::pGEM-T. The complementation strain (Δint::int) was created by transforming the p12133-Pint-cat-12136::pGEM-T plasmid into the 19A-Δint strain using CSP-1 and selecting on chloramphenicol.To construct a complemented 19A ST320 in vivo competition assay for S. pneumoniae infection in mice was conducted as described previously (600 = 0.2–0.6) bacteria were combined at a 1:1 ratio (CFUs) in PBS, and the inoculating doses were confirmed by plating on blood agar plates. Then, 3-week-old female BALB/c mice were challenged intranasally (IN) with 50 μL of bacteria . After 7 days, the mice were sacrificed with CO2, and nasal lavages fluid was collected. The CFUs recovered were determined by serial dilution and plating on blood agar. For the competition experiment between wild-type strain and the integrase deletion mutant, the recovered bacteria were distinguished by replica plating on blood agar containing 250 mg/L spectinomycin. One hundred randomly picked colonies were tested per mouse. A competitive index (CI) was calculated based on the ratio of the competing bacterial strains recovered by nasal lavage, normalized to the ratio of the respective bacteria in the inoculum = (outputtest strain/outputwild type)/(inputtest strain/inputwild type).An eviously . Mid-logt-test. The mouse CI assay was analyzed by a one-sample Student’s t-test (one-tailed) using log-transformed CIs to determine if the indices were significant. A p value less than 0.05 was considered statistically significant. All statistical analyses were performed using SPSS 15.0 for Windows .Data are presented as means ± standard deviation (SD) from more than three independent experiments. Statistical significance was assessed by a two-tailed Student’s p < 0.001)  in mice . In this< 0.001) .HMPREF0837_10256  and HMPREF0837_10294  were significantly higher in 19A ST320 than in 19F ST236 (HMPREF0837_10263 and HMPREF0837_10266) to verify the RNA-seq results. RT-qPCR confirmed an increase in the mRNA levels of these two genes in 19A ST320 compared to the corresponding levels in 19F ST236 (data not shown).The transcriptomic analysis revealed 801 genes that were differentially expressed between the serotype 19A ST320 and 19F ST236 strains, including 641 and 160 genes that were upregulated and downregulated in serotype 19A ST320, respectively . Of note9F ST236 . A RT-qPThere are 34 open reading frames (ORFs) located within the prophage region in 19A ST320 and 19F ST236 . But, thWe hypothesized that there was increased spontaneous prophage induction in 19A ST320, which accounted for the difference in mRNA levels of genes within the prophage between 19A ST320 and 19F ST236. PCR with several combinations of primers was used to assess spontaneous integration and excision of the prophage region in 19A ST320. If the prophage could excise from the bacterial chromosome to form an epichromosomal circular structure, the PCR products would be observed using primer pairs A/B  and C/D (901-bp) . PCR witBacterial cells were harvested at different growth phases, from early log phase to stationary phase, we found that the percentage of the cell population with excised prophage increased with cell growth into late-log phase, but decreased at stationary phase. The average percentage of cells with excised prophage across all culture time points was 17.17 ± 3.19% .integrase gene at the 3′ end   restored prophage excision (HMPREF0837_10263 and HMPREF0837_10266) within the prophage region in the integrase deletion mutant (19A-Δint) were significantly lower than that in 19A ST320 wild type (p < 0.05) (The prophage region of 19A ST320 harbors a putative e 3′ end . The int9A-Δint) . Complemexcision . Also, t < 0.05) .integrase deletion mutant and 19A ST320 wild type were similar (integrase deletion mutant (19A-Δint) of serotype 19A ST320 showed decreased adherence to A549 cells compared to the wild-type strain (p < 0.002), whereas the integrase complementation strain (19A-Δint::int) showed restored cell adherence capacity that was similar to the wild-type strain (p = 0.8) (The growth curves and CFUs in different phases of the  similar . We test similar . The isop = 0.8) .integrase deletion mutant (19A-Δint) to assess the role of prophage excision on colonization. At 7 days post-infection, the isogenic integrase deletion mutant (19A-Δint) was sixfold less competitive than the parent serotype 19A ST320 strain in colonizing the mouse upper airway  with equal CFUs of serotype 19A ST320 (19A-WT) and the isogenic p = 0.9) (ychF was different between strains 19A-WT and 19A-Δint (p < 0.05) . Deletion of ychF in the 19A ST320 strain did not impact growth (data not shown). However, 19A-ΔychF had a lower bacterial adherence ability than 19A-WT (p < 0.01)  to test whether the circular prophage DNA molecule directly modulates bacterial adherence. No difference was found in adhesion ability between the serotype 19A ST320 (19A-WT) and the prophage deletion mutant (19A-Δphage) (p = 0.9) . This re < 0.05) . Transcr < 0.01) . The -359A ST320 .S. pneumoniae isolates in both eastern and western countries  were identified as the specific site of prophage integration. The gene encoding integrase, located at the 3′ junction of the prophage in serotype 19A ST320 stains, catalyzes the site-specific recombination between the prophage recognition site (attP) and a short sequence of bacterial DNA (attB) in serotype 19A ST320 strains. Several phages integrate their genome into the bacterial chromosome and exist in a lysogenic state, or replicate and lyse the bacterial host after induction. There are two lysogenic processes in which bacteria and phage interact: lysogenic conversion and active lysogeny. When the expression of phage-encoded proteins changes the bacterial phenotype and/or contributes to bacterial fitness, this is called lysogenic conversion, which is observed in Corynebacterium diphtheria, with the expression of diphtheria toxin; in E. coli O157:H7, with the expression of shiga toxins; and in Vibrio cholera, with the expression of cholera toxin (comK gene in Listeria monocytogenes and the mismatch repair system (MMR) in Streptococcus pyogenes . As we did not observe any phage structural genes, genes for DNA packaging, or the holing/lysin system for bacterial lysis, the prophage in the 19A ST320 strain did not seem to enter the lytic cycle and release new virions. However, the prophage has the replication protein and primase necessary for DNA replication. Increased mRNA expression of the prophage region in serotype 19A ST320 compared to serotype 19F ST236 is likely due to replication of the circular prophage genome as an autonomous element, just like the prophage CGP3 in pyogenes . The purS. pneumoniae. This decline in the incidence of pneumococcal diseases caused by vaccine serotypes has been offset by substantial increases in both the carriage of and diseases caused by non-vaccine serotypes, which now occupy the niche vacated by vaccine-type pneumococci (The availability and use of pneumococcal conjugate vaccines has dramatically decreased the incidence of invasive diseases caused by vaccine-targeted serotypes . Neverthumococci . Thus, vumococci . Moreoveumococci . The genY-YC, J-TW, T-LL, and Y-CH designed the research, discussed the analysis, and revised the paper. Y-YC, T-HL, and Y-YH prepared materials and performed the experiments. Y-YC, T-LL, and Y-NG analyzed the data. Y-YC and Y-CH wrote the main text of the manuscript. All the authors reviewed and approved the final version of 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.


The kernel row number (KRN) of an ear is an important trait related to yield and domestication in maize. Exploring the underlying genetic mechanisms of KRN has great research significance and application potential.In the present study, N531 with a KRN of 18–22 and SLN with a KRN of 4–6 were used as the recurrent parent and the donor parent, respectively, to develop two introgression lines (ILs), IL_A and IL_B, both of which have common negative-effect alleles from SLN on chromosomes 1, 5 and 10 and significantly reduced inflorescence meristem (IM) diameter and KRN compared with those of N531. We used RNA-Seq to investigate the transcriptome profiles of 5-mm immature ears of N531, IL_A and IL_B. We identified a total of 2872 differentially expressed genes (DEGs) between N531 and IL_A, 2428 DEGs between N531 and IL_B and 1811 DEGs between IL_A and IL_B. A total of 1252 DEGs were detected as overlapping DEGs, and 89 DEGs were located on the common introgression fragments. Furthermore, three DEGs  containing three SNPs associated with KRN were identified using regional association mapping.These results will facilitate our understanding of ear development and provide important candidate genes for further study on KRN.The online version of this article (10.1186/s12870-019-1811-1) contains supplementary material, which is available to authorized users. Zea mays) is widely grown worldwide and is one of the most important food and feed crops with important economic significance . (TIF 81 kb)Additional file 4:Figure S4. Validation of the RNA-Seq results via qRT-PCR. (a) Four genes randomly selected from upregulated DEGs and four genes randomly selected from downregulated DEGs. For each plot, the left columns represent the results in quantitative real-time RT-PCR (qRT-PCR), and the right columns represent FPKM in RNA-Seq. (b) The log2-transformed qRT-PCR expression data are plotted against log2-transformed RNA-Seq data and fit to a linear regression. (TIF 289 kb)Additional file 5:Figure S5. Expression of three candidate genes in different tissues, including the root, stem and leaf, during the three-leaf stage. (TIF 67 kb)Additional file 6:Figure S6. Diagram of the strategy used to mine the proposed candidate genes using a combination of different experiments and methods. (TIF 94 kb)Additional file 7:Figure S7. Diagram of the creation process for the introgression lines (IL_A and IL_B). (TIF 51 kb)Additional file 8:Table S1. Distribution of different genotypes in IL_A and IL_B. (XLSX 11 kb)Additional file 9:Table S2. Reads statistics for N531 and its introgression lines. (XLSX 10 kb)Additional file 10:Table S3. List of the DEGs between N531 and IL_A. (XLSX 170 kb)Additional file 11:Table S4. List of the DEGs between N531 and IL_B. (XLSX 145 kb)Additional file 12:Table S5. List of the DEGs between N531 vs IL_A and N531 vs IL_B. (XLSX 183 kb)Additional file 13:Table S6. List of the DEGs between IL_A and IL_B. (XLSX 107 kb)Additional file 14:Table S7. List of the DEGs among IL_A, IL_B and N531. (XLSX 17 kb)Additional file 15:Table S8. List of the DEGs located in the common introgression fragments between N531 vs IL_A and N531 vs IL_B. (XLSX 21 kb)Additional file 16:Table S9. Genes that may be involved in maize inflorescence development. (XLSX 15 kb)Additional file 17:Table S10. Summary of SNPs associated with KRN. (XLSX 13 kb)Additional file 18:Table S11. Primers used in qRT-PCR. (XLSX 9 kb)


Background The biological diagnosis criteria of the Alzheimer’s disease (AD) suggests that previous work may misclassify the cognitive impairment caused by other factors into AD. Consequently, re-assessing the imaging profile of AD continuum is needed. Considering the high vulnerability of cortical association fibers, we aimed to elucidate the cortical demyelination process in the AD continuum biologically defined.Methods According to the biological diagnosis criteria, we determined the positive amyloid status (A+) as cerebrospinal fluid (CSF) amyloid1–42 < 192 pg/ml, Florbetapir Positron emission tomography (PET) composite standardized uptake value ratio (SUVR) >1.11. Also, the positive Tau status (T+) was determined as p-Tau181 > 23 pg/ml. Based on the cognitive characterization, we further categorized 252 subjects into 27 cognitively unimpaired with normal AD biomarkers , 49 preclinical AD (A+T+), 113 AD with mild cognitive impairment (MCI) (A+T+), and 63 AD dementia (A+T+). We estimated the intracortical myelin content used the T1- and T2-weighted (T1W/T2W) ratio mapping. To investigate the sensitivity of the ratio mapping, we also utilized well-validated AD imaging biomarkers as the reference, including gray matter volume and Fludeoxyglucose PET (FDG-PET). Based on the general linear model, we conducted the voxel-wise two-sample T-tests between the controls and each group in the AD continuum.Results Compared to the controls, the results showed that the preclinical AD patients exhibited decreased T1W/T2W ratio value in the right inferior parietal lobule (IPL); as the disease progresses, the prodromal AD patients demonstrated lower ratio value in bilateral IPL, with hippocampus (HP) atrophy. Lastly, the AD dementia patients exhibited decreased ratio value in bilateral IPL and hippocampus; also, we observed the bilateral temporal cortices atrophy and widespread decreased metabolism in the AD dementia patients. After corrected with gray volume, the results remained mostly unchanged.Conclusion Our study implied the decreased right IPL T1W/T2W ratio might represent early AD-related demyelination in disease continuum. Additionally, we demonstrated that the T1W/T2W ratio mapping is an easy-to-implement and sensitive metric to assess the intracortical myelin content in AD, particularly in the early stage. Concerning AD neuropathologies, extracellular amyloid plaques deposition appears the earliest, followed by intracellular neurofibrillary tangles and downstream neurodegeneration events . In the symptoms . Meanwhiontinuum . SimilarMyelin provides the neuronal basis of high processing speeds for high-level cognitive function . On the On the other side, prior studies have been demonstrated that Fluorodeoxyglucose positron emission tomography (FDG PET) and gray matter (GM) atrophy are reproducible imaging biomarkers for AD continuum . As the The goal of our study was to detect the transformational patterns of the intracortical myelin content as AD progresses. The novelty and highlights of our investigation include that:  adoptingadni.loni.usc.edu). The ADNI was launched in 2003 by the National Institute on Aging (NIA), the National Institute of Biomedical Imaging and Bioengineering (NIBIB), the Food and Drug Administration (FDA), private pharmaceutical companies and non-profit organizations, as a $60 million, 5-year public-private partnership. The primary goal of ADNI has been to test whether serial magnetic resonance imaging (MRI), positron emission tomography (PET), other biological markers, and clinical and neuropsychological assessment can be combined to measure the progression of mild cognitive impairment (MCI) and early Alzheimer’s disease (AD). Determination of sensitive and specific markers of very early AD progression is intended to aid researchers and clinicians in developing new treatments and monitor their effectiveness, as well as lessen the time and cost of clinical trials.Data used in this study was obtained from the Alzheimer’s disease Neuroimaging Initiative (ADNI) database , and other cognitive domains including memory , processing speed , visuospatial , executive , and language ability .Based on cognitive performance, we grouped subjects into cognitively unimpaired subjects, MCI, and dementia patients. Explicitly, we defined the cognitively unimpaired as subjects had CDR score of 0, MMSE between 24 and 30 (inclusive), DM ≥ 9 for subjects with ≥16 years of education; ≥5 for subjects with 8–15 years of education; and ≥3 for years of education ≤7; non-depression  and non-dementia. We defined MCI as subjects preserved daily living activities, non-dementia, and objective cognitive impairment as shown on the DM and CDR score of 0.5. We defined dementia as: patients had MMSE score ≤ 26, CDR ≥ 0.5, and met the NINCDS/ADRDA criteria for probable AD.T-Tau) and phosphorylated tau (p-Tau181), which obtained using the standardized ADNI protocol as previously  since it might represent the non-AD related pathology.Subsequently, we downloaded CSF dataset and identified the levels of Aβ1-42, total Tau ; (b) preclinical AD, consisting of cognitively unimpaired subjects with abnormal Aβ1–42, normal p-Tau181, and positive amyloid status based on PET (A+T+); (c) prodromal AD, consisting of MCI with abnormal Aβ1–42, p-Tau181 and positive amyloid status based on PET (A+T+); (d) AD with dementia, consisting of demented subjects with abnormal Aβ1–42, P-Tau181 and positive amyloid status based on PET (A+T+).Finally, we classified all subjects into four groups according to their cognitive performance and biological profile . ExactlyApolipoprotein (APOE) genotyping for each subject was performed as previously described . In brie18F-AV45. More detailed information can be found online regarding PET scanning protocol: http://adni.loni.usc.edu/wp-content/uploads/2010/05/ADNI2_PET_Tech_Manual_0142011.pdf.Details of the ADNI neuroimaging acquisition protocol are publicly available on the Laboratory of Neuroimaging (LONI) website. Structural images were acquired based on 3D Magnetization Prepared Rapid Acquisition Gradient Echo (MPRAGE) T1W sequence and fast spin-echo/turbo spin-echo (FSE/TSE) sequence. T1W image parameters: voxel size = 1.1 × 1.1 × 1.2 mm3; echo time (TE) = 2.98 ms; repetition time (TR) = 2300 ms. T2W image parameters: TR = 4000 ms; TE = 78 ms; voxel size = 0.9375 × 0.9375 × 4 mm3. The FLAIR images were obtained using an echo-planar imaging sequence with the following parameters: TR = 9000 ms, TE = 90 ms, and TI = 2500 ms. Meanwhile, FDG PET images were acquired by either a 30-min six frame scan acquired 30 to 60-min post-injection or a static 30-min single-frame scan acquired 30 to 60-min post-injection . Amyloid1) in SPM12. Concisely, we firstly aligned the T1W images to the standardized template image. We segmented the standardized T1W images into the compartments of CSF, WM, and GM with the bias correction. Sequentially, we normalized the GM density maps to the MNI space as 1 × 1 × 1 mm3 voxels and smoothed using the 8 mm FWHM Gaussian kernel. Finally, we estimated the modulated GM maps.We processed the T1W images using the Computational Anatomy Toolbox  space. According to the research hypothesis, we extracted the GM components of the T1w/T2w ratio maps based on the segmented normalized T1W image. Before the statistical analyses, we smoothed the T1W/T2W ratio map with a Gaussian kernel of 6 mm full-width at half maximum (FWMH).Specifically, the original T2W image was firstly co-registered to the T1W image through a rigid-body transformation. Next, we separately the subjected T1W and T2W images to bias correction. After correcting for the intensity non-uniformity, the T1W and T2W images were further processed to standardize their intensity by using a linear scaling procedure. After calibrating the T1W and T2W images, their ratio was calculated to generate the calibrated ratio map. To conduct two-sample 18F FDG PET and amyloid PET data from the ADNI database in its most processed format . Specifically, these pre-processed scans were created following co-registration dynamic (to the first frame), averaging frames, spatial re-orientation (AC-PC line), intensity normalization (within subject-specific mask), and smoothing . To exclude the between-subject nuisance variability in tracer uptake, we normalized the intensity of FDG PET by dividing it by the cerebellar pons/vermis; also, we normalized the intensity of amyloid PET by dividing it by the whole cerebellum. Next, the produced standardized uptake values (SUVR) maps entered the followed statistical analyses  may correlate with the demyelination and metabolism abnormalities, we compared the WMH load difference between groups in the current study . For eacous work , 2018.post hoc two-sample T-tests if ANOVA was significant .We conducted the statistical analyses utilizing IBM SPSS 24 statistical software for Windows. Group differences in the age, education level, neuropsychological scores, and CSF were tested by the ANOVA, followed by T-tests to examine the difference between controls and patient groups in the intracortical T1W/T2W ratio mapping, VBM, and FDG PET data. Notably, all imaging analyses were restrained within a standardized group gray matter (GM) mask, created from the CAT12 and SPM Masking Toolbox (p < 0.005 (Z > 2.58), and cluster size thresholds of p < 0.05 , general and multi-domain cognitive abilities score. After multiple comparison correction, some correlation relationship was no longer significant, we still showed these results in the manuscript and labeled them as “uncorrected” for the explorative purpose.To investigate the possible pathological mechanism of imaging metrics, we performed partial correlation analyses among imaging metrics, neuropathological, and neuropsychological data. Specifically, we obtained the mean value of T1W/T2W ratio and FDG PET SUVR from regions showing significant between-group difference below. Based on SPSS, we performed a series of correlation analyses used age, gender, and APOE ε4 status as covariates and Bonferroni correction approach. The variables in correlation analyses included the mean value of T1W/T2W ratio, FDG PET SUVR, amyloid PET SUVR in bilateral inferior parietal lobe and hippocampus regions, CSF data (Aβpost hoc two-sample T-test).We presented the quantitative variables as the mean and standard deviations (SD), and the categorical variable as absolute and relative frequency. There were no statistical differences in term of education level, gender composition, and white matter hyperintensities among four groups. Concerning the pair-wise comparison, preclinical AD only had inferior AVLT performance than controls. As expected, we observed that cognitively unimpaired preclinical AD subjects had lower T1W/T2W ratio value in the right inferior parietal lobule (IPL) than controls . ControlThe Neither preclinical AD nor prodromal AD had decreased metabolism relative to controls . Only thr = −0.15) and right IPL (r = −0.17) T1W/T2W ratio value related to the T-tau level (p < 0.05 uncorrected), the left HP T1W/T2W ratio value related to the Aβ1–42 level . Within the patient groups, we also found that T1W/T2W ratio related to amyloid SUVR in the left  and right HP . However, no correlation relationship between the T1W/T2W ratio and the amyloid SUVR in IPL existed  and right  HP.As for the clinical correlation relationships, within patient groups, we found that T1W/T2W ratio, FDG PET and HP volume correlated with general cognitive and multiple cognitive domains. These results suggested that all three metrics were effective imaging biomarkers. Further, all three metrics correlated with each other. Especially, the T1W/T2W ratio related to the FDG SUVR in term of left , and the inclusion of multiple levels of biological metrics, particularly the neuropathological data. Also, there existed several limitations to this study. First, due to an insufficient sample, no subjects with A + T- biomarker profile included in this study. However, previous similar AD continuum studies have reported that there are biphasic trajectory changes in cortical thickness from A-T- to A + T- until A + T + stage in cognitively intact elderly . TherefoOur study implied that the demyelination of the right IPL might represent the early AD-related abnormality in the disease continuum, showing more sensitivity than both the gray matter atrophy and hypometabolism. Additionally, we proved that T1W/T2W ratio mapping is an easy-to-implement and sensitive measure to assess the intracortical myelin content in the AD continuum.http://adni.loni.usc.edu.Publicly available datasets were analyzed in this study. This data can be found at: XL, KL, QZ, YJ, and PH conceived and designed the study, and analyzed and interpreted the data. SW, ZS, XX, MZ, JX, CW, LK, and JZ critically revised the manuscript for intellectual content and performed the statistical analysis.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.


Drosophila melanogaster pheromonal communication: desat1 affects both the production and the reception of sex pheromones while desat2 is involved in their production in flies of Zimbabwe populations. There is a strong asymmetric sexual isolation between Zimbabwe populations and all other “Cosmopolitan” populations: Zimbabwe females rarely copulate with Cosmopolitan males whereas Zimbabwe males readily copulate with all females. All populations express desat1 but only Zimbabwe strains show high desat2 expression. To evaluate the impact of sex pheromones, female receptivity and desat expression on the incipient speciation process between Zimbabwe and Cosmopolitan populations, we introgressed the Zimbabwe genome into a Cosmopolitan genome labeled with the white mutation, using a multi-generation procedure. The association between these sex-specific traits was determined during the procedure. The production of pheromones was largely dissociated between the sexes. The copulation frequency (but not latency) was highly correlated with the female—but not with the male—principal pheromones. We finally obtained two stable white lines showing Zimbabwe-like sex pheromones, copulation discrimination and desat expression. Our study indicates that the variation of sex pheromones and mating discrimination depend of distinct—yet overlapping—sets of genes in each sex suggesting that their cumulated effects participate to reinforce the speciation process.Sex specific traits are involved in speciation but it is difficult to determine whether their variation initiates or reinforces sexual isolation. In some insects, speciation depends of the rapid change of expression in desaturase genes coding for sex pheromones. Two closely related desaturase genes are involved in  The quality of sensory cues exchanged by sex partners is crucial with regard to sexual isolation and selection . ChemicaOstrinia nubilalis), some populations diverge both for the ratio between two pheromonal isomers (E) and (Z) of the 11-tetradecenyl acetate, and for the male behavioral response to the blends with different (E)/(Z) ratio  ratio . Also, iDrosophila melanogaster, most—but not all—populations show random mating ((desat2) was proposed, based on the presence of a functional desat2 gene in Z, but not in M strains  5,9-heptacosadiene  and codes for the production of C7-desaturated CHs in males (7-tricosene = 7T) and in females  has apparently no, or very little effect, on male copulation . Also, ai) the variation of desat2 expression, (ii) the production of cuticular sex pheromones in males and (iii) the asymmetrical sexual isolation of Z populations remains unclear, we explored the genetic relationship between these traits in both sexes. To mimic a natural process, which may have taken many more generations, we carried out a multi-generations “backcross-selection” procedure in the laboratory to progressively introgress a Z genome into a M line carrying the white mutation (w). We measured, at different time points of this procedure, the relationship between these sex specific traits. This allowed us to evaluate their potential influence on the incipient speciation process observed between D. melanogaster populations. Our final goal consisted to obtain stable ZW lines associating Z-like sex pheromones, copulatory discrimination and desat expression with the w mutation. Such lines could be used to test the effect of transgenes associated with the +w marker.Since the relationship between . M-type flies were transferred every two/three days (and Z-type flies every five to seven days) to avoid larval competition and to regularly provide abundant progeny for testing and breeding. All behavioral experiments were performed under similar conditions.1118 strain which carries the 1118white mutation . In both cases, a split–splitless injector  with helium as the carrier gas (50 cm/sec at 120°) was used. The temperature program began at 120°, ramping at 10°/min to 140°, then ramping at 2°/min to 280° and holding for 10 min. The chemical identity of the peaks was determined using a gas chromatography–mass spectrometry system equipped with a CP Sil 5CB or a polar CP-Wax 58 FFAP column as previously described  diverging between M and Z strains. This simplified CH analysis allowed us to screen in real time during successive generations, the CH profiles of many ZW-BC lines simultaneously sampled. However, the complete CH profiles of parental strains  and of the two stable ZW-IsoP lines  are shown (Table S1).desat1 transcripts  and of the desat2 transcript, we first extracted RNAs from 30 whole 5-day-old flies using the Trizol method (GIBCO BRL) and RNase-free DNase treatment to avoid contamination by genomic DNA . Comparison of fecundity, copulation latency and duration was carried out with a Kruskall-Wallis test with Conover-Iman multiple pairwise comparisons . CH and/or behavioral data were analyzed using Principal Components Anaysis  with standardized values. For qPCR analysis, significant differences in transcript levels ratio between control and sample strain were detected with the Relative Expression Software Tool . Frequencies were compared using a Wilks https://doi.org/10.25387/g3.8230493.The authors affirm that all data necessary for confirming the conclusions of this article are represented fully within the article and its tables and figures. All raw data are available in the annexed "Data_Set.xlsx" file. All supplementary information is included in the 9 Supplemental figure files. Supplemental material available at FigShare: D. melanogaster wild-type strain collected in Zimbabwe (Z30 = Z) into the genome of a wild-type strain from Dijon (Di2) representative of M strains and carrying the white mutation  cuticular pheromones and (ii) mating phenotypes in males and females of parental strains  and in the F1 progeny resulting of their reciprocal crosses .Our genetic procedure consisted to progressively introgress the genome of a For the sake of clarity, our pheromone analysis was mostly based on the measure of the 7T/5T ratio (T-ratio) in males and of the 7,11HD/5,9HD ratio (HD-ratio) in females. Both T- and HD-ratio showed much lower values in Z30 male and female flies  compared to M-type flies . The absolute amounts (in ng) of these compounds, as well as the complete hydrocarbon profile of parental strains, are provided in supplemental information (Table S1).w mutation carried by Di/w and ZW flies causes a visual defect inducing delayed mating latency, our behavioral observations lasted 2 hr. In parallel, and to control for a potential effect induced on mate discrimination by the w mutation, we also tested a second M-type strain with red eyes: the wild-type Canton-S strain (Cs). In the parental crosses, Z30 females showed, as expected, a contrasted mating frequency, which was very low with both M-type males  and high with Z30 males (74%). Differently, both M-type females frequently mated with all males (60–98%), except in Cs x Di/w pairs (38%). F1 females frequently mated with Z30 males (69–85%) and with Cs males (59–83%). With Z30 and Cs females, most F1 males showed very high mating frequencies (73–100%) except for w F1 “Di/w x Z30” males with Z30 females (38%). Based on the fact that the F1 females mated with both types of males, this suggests that the female preference is co-dominant.Our mating experiments always involved pairs of flies . While fertility and fecundity were not affected by copulation status , both parameters were highly correlated in pairs mating within 2 hr or in the following 24h . Z30 females showed a much higher fertility with Z30 males (74%) than with Cs and Di/w males  =234.84) whereas M-type females always showed a very high fertility regardless of the male strain (88–98%) (Table S3). Moreover, Z30 x Z30 pairs copulating during the 2 hr period showed a much lower fecundity  compared to all other pairs involving M-type females =70.03; P < 0.0001; Fig. S3).Both the fertility (ability to leave progeny) and fecundity (number of adult progeny left) were measured in F0 pairs according to their copulation status .As mentioned above, the progeny of the “Di/w x Z30” cross was mated following the genetic procedure designed to establish ZW-BC1 lines with white eyed flies (We obtained 21 ZW-BC1 lines in which we measured during the “A&S session #1”: (1) male and female cuticular pheromones and (2) ZW male copulation with Z30 and Cs females. In particular, the T- and HD-ratio were analyzed during 3 successive generations , but not in F2 and F3 flies . This effect maybe partly explained by the negative correlation between absolute amounts of 7,11HD and 5,9HD, which were higher in F1  than in F2 and F3 flies . Similarly, but to a lesser extent, the amounts of C5- and C7-desaturated CHs showed higher level of correlation in F1 flies than in F2 and F3 flies . The values for T- and HD-ratios are shown in Table S4.The T-ratio showed a wide variation in F1 (1.4-19.0), F2 (2.3-18.5) and F3 males (3.4-20.1). Similarly, the HD-ratio varied in F1 (0.1-47.9), F2 (0.1-4.9) and F3 females (0.2-21.1). Despite the presence of some extreme data points, the variability of both T- and HD-ratios tended to decrease between generations . A significant positive correlation between both T- and HD-ratio was found in F1 , their mating frequency was highly variable with Cs females . However, given that these mating data were not enough discriminant between lines, we selected the three best ZW-BC1 lines only based on their CH phenotypes. Therefore, the #1, #3, #12 lines showing the lowest and most stable T- and HD-ratio values  were used to initiate the BC session #2 (BC2).i.e., Low T-ratio).In summary, this first phenotyping round (ZW-BC1), involving 4 backcrosses between M-type females and ZW male hybrids (Z/M), yielded ZW males which did not yet acquire enough Z-like characteristics to be accepted by Z females and ZW females not accepting M-type males. We therefore selected 3 ZW-BC1 lines based on their T-ratio phenotypes close to Z-type . Each of these six main lines was subdivided into five sublines  representing a total of 30 ZW-BC2 sublines.r = 0.60; P = 0.03; Fig. S5B) strongly decreased in F2 flies . Such decrease maybe due to the low correlation between 7,11HD and 5,9HD and/or null correlation between 7T and 5T in F2 flies . The values for T- and HD-ratios are shown in Table S6.During the “A&S” session #2, both the CH profile and mating performance were analyzed in F1, F2 and/or F3 flies. After screening both CH (on pools) and mating phenotypes (see below) in F1 flies, we only kept the 15 sublines derived from the #1, #3 and B#3 ZW-BC2 lines [1A-1E; 3A-3E; B3A-B3E; The mating performance of ZW-BC2 flies with M-type and Z30 flies substantially varied between lines . Moreover, the copulation frequency in these pairs was negatively correlated with their copulation latency . However, no other significant correlation was found between any other CH-ratio and mating parameter in Z30 x ZW pairs (F2 and F3), or in ZW x Cs pairs . Based on these data, and as indicated above, we kept the 15 sublines derived from the #1, #3 and B#3 main ZW-BC2 lines to initiate the backcross series #3 .III and #3BIII; Table S8) while others produced at least some individuals (or pools) with higher T-ratio (≥5.0). The HD-ratio remained low in several ZW-BC3 sublines including #1BIII and #3BIII, at least until F4 . Each subline was first characterized by its T-ratio, given that this parameter seems to reflect best the proportion of “Z30” genome in ZW recombinant lines. The T-ratio was first measured (at F1 and F2) in two fly pools and subsequently—but only in lines showing a low T-ratio—characterized in individual males . This variation may be due to the increased correlation between the absolute amounts of 7,11HD and (i) 5,9HD or (ii) 5T (both negative) or (iii) 7T (positive) in F3 flies compared to F2 flies .No significant “T-ratio/HD-ratio” correlation was found in F2 or F3 flies, despite a slight increase between these generations , while 1BIII and #3BIII males substantially mated with Z30 females. At F2, #1BIII and #3BIII females showed a Z-like sexual discrimination: no copulation (within 2 hr) with Cs males and a high mating frequency with Z30 males  based both on their Z-like CH profiles and mating discrimination.In summary, after this third round of backcrossing, we selected to ZW-BC3 sub-lines . In each 1W and 3W line, the “F1 to F4” stability of the T-ratio was assessed using individual males  derived from each #1Br = 0.12, P = 0.79) caused by the opposite correlation between 1W and 3W lines  . The correlation between the absolute amounts of single CHs was relatively low and not significant between 5T and either 7,11HD  or 5,9HD . However, a very high correlation was found within the 3W1 line (but not within the 1W14 line) between 7- and 5T  and between 7,11- and 5,9HD .Simultaneous testing of the 8 ZW-IsoP lines (F6-F7) revealed a weak correlation between T- and HD-ratio  and much more frequently with Z30 males  both T- and HD-ratios were not correlated together (r = 0.026), (ii) the copulation frequency of Z30 x ZW-IsoP pair was correlated with HD-ratio (r = 0.524), (iii) the copulation frequency of both ZW-IsoP x ZW-IsoP and ZW-IsoP x Cs pairs was correlated with the T-ratio , but (iv) the copulation frequency of ZW-IsoP x Z30 was not correlated with any CH ratio (r=-0.041 and -0.075).We performed a further cross-examination of CH profile with mating performance in ZW-IsoP lines using Principal Component Analysis . This revealed that while  than with Cs males  whereas ZW-IsoP male fertility was high with both Z30 or ZW-IsoP females . This suggests that mating events occurring after the two-hours observation period were more frequent in ZW-IsoP x ZW-IsoP and ZW-IsoP x Z30 pairs than in ZW-IsoP x Cs pairs.P = 0.037). ZW-IsoP x ZW-IsoP fertile pairs generally produced a low progeny number (<20), but no effect was detected between lines.The fecundity, measured with regard to the genotypes and mating status of pairs , only revealed slight difference. ZW-IsoP x Z30 and Z30 x ZW-IsoP fertile pairs left around 20 and 30 adult progeny per cross, respectively with no difference between IsoP lines. A slight effect of mating status was detected in ZW-IsoP x Z30 pairs: copulating pairs showed a slightly higher fecundity than non-copulating pairs . More precisely, we measured the level of the five desat1 transcripts  and the unique desat2 transcript, in mature flies of both sexes. Males and females of the Z-type lines  showed significantly higher levels of the desat2 transcript compared to both M-type parental strains  in Z-type females compared to M-type females and (ii) in Cs males compared to the four other males.The transcriptional profiles of the white-labeled M genome allowed us to follow, during many generations, the evolution of the relationship between several sex specific traits diverging between D. melanogaster populations. In particular, we found that cuticular pheromones and female sexual receptivity—two sex specific traits potentially involved in incipient speciation—depend of distinct sets of genes. After more than 20 generations of introgression and 10 generations of experimental selection, we obtained two white-eyes lines (1W14 and 3W1) showing the principal Z-type characters: female sexual discrimination, male copulation with Z30 female, male and female desaturated CHs, fecundity and desat1-desat2 gene expression.The experimental introgression of a Z genome into a per se but also on their relative contribution to the pheromonal bouquet, this determining their ratio. Other studies have shown that the ratio between two compounds with potential pheromonal effect, or their relative contribution to a more complex bouquet, can strongly influence insect ability to discriminate between closely related individuals, colonies, sub-species or species  and became positive in IsoP lines . Despite the absence of a global significant correlation, our genetic procedure largely based on the selection of “low T-ratio” in ZW males, allowed us to isolate two stable IsoP lines combining the w mutation with all main Z-like sex specific phenotypes. This suggests that some of the genes  involved in the low T-ratio and in mate discrimination are closely linked. This suggests that these hypothetic “low T-ratio” coding genes could also affect non-pheromonal Z male traits  together with traits underlying female receptivity to Z male traits.Differently, no clear relationship was detected between the mating ability and either the absolute amounts of two principal tricosene isomers 7T, 5T;  or theirD. melanogaster studies revealed the involvement of single genes in female sexual discrimination and receptivity  code for enzymes potentially involved in hydrocarbon biosynthesis  into the genome of our two stable ZW lines to further dissect the mechanisms underlying the incipient speciation process between D. melanogaster populations to better understand the recent evolution and worldwide adaptation of this species.In conclusion, the present study offers a “real-time measure” of the evolution of several sex-specific traits potentially involved in sexual isolation. Our data provide a statistic view on their relative involvement and also on their inter-sex relationship. We are now planning to introduce transgenes targeting chemosensory genes (including


Rf). Although the CMS/Rf system is useful and convenient for economic production of commercial hybrid seed, the molecular mechanisms of CMS occurrence and fertility restoration in cotton are unclear.Cytoplasmic male sterility (CMS) in flowering plants is usually caused by incompatibility between mitochondrial and nuclear genomes, and can be restored by nuclear genes known as restorer-of-fertility (Gh_D05G3392) by cleaving precisely at the 643 nt and 748 nt sites. The further analysis indicated that the sequence variation in the binding regions of Gh_D05G3392 and Gh_D05G3356 may cause a lower cleavage efficiency of the PPR genes by miR7505b and miR7505 in R line, respectively, leading to the up-regulation of the PPR genes and fertility restoration. These results have established their genetic involvement in fertility restoration in the CMS-D2 system.Here, a combined small RNA and transcriptome sequencing analysis was performed on floral buds at the meiosis stage in three-line hybrid cotton system, and differentially expressed microRNAs (DEMs) and their target genes were identified and further analyzed for a possible involvement in CMS and fertility restoration. Totally 10 and 30 differentially expressed miRNA-target gene pairs were identified in A-B and A-R comparison group, respectively. A putative regulatory network of CMS occurrence and fertility restoration-related miRNA-target pairs during anther development were then constructed. The RLM-RACE analysis showed that gra-miR7505b regulates a PPR gene  contains supplementary material, which is available to authorized users. Cotton (Gossypium spp.), one of the world’s most important crops, is a major source of natural fiber materials. Improving cotton cultivars is becoming critical to meet an increased industrial demand for more raw cotton and high-quality cotton fibers. Heterosis refers to the phenomenon in which a hybrid shows higher performance for a trait than both parents. Hybrid breeding, which efficiently exploits heterosis, is thus an important strategy in breeding programs to increase the yield and quality of many important crops, including rice, maize and cotton . One of Brassica juncea [atp1 and atp6 in CMS-D8 as well as cox1 and cox2 in CMS-D2 are putative CMS-associated genes [atpA gene in CMS-D2 line relative to its maintainer line may be involved in CMS occurrence in Upland cotton [CMS in flowering plants, a maternally inherited trait, is characterized by the absence of functional pollen grains  while tha juncea  and peppa juncea . A studyed genes . Furtherd cotton .Rf genes [Rf gene product. Several Rf genes have been cloned in plants such as maize [Rf2, which encodes an aldehyde dehydrogenase that may be involved in the production of plant hormone indole-3-acetyl acetate [Rf genes are reported to encode a pentatricopeptide repeat (PPR) protein. Additionally, the Rf2 gene for Lead type CMS rice encodes a protein containing a glycine-rich domain [Rf3 in maize CMS-S type affects the transcript of orf355-orf77, resulting in fertility restoration in sterile lines [G. harknessii Brandegee (D2) and G. trilobum (DC.) Skovst. (D8) into Upland cotton  nuclear backgrounds [Rf1 gene from G. harknessii (D2) can restore the fertility of both CMS-D2 and CMS-D8, whereas Rf2 gene from G. trilobum only restores CMS-D8 to male fertility [Rf1 and Rf2 gene in cotton functioned sporophytically and gametophytically, respectively, and the two restorer genes are not allelic but tightly linked in 0.93 cM [Rf1-specific was developed based on a candidate PPR gene and could ensure the purity of restorer lines [Rf2 gene including a PPR-AFLP marker [Rf2rf2) and its maintainer line (rf2rf2) showed that the down regulation of starch synthase explains the lack of starch accumulation in sterile rf2 pollen grains in restored plants [Loss of fertility in CMS plants, which exhibits normal vegetative growth, can be restored by nuclear genes known as Rf genes . Fertilias maize , petuniaas maize , radish as maize , 23, ricas maize , 24–26 aas maize . Except  acetate , 28, mosh domain . Furtherle lines . Two maikgrounds , 31. Intertility , 31. Alt 0.93 cM , 31. Furer lines . Wang etP marker . A diffeB. juncea, 47 differentially expressed miRNAs between CMS and maintainer lines were identified that might participate in the regulatory network of CMS occurrence during anther development [B. rapa, 54 conserved and 8 novel miRNA families involved in pollen development [MicroRNAs (miRNAs), a class of endogenous small RNAs approximately 22 nucleotides in length, negatively regulate gene expression via mRNA cleavage or translational repression . Many stelopment . In B. relopment . Totallyelopment . In Uplaelopment . Howeverrf1rf1) and its maintainer line  B and restorer line R line  for the CMS-D2 based three-line hybrid cotton. Through the use of combined small RNA and transcriptome sequencing analyses, we aimed to explore the molecular mechanisms of CMS occurrence and fertility restoration during anther development in three-line hybrid cotton system. Our results should provide a valuable foundation for understanding of CMS in cotton and other crops.Although numerous miRNAs associated with anther development have been identified in many crops, the molecular mechanisms underlying CMS occurrence and fertility restoration in three-line hybrid cotton systems are unclear. Specifically, there is a lack of reports on whether a candidate PPR gene for fertility restoration interacts with miRNA. To systematically investigate miRNAs and target-mediated regulatory network of CMS and fertility restoration in a three-line hybrid cotton system, we constructed small RNA libraries and transcriptome libraries from floral buds of near-isogenic CMS line  can restore the fertility for both CMS-D2 and CMS-D8, and Rf1 gene functioned sporophytically [CMS-D2 is a sporophytic system. According to a genetic study, ytically . Mehetreytically . For valytically , the CMSTo identify transcripts that are involved in CMS occurrence or fertility restoration, RNA-seq libraries from 3 mm floral buds of A, B, and R lines each with three biological replicates were constructed. Totally 9 RNA-seq libraries were constructed for sequencing. Totally 920,746,206 raw reads were obtained from these libraries Table . After fThe FPKMs  of genes was further used to perform a differential expression analysis. For biological replicates, genes with a P-adjusted < 0.05 were assigned as being differentially expressed. Based on this analysis, totally 1913 genes showed significantly differential expression between A and B lines, of which 917 (47.9%) differentially expressed genes (DEGs) were up regulated and 996 (52.1%) DEGs were down regulated in the B line  and cellular component morphogenesis (GO:0032989)  analysis was performed for all DEGs in the three comparison  groups. Among the three comparisons, the A vs. B comparison identifies DEGs related to the male sterile cytoplasm; the A vs. R comparison identifies DEGs related to the functional restorer gene Small RNA libraries representing A, B, and R lines were constructed, resulting in a total of 28,166,751 raw reads obtained. After removing low-quality reads, we obtained 5,972,568 clean reads ranging from 18 to 30 nt in length  from A line, 6,453,537  from B line, and 5,991,695  from R line  were identified as known miRNAs. A total of 370 known miRNAs representing 62 known conserved and 106 non-conserved miRNA families were identified , which was effective evidence of novel miRNAs | ≥ 1), a total of 154 differentially expressed miRNAs (DEMs) (137 known and 17 novel) were identified by comparing the sterile A with fertile B and R lines  were common in A-B and A-R comparisons, of which two cotton-specific miRNAs  were both up-regulated in A line as compared with the B and R line. However, 28 DEMs were only detected in A-B group, among which cotton-specific miRNAs ghr-miR2948-5p, ghr-miR2950 and ghr-miR7495a were all down-regulated in A line. In the A-R group, 82 DEMs were only uniquely detected, of which ghr-miR2949, ghr-miR3476, ghr-miR482 and ghr-miR7504 were all up-regulated in A line relative to R line.Chromosome locations of miRNAs showed that most of the conserved miRNA families are widely distributed on different chromosomes Fig. . For exaRf1 gene is located on Gh_D05 chromosome, and genetic mapping indicated that the nearest SSR marker to Rf1 was BNL3535 with 0.049 cM and NAU3652 on the other side with 0.078 cM [Rf1 region flanked by the two SSR markers, 7 miRNAs  were detected, among which, ghr-miR7500, ghr-miR7491, gra-miR8667, and gra-miR8691 were down regulated in R line, while rgl-miR5139, ghr-miR2948-5p, and cpa-miR8155 were up regulated in B line, as compared with A line. However, the target genes regulated by the 7 miRNAs did not show any significant difference in A-B or A-R comparisons, except for a protein kinase superfamily gene Gh_SAPK2 (Gh_A11G0474) on chromosome A11 (regulated by ghr-miR7491) was up regulated in R line, as compared with A line. These miRNAs within the Rf1 region did not show any sequence variations among the three lines, nor did their target genes. Furthermore, their target genes are not located in the Rf1 region on Gh_D05. Therefore, we conclude that these 7 miRNAs and their targets are not genetically associated with CMS occurrence and fertility restoration.Previous studies indicated that 0.078 cM , 32. IntRf1 region of 0.127 cM between the two flanking SSR markers. However, only 5 PPR genes  were up regulated in R line as expected for its functional and dominant Rf1 allele, as compared with A and B line. While Gh_D05G3357 gene only contains 84 amino acids, the remaining 4 PPR genes  belong to P-class PPR proteins and were selected as candidates for a further analysis. Interestingly, they are all targeted by gra-miR7505b, which is located on homeologous chromosomes Gh_A06 and Gh_D06. In addition, Gh_D05G3356 gene is also targeted by ghr-miR7505, but with no difference in its expression level among A, B and R lines. Since Rf1 is derived from its original CMS cytoplasm donor, i.e., G. harknessii, it is reasonable to believe that the Rf1-regulating small RNA gra-miR7505b is also carried on the D subgenome chromosome, i.e., Gh_D06. Among these 4 PPR genes, even though one or more may be directly responsible for the fertility restoration based on our prior high resolution mapping study [Rf1 gene in R line .Most importantly, 12 PPR genes were identified within the ng study , we specBecause miRNAs regulate many developmental processes by mediating mRNA cleavage or translational repression , target To further understand the biological functions of DEMs in A-B and A-R groups, we carried out GO and KEGG analysis of their target genes | ≥1), 13 differentially expressed targets for 11 DEMs and 47 DEGs for 46 DEMs were identified in A-B and A-R comparisons, respectively  gene precisely at the 643 nt and 748 nt sites. Both the expression of the miRNA-target pair and the RLM-RACE analysis indicate that this miRNA may participate in CMS occurrence or fertility restoration during anther development by silencing their corresponding targets .To validate the cleavage sites, gra-miR7505b-PPR pairs were chosen for an RLM-RACE analysis. Similar to the bioinformatics prediction result, the RLM-RACE analysis showed that gra-miR7505b cleaves the PPR , the SNP (“C” in A/B to “T” in R line) at 785 nt caused a mismatch with gra-miR7505b in A and B line. In position 900 nt, a SNP (“C” in R line and “T” in A and B line) caused a mismatch with ghr-miR7505 in the 3′ end of its mature sequence in R line  synthesis . In cott. juncea , which sAth-miR160a-5p was predicted to target the ARF17 transcript. Several studies has indicated that ARFs are key regulators of gene expression response to auxin and floral organ formation , 56, 57.The cotton-specific ghr-miR2949b was predicted to act as a regulator of sugar/nucleotide transporter protein (STP). According to a previous study, pollen of CMS lines lacks soluble sugars , with moRf1 gene region on chromosome D5. Further studies should be focused on how some of these miRNA-target pairs are molecularly regulated by PPR genes, leading to pollen development and male fertility restoration.However, it is important to recognize that the above miRNA-target pairs do not have a direct genetic link to fertility restoration for the CMS-D2 system, i.e., there is no genetic causative relationship between them and fertility restoration, because none of them is located within the In this study, we attempted to reveal the molecular mechanisms involved in CMS occurrence and fertility restoration by combining small RNA and transcriptome sequencing of floral buds among CMS, maintainer, and restoration lines. Among several differentially expressed miRNA-target pairs in A-B and A-R groups identified for the first time, the miR7505b/miR7505 and two PPR gene targets are genetically related to fertility restoration in CMS-D2 system. Our new findings will serve as a valuable foundation to shed light on the miRNA-mediated regulatory network of CMS occurrence and fertility restoration.1 plants to construct a BC8F1 population. From this segregating population, the sterile and fertile plants were selected as the CMS-D2 A line and restorer line ®, respectively [S(rf1rf1), while maintainer line (B) harbors normal fertile Upland cotton cytoplasm and has the same nuclear allelic composition N(rf1rf1). The restorer line ® is homozygous for dominant  fertility restorer alleles S(Rf1Rf1) to allow recovery of fertility in CMS-D2 cotton plants in a cross of A × R. The three lines were planted under normal production conditions. For sample collection as described in previous studies [The CMS-D2 three-line hybrid cotton system was developed at the Cotton Research Institute (CRI), Chinese Academy of Agricultural Science (CAAS). In previous study, the CMS line with the CMS-D2 cytoplasm was crossed with the restorer line, and the maintainer line with normal fertile Upland cotton (AD1) cytoplasm as recurrent male parent to backcross with the Fectively . The CMS studies , 18, eac−) followed by PCR amplification. Fragments corresponding to 140 to 160 bp  in the PCR product mixture were separated and purified by 8% polyacrylamide gel electrophoresis. The transcriptome libraries were generated using a NEB Next Ultra RNA Library Prep kit for Illumina following manufacturer’s recommendations. Both small RNA and transcriptome sequencing were performed on an Illumina Hiseq 2500/2000 platform.Extraction of total RNA was performed using Spectrum™ Plant Total RNA kit according to manufacturer’s instructions. Equal amounts of RNA from three biological replicates were used to construct small RNA libraries  and transcriptome libraries . Small RNA libraries were generated as follows: 3′ and 5′ adaptors were ligated to small RNA, with first-strand cDNA then synthesized using M-MuLV reverse transcriptase (RNase Hhttp://www.mirbase.org/index.shtml) without mismatches to identify known/conserved miRNAs. The remaining unknown reads were used to predict novel miRNAs using miREvo [http://www.cottongen.org) using TopHat after obtained clean reads. The raw sequence data of transcriptome and small RNA can be found in the National Center for Biotechnology Information (NCBI) under accession number SRX3421007 and SRX3422274, respectively.Clean reads were obtained from the raw reads by removing low-quality reads, 5′ adaptor contaminants, reads containing poly-N, A, T, G or C. Clean reads comprising 18 to 30 nt were mapped to the TM-1 reference genome using Bowtie , 61. Onlg miREvo  and mirdg miREvo . The mRN1.5 (sample1/sample2)| ≥ 1 and P ≤ 0.05. For transcriptome, gene expression were computed by summing the FPKMs of transcripts in each sample using Cuffdiff v2.1.1 [P-value < 0.05 between two lines were assigned as differentially expressed.MiRNA expression levels were normalized based on transcripts per million (TPM) using the following formula : normalif v2.1.1 , and thehttp://plantgrn.noble.org/psRNATarget/). GO enrichment analysis was performed with GOseq package [Candidate targets of miRNAs were predicted with psRNATarget ( package  and KEGG package .Ubiquitin 6 (GhUBQ6)  was reference gene for normalization. All reactions were performed with three biological replicates, and miRNA relative expression levels were calculated using the 2−ΔΔCt method.QRT-PCR was performed to evaluate the validity of transcriptome and small RNA sequencing results. MiRNAs and total RNAs were extracted from floral buds and reverse transcribed to cDNA using TransScript miRNA First-Strand cDNA Synthesis SuperMix kit  and PrimeScript RT reagent kit , respectively, following the manufacturer’s guidelines. MiRNA-specific primers were designed according to the mature miRNA sequence , following the manufacturer’s guidelines. Gene-specific 5′ primer and 5’ RACE gene-specific nested primer were designed to amplify the cleaved transcripts. The 5’ RACE products were cloned with pEASY-T1 vector  and sequenced. Additionally, specific primers were designed to clone targets for sequence analysis. The PCR products were linked with pEASY-T1 vector and selected 8 clone in every sample to sequence. The MEGA7 was used for sequence alignment. All primers used above are listed in Additional file Additional file 1:Table S1. Information of differentially expressed genes in A, B, and R lines. (XLSX 488 kb)Additional file 2:Table S2. Information of conserved and non-conserved miRNA family in A, B, and R lines. (XLSX 24 kb)Additional file 3:Table S3. Information of novel miRNA in A, B, and R lines. (XLSX 12 kb)Additional file 4:Table S4. Information of differentially expressed miRNAs in A-B and A-R compare group. (XLSX 19 kb)Additional file 5:Table S5. Annotation of predicted target genes for known and novel miRNAs. (XLSX 661 kb)Additional file 6:Table S6. GO and KEGG analysis of predicted target genes for DEMs in A-B and A-R compare group. (XLSX 403 kb)Additional file 7:Table S7. Information of differentially expressed miRNA-target pairs in A-B and A-R compare group. (XLSX 12 kb)Additional file 8:Table S8. Primers for quantitative RT-PCR and RLM-5’ RACE and sequence variation analysis. (XLSX 9 kb)


Salmonella enterica sv Minnesota rough strains R5 and R7 (rough mutant chemotypes Rc and Rd1) have been determined. The structures reveal that hSP-D specifically and preferentially targets the LPS inner core via the innermost conserved Hep-Kdo pair with the flexibility for alternative recognition when this preferred epitope is not available for binding. Hep-Kdo binding is achieved through calcium dependent recognition of the heptose dihydroxyethyl side chain coupled with specific interactions between the Kdo and the binding site flanking residues Arg343 and Asp325 with evidence for an extended binding site for LPS inner cores containing multiple Kdo residues. In one subunit of the R5-bound structure this preferred mode of binding is precluded by the crystal lattice and oligosaccharide is bound through the terminal inner core glucose. The structures presented here thus provide unique multiple insights into the recognition and binding of bacterial LPS by hSP-D. Not only is it demonstrated that hSP-D targets the highly conserved LPS proximal inner core Hep-Kdo motif, but also that hSP-D can recognise either terminal or non-terminal sugars and has the flexibility and versatility to adopt alternative strategies for bacterial recognition, utilising alternative LPS epitopes when the preferred inner core Hep-Kdo disaccharide is not available for binding.The crystal structures of a biologically and therapeutically active recombinant homotrimeric fragment of native human SP-D (hSP-D) complexed with the inner core oligosaccharide of the  The innate immune protein human surfactant protein D (hSP-D) is a collagenous C-type lectin prototypically associated with the lung surfactant but now identified throughout the human body . SP-D rein vivo and in vitro [Numerous studies of the binding of simple sugars to both hSP-D and rfhSP-D, a recombinant homotrimeric native fragment comprised of the neck region plus three carbohydrate recognition domains (CRDs) which exhibits significant biological and therapeutic activity in vitro –10 have in vitro –13 have in vitro –18). In Escherichia coli, Salmonella enterica serovar Minnesota and Haemophilus influenzae [H. influenzae Eagan strains inner core LPS by a recombinant fragment of native hSP-D (rfhSP-D) [A number of studies of gram-negative bacteria, including fluenzae ,20 have fluenzae –16. Our rfhSP-D) ,21 revearfhSP-D) ,16. FurtrfhSP-D) .Salmonella enterica is a gram-negative bacterium that causes widespread outbreaks of gastroenteritis and bacteraemia in humans, with the most severe S. enterica Typhi infections causing enteric fever , L-glycero-D-manno-heptose (Hep), α-D-glucose (Glc), α-D-galactose  and N-acetyl-D-glucosamine (GlcNAc), and the antigen-specific O-oligosaccharides that extend in repeats away from the bacterial cell surface [Salmonella species the inner and, to a large extent, the outer core oligosaccharides are a conserved epitope [n et al. ). Serovan et al. –26. The  surface . In Salm epitope .S. enterica Minnesota lack the O-antigen, leaving the core sugar residues exposed to the immune system leading to the possibility of recognition by SP-D in a similar manner to that reported for H. influenzae Eagan 4A [Rough mutants of Eagan 4A . While tEagan 4A . The R5 Eagan 4A –33.H. influenzae Eagan 4A LPS [S. enterica Minnesota strains R5 and R7provide the opportunity to investigate, through crystal structure analysis, host recognition of more complex natural LPS structures with inner cores containing multiple Kdo and Hep alongside a terminal Glc in the R5 strain. Here we show that hSP-D recognition of Salmonella enterica sv Minnesota rough strains R5 and R7 (rough mutant chemotypes Rc and Rd1) LPS occurs preferentially through the inner core Hep-Kdo, with hSP-D having the flexibility and versatility to target the terminal glucose of R5 when the preferred epitope is not accessible for binding.While the n 4A LPS  includesSalmonella enterica sv. Minnesota R5 (Enzo Lifesciences-ALX-581-017-L002) and R7 (Enzo Lifesciences-ALX-581-018-L002) mutants were prepared under mild hydrolysis conditions with 2% acetic acid, in accordance with the method previously published by Masoud et al. [The oligosaccharide (delipidated) portions of d et al.  and our d et al. . For thed et al. , making A recombinant homotrimeric fragment of human SP-D (rfhSP-D) was expressed and purified as described previously ,6,14. Ea2 to give 10mM, and after dialysis (for the R5 studies) into 50 mM tris(hydroxymethyl)aminomethane (Tris), 10 mM CaCl2, 150 mM NaCl, 0.02% NaN3 at pH7.4. Protein concentrations used were 7.7–8.9 mg/ml. Native crystals of rfhSP-D were grown in sitting drops using screens around conditions previously reported [The endotoxin treated rfhSP-D at 8.8 mg/ml in PBS was used for crystallisation both directly (for the R7 studies), following addition of CaClreported . Those freported  to proviMOSFLM [AIMLESS, TRUNCATE, SORTMTZ and, where necessary, REINDEX [COOT [REFMAC5 [ProDRG [Data for the rfhSP-D R5 and R7 complexes were collected at 100 K at Diamond Light Source, Oxfordshire, on beamlines I04-1 and I03, respectively, using Dectris Pilatus 6M hybrid photon counting detectors. Integrated intensities were calculated using the program MOSFLM . The 1.6MOSFLM ) was use REINDEX . Model bEX [COOT  and refi[REFMAC5 , availab [ProDRG  followin [ProDRG . The coo [ProDRG  and the The trimeric rfhSP-D structures comprising three carbohydrate recognition domains (CRDs) and the α-helical coiled-coil neck region have been refined to 1.65 Å and 1.75 Å resolution for the R5-bound and R7-bound structures respectively. In all but one of the CRDs the structures reveal oligosaccharide ligand in the Ca1 binding pocket. No ligand is present in subunit A of the R7-bound structure while the mode of oligosaccharide recognition in subunit A of the R5-bound structure differs from the Hep-Kdo binding seen in subunits B and C of both structures.H. influenzae Eagan 4A structure (pdb entry 4E52) but here the 5-membered ring and all substituents are fully defined in the electron density , SP-D conserved Arg349 and Glu333 . The relIn both structures and for both modes of binding (Ca1 coordination of HepI or GlcI) additional direct interactions between the protein and bound oligosaccharide are dominated by the binding-pocket flanking residues Arg343 and Asp325 . For botS. enterica Minnesota LPS from deep rough mutant strains reveal SP-D targeting of the proximal inner core Hep-Kdo motif through calcium-dependent binding of the  Hep supplemented by interaction of both Hep and Kdo with the binding site flanking residues Arg343 and Asp325. The structures also provide a definitive demonstration for the first time that hSP-D has the versatility to recognise alternative LPS epitopes when the Hep-Kdo inner core unit is inaccessible. This flexibility and versatility of LPS recognition is supplemented by evidence of an extended recognition surface that interacts with the LPS inner core oligosaccharide. The Kdo structure itself provides the first direct confirmation that mild hydrolysis removal of the lipid A from intact LPS exhibiting 4-KdoI substituted KdoII is accompanied by the elimination of KdoII from KdoI C4 [The structures of rfhSP-D, a biologically active recombinant native fragment of hSP-D, bound to inner core structures of  KdoI C4 ,45 in a  KdoI C4 . This isH. influenzae Eagan 4A –rfhSP-D structure [In all subunits where the carbohydrate binding site is openly accessible in the crystal , HepI is coordinated to calcium Ca1 as reported for the tructure  with Kdotructure , this reThe R5 bound structure  reveals S. enterica sv. Minnesota R5 and R7 LPS oligosaccharides (Salmonella enterica mutants with more extended cores and which are recognised by SP-D [Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae which are known to be recognised by rfhSP-D [The full charides  are know by SP-D  can also rfhSP-D .Previous studies of the hSP-D/LPS interaction have shown the importance of Asp325 and Arg343 as oligosaccharide ligand selection mediators, flanking the binding site and directing calcium recognition –17, alon


CTLA-4 +49A/G (rs231775), PTPN22 +1858G/A (rs2476601), and MBL2 -550 (H/L) (rs11003125), -221(X/Y) (rs7096206) and exon 1 (A/O) in women from northeastern Brazil to determine whether polymorphisms within these key immune response genes confer differential susceptibility to clinical conditions in TS. A case-control genetic association study was performed, including 86 female TS patients and 179 healthy women. An association was observed for the A/G genotype of CTLA-4 +49A/G in TS patients . In addition, an association between the CTLA-4 G/G genotype and obesity was detected in TS patients . Regarding, the -550(H/L) polymorphism in the MBL2 promoter, the frequency of the H/L genotype was significantly higher in the TS group than healthy controls . The H/H genotype indicated a protective effect in TS patients . No differences were observed in the distribution of -221(X/Y), MBL2 exon 1 variants, and PTPN22 +1858G/A in any assessed groups. CTLA-4 variants are potentially involved in obesity in this cohort of TS patients from northeastern Brazil.Turner syndrome (TS) is characterized by a set of clinical conditions, including autoimmune/inflammatory diseases and infectious conditions, that can compromise a patient’s quality of life. Here we assessed polymorphisms in  Turner syndrome (TS) is one of the most common chromosomal abnormalities in humans and is characterized by the presence of one X chromosome and total or partial loss of the second sex chromosome. TS is estimated to affect 1 in every 2500 live female births (Some studies have reported increased levels of autoantibodies (anti-thyroid peroxidase and anti-glutamic-aciddecarboxylase) in TS patients and an increased risk of developing a range of autoimmune diseases, such as Hashimoto’s thyroiditis, type I diabetes mellitus, celiac disease, Crohn’s disease, ulcerative colitis, juvenile rheumatoid arthritis, Addison’s disease, autoimmune hepatitis, psoriasis, vitiligo, and alopecia (tyrosine-protein phosphatase non-receptor type 22 gene (PTPN22), cytotoxic T-lymphocyte-associated protein 4 gene (CTLA4), and mannose-binding lectin (MBL2) (PTPN22 rs2476601 has been assessed in Brazilian TS patients from São Paulo (Southeast region) , ranging from 0.1 to 33 years. Clinical data shown in the Clinical diagnosis of 86 TS patients was confirmed by chromosome analysis in peripheral blood leucocyte. Karyotypes found were as follows: 45,X ; 45,X/46,X,i(Xq) ; 46,X,i(Xq) ; 45,X/46,XY ; 45,X/46,X,r(X) ; other chromosomal constitutions summed 13 individuals (15.12%).PTPN22 + 1858G/A (rs2476601) at codon 620, CTLA-4 +49A/G (rs231775) within codon 17 in the first exon and MBL2 promoter region -550(H/L) (rs11003125), -221(X/Y) (rs7096206). Genotyping was performed using TaqMan SNP genotyping assays and Taqman Universal Master Mix  according to manufacturer instructions. SNP assessment within MBL2 exon 1 (A/O) was performed using SYBR Green  as previously described (MBL2 genotyped samples were sequenced in order to double-check the Melting Temperature assay (MTA) results. We found 100% concordance between sequenced samples and the MTA results.Genomic DNA was extracted from whole blood using IllustraTM Blood GenomicPrep Mini Spin Kit  according to manufacturer’s instructions. SNPs selection was based on minimum allele frequency (MAF) of 10% and/or SNP consequence/function upon gene action. A total of five SNPs were selected distributed as follows 2 or Fisher’s exact test. Odds Ratio (OR) and 95% Confidence Intervals (CI) were also calculated and a p-value < 0.05 was considered statistically significant. Combined alleles for the PTPN22 and CTLA-4 genes, haplotypes for the MBL2 gene and a possible association of these combined alleles and haplotype with clinical conditions in TS patients were also assessed. Combined genotypes for the MBL2 gene were assessed by Arlequin version 3.1 software (2 tests) power analysis was performed with the G* Power software , with α error probability of 0.05Statistical analyses were carried out using SNPStats available at http://bioinfo.iconcologia.net/SNPstats_web and R software (https://www.r-project.org/). Hardy–Weinberg equilibrium was tested for each polymorphism by comparing observed with expected frequencies using chi-square (χ2) tests. Differences in allele and genotype frequencies from each studied polymorphism in patients and controls were assessed using χPTPN22 rs2476601 (G > A) and CTLA-4 rs231775 (A > G) among TS patients and healthy controls are summarized in p > 0.05) was observed in both SNP distributions, and no significant differences were found in the allele and genotype frequencies of these variants in both groups  in any of the studied groups. A lower frequency of the A allele and G/A genotype was observed in both TS patients and controls  (Table S1). The power was 80.7% (α-error = 5% confidence) to detect a medium effect size (w=0.3) for PTPN22 genotypes in both patients and controls.We did not detect the homozygous A/A genotype of controls . No signCTLA-4 rs231775, significantly different distributions of genotype frequencies were observed in TS patients compared to the control group. An association was detected for the A/G genotype , indicating a differential distribution for this SNP in TS patients. The power was 99% (α-error = 5% confidence) to detect a medium effect size (w=0.3) for CTLA4 genotypes comparing patients and controls. Furthermore, when assessing the clinical features of TS and the allele and genotype distribution, we detected an association between the CTLA-4 allele (recessive model: A/A—A/G vs. G/G) and obesity in TS patients  (Table S2).Concerning PTPN22 and CTLA-4 and frequencies for both groups are given in Table S3).The combined alleles of MBL2 genotype and allele distributions in TS patients and controls are given in MBL2 polymorphisms were in HWE in the TS and control groups, except for rs11003125 (-550 H/L) in the TS group (p < 0.05). The H/L genotype frequency was significantly higher in TS patients than controls . Furthermore, the H/H genotype indicated a protective effect in TS patients compared to healthy controls . Again, this result cannot be fully explained due to an absence of HWE in this specific group. The power was 99.9% (5% confidence) to detect a medium effect size (w=0.3) for -550 MBL2 genotypes in both groups.The MBL2 rs7096206 (-221 X/Y), no differences in allele or genotype frequencies were observed between TS patients and controls. The power was 30.5% (5% confidence) to detect a medium effect size (w=0.3) for -221 MBL2 genotypes when comparing patients and controls. Furthermore, no significant differences were observed in the genotype and allele frequencies of exon 1 variants between TS patients and controls. The power was 91.2% (5% confidence) to detect a medium effect size (w=0.3) for exon 1 MBL2 genotypes when comparing TS patients and controls. In patients with TS, no SNP in the MBL2 gene or promotor region was associated with clinical characteristics (Table S4).Regarding MBL2 -550 and -221 promoter region and exon 1 polymorphisms are given in Table S5).The frequencies of haplotypes and combined genotypes originating from linkage disequilibrium between the Table S6).Genotypes were classified as high ; low ; and deficient producers of MBL . Significant differences were not found between the evaluated groups. Moreover, no significant difference was observed among combined genotypes and the clinical data of TS patients  and the development of autoimmune diseases in TS in another cohort from Southeast Brazil. The frequency of the A allele in women with TS (1.1%) and healthy controls (3.6%), as well as the heterozygote genotype in both groups , was lower in our study than that of Our results differ from This difference between studies could be due to variation in allele frequencies of some disease-associated SNPs in different ethnic groups (PTPN22 rs2476601 (G > A) and other clinical features in TS patients, such as obesity, as well as dyslipidemia, given its role in modulating inflammatory conditions. As obesity is a disease characterized by chronic mild inflammation, the concentration of acute phase proteins and cytokines associated with inflammation are higher in obese individuals compared to normal weight individuals .The CTLA-4 +49A/G variant has been associated with different diseases, and G/G individuals may possess CTLA-4 protein with a weak suppression function compared to individuals with the A/A genotype  polymorphism and different clinical conditions in TS patients. This MBL2 promoter variation has a significant down-regulating effect on the serum MBL concentration, leading to ineffective clearance of apoptotic cells and the spread of self-antigens, permitting an immune response toward autoimmunity and tissue damage (Y variant is associated with high serum MBL expression (No association was found between the p < 0.05), revealing a different distribution in both groups.On the other hand, our results revealed significant differences regarding the -550  promoter polymorphism between TS patients and controls  variant and clinical data of the TS group.Notably, in our study population, the -550 H/L In summary, even though our study is a pilot study, due to limited number of TS patients and controls included, our results indicate a differential distribution for some polymorphisms within key inflammation-regulating genes in TS patients. The understanding of how key immune genes and its variants are related in TS might help in future therapy strategies. These findings may open up a new potential line of research to improve life’s quality in these individuals.


Apoptosis inhibition is a critical strategy of mycobacteria facilitating its survival in macrophages, but the underlying mechanism is not completely understood. In this study, we found that Rv3033, a secreted virulence factor of mycobacteria, played an important role in bacillary survival within macrophages. Forced over-expressed of Rv3033 in macrophages could efficiently resist mycobacteria-induced early and late apoptosis, accompanied with the obvious increased cellular bacterial burden. By exploring the underlying mechanism, we found that Rv3033 efficiently repressed the intrinsic (caspase-9 meditated), but not the extrinsic (caspase-8 mediated) apoptotic pathway in mycobacteria-infected macrophages. And this repression relied on the orchestrating blockade of both mitochondrial cytochrome c release and endoplasmic reticulum (ER) stress PERK branch activation. Our study uncovered a novel function of mycobacterial virulence factor Rv3033 as an anti-apoptotic protein, which may provide a new target for tuberculosis (TB) treatment. Tuberculosis is an infectious disease caused by the intracellular pathogen mycobacteria, which results in 1.7 million deaths and 6.3 million new cases annually worldwide .As the first line of host defense, macrophages are key immune cells in resistance against mycobacteria –5. Long-In 2005, Rengarajan and colleagues screened a genome-wide mycobacteria transposon mutant library and established several candidate factors that are essential for mycobacteria survival in macrophages . Based oIn this study, we found that Rv3033 had higher expression in the virulent strain H37Rv than the avirulent strain BCG and H37Ra, could suppress mycobacteria-induced macrophage apoptosis and enhance the bacillary survival. In addition, this inhibitory effect relied on the repressing intrinsic apoptosis by orchestrating blockade of mitochondrial cytochrome c release and endoplasmic reticulum (ER) stress PERK branch activation. In conclusion, this study revealed the anti-apoptosis role of Rv3033 in mycobacteria-infected macrophages, and modulating Rv3033 expression might represent a new therapeutic target for controlling TB.2. Mice were purchased from the Experimental Animal Center of the Chinese Academy of Sciences .All animal-related experimental procedures were performed in accordance with the guidelines for the Care and Use of Laboratory Animals . The guidelines were approved by the Ethics Committee of Soochow University. BMDMs were prepared as previously described (M. tuberculosis strains BCG (ATCC35733), H37Ra and H37Rv were grown in Middlebrook 7H9 broth medium supplemented with 10% ADC, 0.5% glycerol, and 0.05%Tween-80 at 37°C. M. smegmatis mc2155 (Ms), Ms::Vector, Ms::Rv0928, Ms::Rv1096, Ms::Rv3033, and Ms::Rv3369 were grown in Luria-Bertani medium supplemented with 0.05% Tween-80.RAW264.7 and HEK293T cells (ATCC) were cultured in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 1% penicillin- streptomycin. Cells were cultured in a humidified incubator at 37°C and 5% COescribed . Briefly*g for 15 min to generate pellets isolation, then were denatured by heating for 30 min at 100°C.For mycobacterial pellets isolation, BCG, H37Ra, and H37Rv were first grown in 7H9 medium to mid-log phase and then sub-cultured into the 7H9 media at a starting OD600 of 0.1. Cultures were harvested when they reached an OD600 of 1.0 and were centrifugation at 8200The construction of recombinant strains was performed using Ms. The Rv0928, Rv1096, Rv3033 and Rv3369 genes (Tuberculist database) were cloned into the pMV261 vector in frame with a C-terminal flag-tag. All constructed gene were electroporated into Ms. The selected pMV261 (MS::Vector) and pMV261-Rv0928, Rv1096, Rv3033, and Rv3369  were cultured in Luria-Bertani medium supplemented with 0.05%Tween-80 containing 50 μg/ml kanamycin. These recombinant strains were identified by western blot using an anti-flag antibody. The following primers were used:Ms::Rv0928 Forward: 5′-CCGGAATTC TTGAAACTCAACCGATTTGGTGCMs::Rv0928 Reverse: 5′-CAGAAGCTTTCACTTGTCGTCATCGTCTTTGTAGTCG GCGATCG CGTTGACMs::Rv1096 Forward: 5′-CGCGGATCCGTGCCGAAGCGACCCGACMs::Rv1096 Reverse: 5′-CCCAAGCTTTTACTTGTCGTCATCGTCTTTGTAGTCCATCGCACC GTTATTTGGCCCMs::Rv3033 Forward: 5′-CCGGAATTC ATGGCTCACTCGATCGTTCGMs::Rv3033 Reverse: 5′-CAGAAGCTTCTACTTGTCGTCATCGTCTTTGTAGTCCTCGGGGTG GTCATCGAMs::Rv3369 Forward: 5′- CCGGAATTCATGTGGGCAGGCTACCGTTGMs::Rv3369 Reverse: 5′-CAGAAGCTTTCACTTGTCGTCATCGTCTTTGTAGTCGCCCGTGGG CGTC.2 in a humidified atmosphere. After reaching 70–80% confluency, 293T cells were co-transfected with the retroviral expression vector pMSCV-eGFP-Rv3033 and packaging vector pcl-Ampho using Lipofectamine 3000 (Invitrogen). Culture supernatants were harvested at 48 and 72 h, filtered with a 0.45-μm pore size filter and concentrated with ultracentrifugation at 144000*g for 2 H. The viruses were resuspended in PBS and stored at −80°C.The Rv3033 gene (Tuberculist database) was amplified by polymerase chain reaction (PCR) using H37Rv genomic DNA as a template. The PCR primers (pMSCV-eGFP-Rv3033Forward:5′-CCGCTCGAGATGGATTACAAGGATGACGACGATAAGGCTCACTCGATCGTTCGCACG Reverse: 5′-AAGGTTAACCTACTCGGGGTGGTCAT CGA) contained XhoI and HpaI restriction enzyme sites. The PCR products were digested and ligated into a pMSCV-eGFP retroviral vector to form pMSCV-eGFP-Rv3033. HEK293T cells were cultured in 10 cm dishes at 37°C and 5% CORAW264.7 cells were infected with viral supernatants collected from HEK293T cells transfected with retroviral constructs. After 72 h, GFP-positive cells were sorted by flow cytometry, and Rv3033 expression was verified by western blot analysis. RAW264.7 cells infected by pMSCV-eGFP and pMSCV-eGFP-Rv3033 viruses were named RAW-Vector and RAW-Rv3033 cells, respectively. Then the viability of RAW-Vector and RAW-Rv3033 cells were detected by CCK8 assay.The CFU assay was performed as previously described . BrieflyRAW264.7 cells were seeded into 6-well plates, and adherent monolayers were infected with Ms::Vector, Ms::Rv0928, Ms:: Rv1096, Ms:: Rv3033, or Ms:: Rv3369 (MOI = 10) for 48 h. The apoptotic cells were measured by FACS using an Annexin V/PI kit . BMDM cells were seeded into 6-well plates, and adherent monolayers were infected with Ms::Vector or Ms:: Rv3033 (MOI = 10) for 48 h. The apoptotic cells were measured by FACS using an Annexin V/PI kit . RAW-Vector and RAW-Rv3033 cells were seeded into 6-well plates, and adherent monolayers were infected with BCG (MOI = 10) with or without pretreatment for 1 h with TUDCA (500 μg/ml) for 36 h. The apoptotic cells were measured by FACS using Annexin V-7-AAD kit . RAW-Vector and RAW-Rv3033 cells were seeded into 6-well plates, and adherent monolayers were infected with H37Ra for 36 h. The apoptotic cells were measured by FACS using Annexin V/7-AAD kit .in situ Cell Death Detection Kit, TMR red (Roche). Briefly, cells were washed three times with PBS, fixed with 4% paraformaldehyde for 1 h and permeabilized with 0.1% Triton-X-100 for 2 min on ice. TdT enzyme was used for BrdUTP incorporation into DNA nicks at 37°C for 1 h. The cells were washed twice with PBS and incubated with DAPI for 15 min at room temperature. Cells were washed again, analyzed using a fluorescence microscope and counted.RAW264.7 cells were seeded into 6-well plates, and adherent monolayers were infected with BCG or H37Rv (MOI = 10) for 24 h. TUNEL staining was performed using an RAW-Vector and RAW-Rv3033 cells were infected with BCG at an MOI of 10 for 24 h. Whole cell lysates and mitochondria were obtained with a cell mitochondria isolation kit according to the manufacturer's protocol .Cells were harvested and lysed in lysis buffer for 30 min on ice. Equal amounts of protein from each sample were separated by SDS–PAGE and electro-blotted onto PVDF membranes. The membrane was blocked with 5% dry milk for 2 H and incubated with primary antibodies overnight at 4°C. The membranes were then incubated with relevant secondary antibodies at room temperature for 1 h and visualized using ECL  according to the manufacturer's instructions. Bands were quantified by ImageJ image software using β-tubulin as internal controls. The primary antibodies used in the present study were anti-caspase-3, anti-caspase-8, anti-caspase-9, anti-caspase-12, anti-Bip, anti-Chop, anti–phospho (Ser-51)-eIF2α, anti-cytochrome c, anti-Bax, anti-β-tubulin , anti-Rv3033.RAW-Vector and RAW-Rv3033 cells were seeded into 6-well plates, and adherent monolayers were infected with BCG (MOI = 10) for 12 h. Total RNA was extracted by TRIzol reagent. NGS was sequencing by Genewiz.post hoc test or two-way ANOVA was used for statistical analysis. *p < 0.05; **p < 0.01; ***p < 0.001.The data are represented as the mean ± SEM and were analyzed using GraphPad Prism software. One-way ANOVA followed by Turkey's Macrophage line RAW264.7 cells were infected with distinct virulent mycobacteria (virulent H37Rv or avirulent BCG) for 24 h, and cell death was evaluated by CCK8, TUNEL, and western blot assays. As shown in Figures M. smegmatis individually, we found that compared with other recombinant bacteria, Rv3033 possessed the most potential anti-apoptotic function, evidenced by the lowest macrophage apoptosis level post M. smegmatis infection , early  as well as late  apoptotic rations  were established. Following infected with BCG, the apoptotic cell percentages were assessed by Annexin V /7-AAD staining assay. As shown in Figure Apoptosis could be caused by two main pathways: caspase-9-mediated intrinsic pathway and caspase-8-mediated extrinsic pathway, both of which have been reported to be activated post mycobacteria infection . These tCytochrome c release from mitochondria to cytosol is a canonical trigger for intrinsic apoptosis. To verify whether Rv3033 affected mitochondrial-mediated intrinsic apoptosis after mycobacteria infection, we tested the translocation of the mitochondrial apoptosis-related markers Bax and cytochrome c from the mitochondria to cytosol. As shown in Figures Several studies have indicated that intrinsic apoptosis could also be mediated by ER stress after mycobacteria infection , 24–27. TB is a highly infectious disease that has become a major threat to public health. Currently, although a cocktail of first-line drugs, including isoniazid (INH), rifampin, pyrazinamide (PZA), and ethambutol are effective during the active phase of TB infections , the effM. smegmatis individually, we found that Rv3033 behaved as the most potent macrophage apoptosis inhibitor and could potently facilitating intracellular mycobacteria survival.Rv3033, a 19 kDa mycobacterial protein, was first identified by the complete mycobacteria genomic sequencing in 1998 , while iApoptosis can be induced by extrinsic (caspase-8 mediated) and intrinsic (caspase-9 mediated) pathways, which all play effective roles in eliminating mycobacteria in macrophages –13, 30. The canonical intrinsic apoptosis is relied on mitochondrial release of pro-apoptotic factor cytochrome c and apoptosis inducing factor (AIF). Following interacting with the apoptotic protease activating factor-1 (Apaf-1), cytochrome c drives the apoptosome assembly and caspase-9 activation, leading to apoptotic DNA fragmentation via cleaving several target proteins . To oppoBesides of mitochondrial pathway, recently ER stress is also found as a novel apoptosis pathway in mycobacteria-infected macrophages , 27, 35.In conclusion, we identified a novel function of the mycobacterial virulence factor Rv3033 as facilitating latent infection by inhibiting macrophage intrinsic apoptosis. This function relied on simultaneously blockade of both mitochondrial cytochrome c release and endoplasmic reticulum (ER) stress PERK branch activation. This might make Rv3033 as a more promising therapeutic target to control the latent mycobacteria infection.SX conceived this study. YY revised the manuscript. YD designed the experiments. WZ performed the experiments, analyzed the results, and drafted the manuscript. QL constructed the Ms::Rv1096 strain and assembled the Figure 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.