Lactuca sativa L.) is one of the most important leafy vegetable that is consumed during its vegetative growth. The transition from vegetative to reproductive growth is induced by high temperature, which has significant economic effect on lettuce production. However, the progression of floral transition and the molecular regulation of bolting are largely unknown. Here we morphologically characterized the inflorescence development and functionally analyzed the FLOWERING LOCUS T (LsFT) gene during bolting regulation in lettuce. We described the eight developmental stages during floral transition process. The expression of LsFT was negatively correlated with bolting in different lettuce varieties, and was promoted by heat treatment. Overexpression of LsFT could recover the late-flowering phenotype of ft-2 mutant. Knockdown of LsFT by RNA interference dramatically delayed bolting in lettuce, and failed to respond to high temperature. Therefore, this study dissects the process of inflorescence development and characterizes the role of LsFT in bolting regulation in lettuce.Lettuce ( Lactuca sativa L.) is one of the most important leafy vegetables that cultivated worldwide and consumed throughout the year , which is situated at the tip of the shoot apex and is comprised of a pool of stem cells that continuously divide and replenish themselves . During Arabidopsis, floral transition is regulated by six genetic pathways that incorporate important endogenous and environmental cues, including vernalization, photoperiod, gibberellin (GA), autonomous, ambient temperature and age pathways (FLOWERING LOCUS T (FT), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) and LEAFY (LFY) (FT belongs to the phosphatidylethanolamine-binding protein (PEBP) family, which consists of six members in Arabidopsis-48pc: FT, TERMINAL FLOWER 1 (TFL1), ARABIDOPSIS THALIANA CENTRORADIALIS (ATC), BROTHER OF FT AND TFL1 (BFT), MOTHER OF FT AND TFL1 (MFT) and TWIN SISTER OF FT (TSF)  to activate downstream genes such as APETALA1 (AP1) and LEAFY (LFY)  . FT beloFT (TSF) . FT is pFY (LFY) . In Arab in TFL1 . Homolognditions . In sunfog HaFT4 . The putbidopsis . HoweverLsFT in nine lettuce varieties with different bolting times. Further, knockdown of LsFT by RNA interference resulted in significant delay in lettuce bolting and LsFT knockdown lines failed to respond to high temperature, indicating the important role of LsFT in regulating bolting in lettuce.Herein, we investigated the histological and morphological features of capitulum development in lettuce, and explored the expression of Lactuca sativa L.) varieties S24, S43, S7, S1, S3, S8, S28, S26, and S39 were selected from 705 lettuce collection for their different bolting times and were grown in the Beijing University of Agriculture Experimental Station under standard greenhouse conditions. Pest control and water management were performed according to standard practices. For morphological characterization and transgenic analysis, lettuce variety S39 (bolting sensitive) was cultivated in a growth chambers at 25/15°C (day/night), and using a 16 h day/8 h night with a photon flux density (PFD) of 200 μmol photons m-2s-1. The wild type and LsFT-RNAi lettuce were planted in a growth chambers at 25/15°C (day/night) under a 16 h day/8 h night cycle. The Arabidopsis ft-2 mutant was obtained from The Arabidopsis Information Resource2. All Arabidopsis plants were grown in soil at 22°C under a 16 h/8 h light/dark cycle in the growth chambers.The leafy lettuce .The shoot apex, inflorescence and florets of different developmental stages were dissected from lettuce S39 under a light microscope . After dissection, samples were fixed in formaldehyde-acetic acid-ethanol (FAA) overnight, and then critical-point dried in liquid COLsFT (Lsat_1_v5_gn2_17881.1), LsAP1 (Lsat_1_v5_gn3_97021.1), LsAP3 (Lsat_1_v5_gn3_75340.1) and LsLFY (Lsat_1_v5_gn4_ 84380) were obtained by homologous alignment in the lettuce website3. AP1, AP3, and LFY were the downstream genes of FT . A TIANGEN reverse transcriptase kit  was used to synthesize cDNA. Sequence information of es of FT . Coding Arabidopsis using Quick RNA isolation Kit . The RNA of wild type and LsFT-RNAi lines were extracted from the fourth leaves at 25 days. A TIANGEN reverse transcriptase kit (Tiangen) was used to synthesize cDNA. An ABI PRISM 7500 Real-Time PCR System  was used for quantitative real-time PCR (qRT-PCR) experiments. Three biological replicates and three technical replicates (3 × 3) were performed for each gene. Lettuce 18srRNA (Gene Bank accession number HM047292.1) and Arabidopsis ACTIN2 (Gene Bank accession number AT3G18780.2) genes were used as internal controls to normalize expression data. The gene-specific primers are listed in Supplementary Table Total RNA was extracted from different tissues of lettuce or LsFT cDNA was amplified and introduced into the XbaI and SmaI sites fused with GFP in a pUC-19 vector. The 35S promoter was used for directing the expression of fusion gene. Subcellular localization of LsFT by fusion with green fluorescent protein (GFP) in the C-terminal region The bombardment of onion epidermal cells was performed as previously described (The open reading frame (ORF) of escribed , and imaLsFT-RNAi lines were grown for 28 days in the growth chamber at 25°C/15°C (16 h day/8 h night), and then moved to the growth chamber at 35°C /25°C (16 h day/8 h night) for heat treatment. Upon 1, 2, 3, and 4 Day after heat treatment, the fourth leaves were harvested at 10 am in the morning and frozen in liquid nitrogen, and stored at -80°C until further use.Lettuce varieties S24, S43, S7, S1, S3, S8, S28, S26, S39 were used for heat treatment. The lettuce plants were grown in the growth chamber at 25°C /15°C (16 h day/8 h night) for 28 days, and then moved to the growth chamber at 35°C/25°C (16 h day/8 h night) for heat treatment. After 48 h, the fourth leaves were cut off and frozen in liquid nitrogen, and stored at -80°C until further use. The WT and The amino acid sequences of LsFT (Lsat_1_v5_gn_2_17881.1), HaFT2 (GQ884982), HaFT4 (GQ884984), CsFTL1 (AB679270), CsFTL2 (AB679271), CsFTL3 (AB679272), OsRFT1 (AB426873.1), OsHd3a (AB052944), AtFT (AT1G65480.1), AtTSF (AT4G20370.1), CiFT (AB027456), BvFT1 (HM448910.1), BvFT2 (HM448912.1), PnFT2a (AB109804.1), PnFT4a (AB369074.1), GhFT1 (HM631972), MdFT (AB161112) were obtained from the National center for Biotechnology Information website. The phylogenetic tree was constructed using the Neighbor-Joining (NJ) method with default parameters in the MEGA 6.0 software. The numbers next to nodes are 1,000 bootstraps .LsFT overexpression construct, full length LsFT CDS were amplified and cloned into the binary vector pBI121 through XbaI and SmaI sites. The construct was then introduced into Agrobacterium strain C58 by electroporation and transformed into Arabidopsis ft-2 mutant plants using the floral-dip method /SwaI(3′ end) and BamHI(5′ end)/SpeI(3′ end) sites, respectively. Primers containing restriction enzyme cutting sites are listed in Supplementary Table LsFT-RNAi construct and empty pFGC1008 vector were then delivered into Agrobacterium by electroporation.To obtain Agrobacterium tumefaciens cells with an optical density (OD) of 0.2–0.3 for 13 min. Next, the cotyledons were placed on a piece of filter paper on top of co-cultivated medium and co-cultivated at 28°C (dark) for 2 days  in conjunction with DNase I (Qiagen) for removing potential DNA contamination. The Target Amp 2-Round Aminoallyl-aRNA Amplification Kit 1.0 (Epicentre Biotechnologies) was used for RNA amplification following the manufacturer’s protocol. RNA abstracted from LCM was subjected to two rounds of amplification to yield 35–55 μg of RNA per sample. Next, RNA-Seq were made using NEB Next Ultra Directional RNA Library Prep Kit for Illumina . The libraries were pooled and sequenced in one lane with 100 bp paired-end reads on the Illumina HiSeq 2000. Sequencing data were deposited to the Sequence Read Archive (SRA) at the National Center for Biotechnology Information (NCBI) with accession number Figures 1A–D). Generally, the bolting stage starts from the 7–8 leaf stage, and the inflorescence stage starts upon the shoot producing 10–12 elongated internodes. The lettuce capitula are complex inflorescences. Each capitulum is made up of 15–25 ray florets. Each ray floret is composed of a ligule corolla with five fused petals, five fused anthers that form a tube surrounding the style and the bipartite stigma, a modified calyx called a pappus, and an ovary that produces one seed after pollination . The blossom of each capitulum lasts for 1–2 h, and seeds are ready for harvest 2 weeks after anthesis.To dissect the process of flowering in lettuce, a bolting-sensitive variety S39 was used and grown in a growth chamber at 25/15°C (day/night). The whole life cycle of S39 takes about 130 days, in which four critical developmental stages can be observed: vegetative stage (0–35 DAP [days after planting]), bolting stage (35–75 DAP), inflorescence stage (75–95 DAP), and flowering stage (95–125 DAP)  with a flat shoot apical meristem (SAM) . Stage 4 (45 DAP) highlights the development of involucre primordium from inflorescence meristem (IM) , and stage 8 (95 DAP) marks the ovary development which located in the base of each floret and gives rise to the dry seed measuring 3–4 mm long at maturity .To characterize the floral transition progression in lettuce, we chose the S39 line growing in the growth chamber at 25/15°C (day/night) for observation. Histological sections and morphological features were recorded by optical microscope and scanning electron microscope (SEM) over time. As shown in FT gene plays a key role in promoting flowering in several plant species, and that overexpression of the putative lettuce FT homolog, LsFT, promotes flowering in wild-type Arabidopsis  analysis . LsFT was shown to be highly expressed in the mature leaf , which was consistent with previous findings . To explore whether the expression of LsFT correlates with bolting in lettuce, qRT-PCR of LsFT were performed in the mature leaves of nine lettuce varieties, which were selected from 705 lettuce varieties with different bolting times . Specifically, the nine lettuce varieties were divided into three groups: late bolting , middle bolting  and early bolting . The days to bolting (the days to the first visible elongated stem) in the growth chamber were as follows: S24 (75 days), S43 (58 days), S7 (55 days), S1 (50 days), S3 (48 days), S8 (46 days), S28 (43 days), S26 (42 days), S39 (38 days). As shown in Figure 2D, the expression of LsFT was negatively correlated with the bolting time in lettuce. In the earliest bolting variety S39, LsFT transcript accumulation was 85-folds higher than that in the latest bolting variety S24 under normal conditions . Upon heat treatment for 48 h, expression of LsFT was promoted in all lettuce varieties, consistent with previous studies . For example, expression of LsFT increased 2.5-folds in the latest bolting variety S24, while 13-folds in the earliest bolting line S39. Consequently, heat treatment significantly promoted bolting in the early bolting varieties, while no dramatic difference were observed in the middle- or late-bolting lines , suggesting the important role of LsFT during heat promoted bolting in lettuce.Previous studies shown that bidopsis . Phylogebidopsis . Unlike bidopsis , sequencfindings . Subcell studies . HoweverLsFT plays an equivalent role in flowering regulation as Arabidopsis FT, ectopic expression of LsFT under the 35S promoter in the Arabidopsis ft-2 mutant was performed. A total of 30 transgenic lines were obtained and all lines flowered earlier than the ft-2 mutant plants . Based on the severity of the phenotype, transgenic lines could be divided into three classes: Class 1 such as OV-14 line is the strongest, which flowerings 9–11 days earlier than wild-type (WT). Class 2 such as OV-6 line is the moderate, which flowerings 5–7 days earlier than WT. Class 3 such as OV-1 line is the weakest, which flowerings at about the same time as WT and 9–11 days earlier than ft-2 mutant plants . Interestingly, qRT-PCR analyses showed that the severity of the phenotype positively correlated with the expression of LsFT in the transgenic lines , suggesting that LsFT can fully replace the function of FT in promoting flowering in Arabidopsis.To explore whether LsFT in lettuce, we optimized the Agrobacterium-mediated transformation in lettuce . Seven independent LsFT-RNAi lines were obtained and the expression of LsFT decreased to 29–56% in the transgenic lines . Three lines with different expression levels  were chosen for further characterization. All of the three transgenic lines bolted later than the empty vector control (CK), and the degree of delay positively correlated with the level of LsFT knockdown . For example, the number of days to bolting was 45, 55, and 75 days in lines Ri-22, Ri-9 and Ri-63, respectively, whereas the CK plants took only 38 days to bolting . Similarly, the appearance of the first floral bud and the first opening flower were significantly delayed in the transgenic lines . These data demonstrated that the decreased LsFT expression in the transgenic lines resulted in delayed bolting in lettuce. Next, we examined the transcript abundance of the putative homologs of the FT downstream genes, LsAP1, LsAP3, and LsLFY in the transgenic lines  . Furthermore, we explored the response of LsFT knockdown lines to heat treatment. In the CK plants, the expression of LsFT was significantly increased upon heat treatment, concomitant with precocious bolting. However, LsFT knockdown lines failed to respond to heat treatment, with respect to both LsFT expression and the number of days to bolting .To examine the function of ic lines . As compFigure 1). Further, we found that LsFT was highly expressed in mature leaves, and subcellular localization showed that LsFT was localized to the nucleus . Expression of LsFT negatively correlated with bolting in different lettuce varieties and was promoted by heat treatment . Overexpression of LsFT could rescue the phenotype of Arabidopsis ft-2 mutant , and knockdown of LsFT by RNAi dramatically delayed bolting in lettuce . Moreover, LsFT knockdown lines failed to respond to heat treatment, suggesting that LsFT may play a role during the heat-promoted bolting in lettuce.In this study, we characterized the structure of lettuce capitulum and identified the eight developmental stages during floral transition in lettuce  are found in Chrysanthemum and Heliantheae, while three types of florets  are identified in Gerbera hybrid . Here, wFigure 1), similar to that in monocot species such as rice and maize  SAMArabidopsis, tobacco, rice and cotton indicated that FT-like proteins were localized in both the cytoplasm and the nucleus , which is similar to that in grapevine  (FLOWERING LOCUS C (FLC) acts antagonistically to the photoperiod pathway by repressing the key floral integrators, FT and SOC1 . Such discrepancy may lie in the expression level of LsFT in different transgenic lines.Previous studies have shown that  species . In ArabANS (CO) . FLOWERIand SOC1 . Within n leaves . Overexpnditions . Lettucebidopsis . Our resLsFT negatively correlated with bolting time in different lettuce varieties, and the expression of LsFT was increased much higher in early bolting lines than that in late-bolting lines upon heat treatment . Knockdown of LsFT by RNAi resulted in delayed bolting in lettuce and failed to respond to heat treatment , suggesting the important role of LsFT during bolting regulation in lettuce ATG motif that is present in tandem within the FT promoter (FLC and SHORT VEGETATIVE PHASE (SVP), form a complex to repress FT expression through binding to the DNA regions within the proximal FT promoter and the first intron that contains CArG boxes (LsFT promoter in the future, which will shed light on the specific functions of LsFT during heat response in lettuce. Despite bolting is generally followed with flowering in lettuce, bolting and flowering are two separate processes and may be regulated by different mechanisms. More studies using floral markers such as LsAP1 and LsLFY are needed to differentiate lettuce bolting and flowering in the future.Further, we found that the expression of  lettuce . In lettpromoter . The MADrG boxes . TherefoWe thank members of the Zhang and Wang Labs for discussions and help with techniques. We also thank BioMed Proofreading LLC for proofreading this manuscript. ZC, XZ, and QW conceived and designed the experiments. ZC, YH, KN, YD, SY, WZ, CL, and XJ performed the experiments. ZC, YH, KN, and YD analyzed the data. DG and RL uploaded the database. ZC, XZ, and QW wrote the paper. All authors read and approved the final 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.


Wnt-signaling and thereby promotes the development of breast cancer. To test this hypothesis, we evaluated the correlations between expression of multiple inflammatory cytokines and Wnt pathway downstream genes in mammary tissues from women (age ≥ 50) undergoing reduction mammoplasty. Moreover, we specifically examined the role of tumor necrosis factor-α (TNF-α), an important proinflammatory cytokine associated with obesity and a possible modulator of the Wnt pathway. The regulatory effects of TNF-α on Wnt pathway targets were measured in an ex vivo culture of breast tissue treated with anti-TNF-α antibody or TNF-α recombinant protein. We found that BMI was positively associated with the secretion of inflammatory cytokines IL-1β, IL-6 and TNF-α, all of which were negatively correlated with the expression of SFRP1. The transcriptional expression of Wnt-signaling targets, AXIN2 and CYCLIN D1, were higher in mammary tissue from women with BMI ≥ 30 compared to those with BMI < 30. Our ex vivo work confirmed that TNF-α is causally linked to the up-regulation of active β-CATENIN, a key component in the Wnt pathway, and several Wnt-signaling target genes . Collectively, these findings indicate that obesity-driven inflammation elevates Wnt-signaling in mammary tissue and thereby creates a microenvironment conducive to the development of breast cancer.Epidemiological studies have convincingly suggested that obesity is an important risk factor for postmenopausal breast cancer, but the mechanisms responsible for this relationship are still not fully understood. We hypothesize that obesity creates a low-grade inflammatory microenvironment, which stimulates  These immune cells, as well as enlarged adipocytes, are responsible for the production of pro-inflammatory cytokines such as IFN-γ, IL-1β, IL-6, IL-8, and TNF-α , 10. In Wnt/β-catenin cell signaling pathway is a critical regulatory pathway of tumorigenesis that controls cell proliferation, migration, and differentiation [APC) in the Wnt-signaling cascade is a major contributing factor for familial colorectal cancers [APC mutations and mutations in the β-catenin gene in breast cancer are very rare [Wnt-signaling abnormalities associated with 60% breast tumors [Wnt-signaling pathway may be due to mediators other than the mutation of Apc in the development of breast cancer.The ntiation , 12. In  cancers , but it ery rare , 15, dest tumors , 17. TheWnt-signaling, particularly in mammary tissue. Recently, data from our laboratory [GSK3β, a key component of the Wnt pathway in the colon, and this association is also manifested in an in vivo model of gastral cell lines [Wnt target genes in mammary tissue from women with a variety of BMI. We further demonstrated the causal role of TNF-α in the regulation of Wnt target gene expression in an explant culture of mammary tissue treated with anti-TNF-α antibody or TNF-α recombinant protein.To date, there are limited empirical data to delineate how obesity-promoted inflammation activates the pro-tumorigenic boratory  indicatell lines . In the Exp. I, 2 (range: 21 – 29), and 16 subjects had BMI ≥ 30 with an average of 36.0 kg/m2 (range 30–48). There were no differences in age between the two groups (57.2 vs 56.0y old respectively). In the second explant culture study , and 5 subjects had BMI ≥ 30 with an average of 36.3 kg/m2 (range 30–50). There was also no difference in age.As described in the following Materials and Methods section, due to the fact that subjects who underwent reduction mammoplasty were mainly obese, subjects were categorized into 2 groups: BMI ≥ 30 group and BMI < 30 group. In the first association study Exp. I, , of the Exp. II, , of the Exp. I, significantly higher concentrations (p < 0.05) of IL-1β, IL-6 and TNF-α were found in breast tissue samples from obese women  units increase in BMI, the protein level of IL-1β, IL-6 and TNF-a was significantly increased by 0.055 (p = 0.003), 0.495 (p < 0.001) and 0.0085 (p = 0.020) ng/mg of those cytokines, respectively  for those 26 tissue homogenates in n Figure . Linear y Figure .Wnt-signaling cascade, including 6 Wnt ligands and Wnt antagonists, 3 signaling transduction genes, and 7 Wnt downstream target genes, was measured in the 26 samples in Exp. I  and AXIN2 (p < 0.05), with marginal decrease of the expression for SFRP1 (p = 0.0578) and increase for JNK1 (p = 0.0582) for the individuals with BMI ≥ 30  with the expression SFRP1, as indicated by an increased DCt .When Pearson's correlation analyses were performed between IL-1β, IL-6 and TNF-α, the cytokines whose concentrations were identified to be altered in an obese state and those t Figure . In addiWnt-signaling, in the explant study (Exp. II), we treated fresh mammary tissues with anti-TNF-α antibody or TNF-α recombinant proteins. We assumed mammary tissue from obese women might have an elevated level of inflammation, whereas mammary tissue from women with BMI < 30 might have a low level of inflammation. Therefore, we treated samples from obese women with anti-TNF-α antibody but treated samples from women with BMI < 30 with TNF-a recombinant proteins, with the intention to detect the impact of TNF-α, which otherwise might not be detected if opposite treatments were applied. The expression of Wnt-signaling pathway downstream genes was subsequently meaured. We found that treatment with anti-TNF-α antibody for those mammary tissues from women with BMI ≥ 30 diminished the expression of CYCLIN D1 and COX2 (p < 0.05) in obese individuals, whereas treatment with TNF-α recombinant protein in samples from individuals with BMI < 30 significantly increased CYCLIN D1 expression (p < 0.05) and marginally increased AXIN2 expression (p = 0.082), but decreased P53 expression (p < 0.05)  . The chaWnt pathway downstream genes, which collectively indicates the elevation of Wnt-signaling by TNF-α, we further examined the accumulation of active β-CATENIN, a key indicator of the activation of the canonical Wnt-signaling pathway  with a tendency for upregulation of JNK1 (p = 0.0582). We also observed that the expression of SFRP1 was marginally increased (p = 0.0579) in obese subjects, but it significantly correlated (p < 0.05), in a negative fashion, with all 3 inflammatory cytokines, IL-1β, IL-6 and TNF-α, which were identified to be significantly increased in an obese state. C-MYC and CYCLIN D1 are generally considered as critical Wnt pathway downstream genes. We did not see alterations in C-MYC expression, but it is not surprising since C-MYC is also well-known to be influenced by a number of factors other than Wnt-signaling. For instance, C-MYC has been proposed as a putative target gene of signal transducer and activator of transcription 5 (STAT5) [AXIN2 is considered as a more direct target of the Wnt pathway and it is therefore a common readout for Wnt-signaling activation [Wnt-signaling target genes indicated its elevation in obese subjects in response to obesity-driven inflammation  . AXIN2 itivation , 32. In Wnt-signaling targets in mammary tissue, we examined one of the most critical inflammatory cytokines, TNF-α, a cytokine consistently elevated in obese individuals and also a known activator of the Wnt pathway [Wnt-signaling by anti-TNF-α antibody, but an upregulation of Wnt-signaling by TNF-α recombinant protein, as indicated the expression of Wnt pathway downstream genes and the accumulation of β-CATENIN , we examined the association between inflammatory cytokines and the expression of Wnt-signaling target genes in mammary tissue from women with age ≥ 50y and BMIs ranging from 21 to 48. Samples were selected from the tissue bank at the Biospecimen Resource and Molecular Analysis Facility in the Baystate Medical Center. The inflammatory cytokine profile and the expression of Wnt-signaling targets were measured using an electrochemiluminescence assay and real-time PCR respectively.In the first association study (ex vivo tissue culture study (Exp. II), we investigated the causal role of TNF-α in the regulation of Wnt-signaling using an explant culture of fresh mammary tissue collected from women who underwent elective breast reduction surgery. Mammary tissue was harvested and immediately minced and placed on Surgifoam gelatin sponges  in 60 mm tissue culture dishes containing 2 mL of whole organ culture medium and maintained for 24 h in 5% CO2. Tissues obtained from obese women (BMI < 30) were treated with 2.5 μg/ml anti-TNF-a antibody , whereas tissues from women with BMI < 30 were treated with TNF-α recombinant protein . Explant tissue was then flash frozen in liquid nitrogen and stored at −80°C for subsequent real-time PCR assay for Wnt pathway targets.In the second Exp. I) were collected and homogenized in a lysis buffer (10-20 mg of tissue per 400 μL of buffer) with phosphatase and protease inhibitors  added to inhibit degradation. The lysis buffer was prepared according to the protocol for the electrochemiluminescence assay . The lysate was made from a Tris lysis buffer consisting of 150 mM NaCl, 20 mM Tris, 1 mM EDTA, 1 mM EGTA and 1% Triton-X-100 with pH adjusted to 7.5. Samples were homogenized and centrifuged at 4°C at 12,000 rpm for 30min, and then the protein fraction was collected. The total protein concentrations were determined using a commercially available PierceTM BCA Protein Assay Kit  and the SpectraMax microplate reader . Six inflammatory cytokines, IL-1β, IL-2, IL-6, IL8, IFNγ and TNF-α, were measured using the QuickPlex SQ 120 imager . Assays were performed according to the manufacturer's instructions and cytokines are expressed as ng of cytokine per milligram of total protein. All standards and samples were measured in duplicate.A total of 26 mammary tissue samples . Primer sequences were listed in Supplementary Data , and cDNA was synthesized with SuperScript III . Real-time PCR was performed on the ViiAary Data .For immunoblot, protein were run on a SDS-PAGE gel and transferred onto PVDF membranes, which were then incubated with specific primary antibodies followed by secondary antibodies for detection. Anti-Active-β-catenin was purchased from EMD Millipore . The epitope corresponds to amino acid residues 36–44 of β-catenin antibody, and specifically recognizes active form of β-catenin with dephosphorylation on Ser37 or Thr41.p < 0.05 with a False Discovery Rate (FDR) cutoff of q < 0.25 applied. Pearson's correlation was performed to determine the association between expression of the Wnt pathway specific genes and inflammatory cytokines. For the analysis of the explant culture study (Exp. 2), a paired T-test was used, and also when a multiple comparison was performed, significance was accepted with p < 0.05 and a FDR cutoff of q < 0.25. The expression of each gene was normalized to the housekeeping gene GAPDH (ΔCt = Cttarget gene-CtGAPDH). Statistical analyses were performed based on differences in ΔCt between individuals with BMI < 30 and individuals with BMI ≥ 30 and relative expression is reported as 2−ΔΔCt, where ΔΔCt = ΔCtBMI≥30−ΔCtBMI<30.Data are expressed as means ± SEM, and statistical analysis was performed using SAS . Because the population of women undergoing reduction mammoplasty typically have high BMIs, we combined the normal (BMIs between 18–25) and the overweight (BMIs between 25–30) women together and grouped subjects into two categories: samples from individuals with a BMI < 30 or BMI ≥ 30. For the data analysis in the association study (Exp. I), a linear regression analysis was performed to examine the production of inflammatory cytokines in response to BMI. For the Wnt pathway gene expression profile, significance was accepted when


High production cost of bioplastics polyhydroxyalkanoates (PHA) is a major obstacle to replace traditional petro-based plastics. To address the challenges, strategies towards upstream metabolic engineering and downstream fermentation optimizations have been continuously pursued. Given that the feedstocks especially carbon sources account up to a large portion of the production cost, it is of great importance to explore low cost substrates to manufacture PHA economically.Escherichia coli was metabolically engineered to synthesize poly-3-hydroxybutyrate (P3HB), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB), and poly (PHBV) using acetate as a main carbon source. Overexpression of phosphotransacetylase/acetate kinase pathway was shown to be an effective strategy for improving acetate assimilation and biopolymer production. The recombinant strain overexpressing phosphotransacetylase/acetate kinase and P3HB synthesis operon produced 1.27 g/L P3HB when grown on minimal medium supplemented with 10 g/L yeast extract and 5 g/L acetate in shake flask cultures. Further introduction succinate semialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase, and CoA transferase lead to the accumulation of P3HB4HB, reaching a titer of 1.71 g/L with a 4-hydroxybutyrate monomer content of 5.79 mol%. When 1 g/L of α-ketoglutarate or citrate was added to the medium, P3HB4HB titer increased to 1.99 and 2.15 g/L, respectively. To achieve PHBV synthesis, acetate and propionate were simultaneously supplied and propionyl-CoA transferase was overexpressed to provide 3-hydroxyvalerate precursor. The resulting strain produced 0.33 g/L PHBV with a 3-hydroxyvalerate monomer content of 6.58 mol%. Further overexpression of propionate permease improved PHBV titer and 3-hydroxyvalerate monomer content to 1.09 g/L and 10.37 mol%, respectively.The application of acetate as carbon source for microbial fermentation could reduce the consumption of food and agro-based renewable bioresources for biorefineries. Our proposed metabolic engineering strategies illustrate the feasibility for producing polyhydroxyalkanoates using acetate as a main carbon source. Overall, as an abundant and renewable resource, acetate would be developed into a cost-effective feedstock to achieve low cost production of chemicals, materials, and biofuels.The online version of this article (10.1186/s12934-018-0949-0) contains supplementary material, which is available to authorized users. Food and agro-based renewable bioresources, such as xylose , glucoseEscherichia coli [Cryptococcus curvatus [Clostridium sp. [Halomonas boliviensis [Moorella thermoacetica from carbon dioxide [Acetate is the second simplest carboxylic acid, and many microorganisms can utilize acetate as alternative carbon source for cell growth. The acetate assimilation species include hia coli , Cryptoccurvatus , Clostridium sp. , Halomoniviensis , and so  dioxide , 17. In  dioxide , 19. In C. curvatus [Yarrowia lipolytica [E. coli fermentation, recombinant E. coli strains were constructed to use acetate for the production of free fatty acids [Oleaginous yeasts including curvatus  and Yarrpolytica  have beety acids  and succty acids . TherefoE. coli were engineered to produce poly-3-hydroxybutyrate (P3HB), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB), and poly (PHBV) using acetate as a main carbon source  medium at 37 °C and 200 rpm. LB medium contained 5 g/L yeast extract, 10 g/L Bacto tryptone, and 10 g/L NaCl. To maintain plasmid stability, 100 μg/mL ampicillin and/or 50 μg/mL kanamycin was added to the media when required.For construction of recombinants and plasmid amplification, ® High-Fidelity DNA Polymerase from New England Biolabs  was employed for PCR reactions. Plasmid isolation and DNA purification kits were purchased from Biomed . Restriction enzymes and DNA modifying enzymes were purchased from New England Biolabs.Strains and plasmids used in this study are listed in Table pta-ackA encoding phosphotransacetylase/acetate kinase and acs encoding acetyl-CoA synthetase were amplified from E. coli genomic DNA using primers pa1F/pa1R and acsF/acsR, respectively. The PCR products were purified and digested with HindIII/XbaI and inserted into the corresponding restriction sites of pEn to construct pEn-pta-ackA and pEn-acs, respectively. Moreover, Ppdc promoter and gene coding sequence were removed from pEn-derived plasmids and subcloned into kanamycin resistance carrying broad-host-range cloning vector pBBR1MCS-2 to generate pMCS-pta-ackA and pMCS-acs, respectively. Primers pa2F/pa2R were designed to amply pta-ackA from pEn-pta-ackA. The PCR product was digested with NheI/XhoI and cloned into the sucD-4hbD expression vector pMCSH5 cut with the same enzymes to form pMCSH5-pta-ackA. Primers prpPF/prpPR were designed to amply prpP encoding propionate permease from Ralstonia eutropha H16 genomic DNA. The PCR product was digested with BamHI/HindIII and cloned into pEn to generate pEn-prpP. Subsequently, Ppdc-prpP was excised from pEn-prpP using SpeI/XhoI and cloned into pMCS-pta-ackA digested with NheI/XhoI to generate pMCS-pta-ackA-prpP.The To prepare the seed cultures, 10 μL of glycerol stock was inoculated into 20 mL of LB medium and cultivated at 37 °C and 200 rpm for 12 h. For biopolymer producing shake flask studies, 4% (v/v) of the seed culture was used to inoculate into 500 mL shake flasks containing 50 mL of medium and cultured at 37 °C and 200 rpm for 48 h.phaCAB expression plasmid pBHR68, were co-transformed into E. coli JM109 to generate P3HB producing strains. Three different kinds of fermentation media were employed for P3HB production: LB, terrific broth (TB), and minimal medium (MM). TB medium contained 24 g/L yeast extract, 12 g/L Bacto tryptone, 2.31 g/L KH2PO4, and 12.54 g/L K2HPO4. MM medium comprised 2 g/L NH4Cl, 5.0 g/L (NH4)2SO4, 6.0 g/L KH2PO4, 8.4 g/L MOPS, 0.5 g/L NaCl, 0.24 g/L MgSO4, 0.002 g/L Na2MoO4 and 1 mL/L trace element solution. The trace elements solution contained (g/L): FeCl2·4H2O 3.6, CaCl2·2H2O 5.0, MnCl2·2H2O 1.3, CuCl2·2H2O 0.38, CoCl2·6H2O 0.5, ZnCl2 0.94, H3BO3 0.0311, Na2EDTA·2H2O 0.4, and thiamine-HCl 1.01. When necessary, MM medium was supplemented with a certain amount of yeast extract.The plasmids including pBBR1MCS-2, pMCS-pta-ackA, and pMCS-acs, each in combination with phaCAB-orfZ expression plasmid p68orfZ, were co-transformed into E. coli JM109SG to generate P3HB4HB producing strains. The resulting recombinants were cultivated in MM medium supplemented with 10 g/L yeast extract and 5 g/L acetate for P3HB4HB accumulation. When necessary, α-ketoglutarate or citrate was added as a second carbon source at a concentration of 1 g/L.Plasmids pMCSH5 and pMCSH5-pta-ackA, each together with pct-phaCAB expression plasmid p68-pct-CAB, were co-transformed into E. coli JM109 to generate PHBV producing strains. The resulting recombinants were cultivated in MM medium supplemented with 10 g/L yeast extract, 5 g/L acetate, and 1.5 g/L propionate for PHBV production.Plasmids pBBR1MCS-2, pMCS-pta-ackA, and pMCS-pta-ackA-prpP, each together with g for 10 min. The cell pellets were washed twice with distilled water and then lyophilized for 12 h for cell dry weight (CDW) measurement. Lyophilized cells were subjected to methanolysis at 100 °C for 4 h in the presence of 3% (v/v) H2SO4. Intracellular polymer content (wt%) and composition was analyzed using gas chromatography (GC) (Hewlett-Packard 6890) equipped with a capillary column HP-5  and a flame-ionization detector. PHBV and γ-butyrolactone purchased from Sigma-Aldrich  were used as analysis standards. Residual cell weight (rCDW) is defined as non-PHA cell mass. rCDW in each sample was determined by substracting the PHA concentration measured by GC analysis from CDW.Bacterial cells were harvested by centrifugation at 8000® HPX-87H, BioRad) and a refractive index detector . A mobile phase of 7 mM H2SO4 at a 0.6 mL/min flow rate was used.For acetate detection, the supernatant of culture broth was filtered through a 0.2-μm syringe filter and stored chilled for high-performance liquid chromatography (HPLC) analysis equipped with an ion exchange column  consumed all the acetate and yielded 1.49 g/L CDW with 0.22 g/L P3HB accumulation, which indicated that P3HB could be produced from acetate, yet the P3HB titer was much lower than that obtained from glucose [To investigate the possibility of P3HB production using acetate as a main carbon source in  glucose . TherefoE. coli, there are two potential routes for the generation of acetyl-CoA from acetate: phosphotransacetylase/acetate kinase (pta-ackA pathway) and AMP-forming acetyl-CoA synthetase (acs pathway)  consumed 5 g/L acetate and produced 0.66 g/L P3HB. The P3HB production titer of was roughly four times of that produced by control strain JM109 (pBHR68 + pBBR1MCS-2). By contrast, the overexpression of acs gene has little effect on improving P3HB accumulation  Fig. . These tynthesis , 33. It  pathway , 29. TheE. coli JM109 (pBHR68 + pMCS-pta-ackA) and control strain JM109 (pBHR68 + pBBR1MCS-2) were cultivated in shake flasks with LB, TB, and MM medium to evaluate the effects of medium composition on cell growth and P3HB accumulation  reached 1.03 g/L, and P3HB concentration was only 0.02 g/L. The CDW and P3HB accumulation increased gradually with the increase of yeast extract concentration, although the intracellular P3HB content did not change obviously. The addition of 10 g/L yeast extract yielded 3.02 g/L CDW, containing 1.27 g/L P3HB, which was the highest P3HB production titer obtained in this study. The production yield was 0.25 g P3HB/g acetate, which was 35% of the maximal theoretical yield. It has been shown that yeast extract contributes a large amount of amino acids for biomass growth, thus the addition of complex nitrogen sources such as yeast extract to defined medium saved NADPH for amino acids synthesis and increased intracellular NADPH level as well as NADPH/NADP ratio [E. coli [Next, different concentration  of yeast extract were added to MM medium to study its potential for enhancing cell growth and P3HB accumulation  using MM medium supplemented with 10 g/L yeast extract and different concentration  of acetate to evaluate the effect of acetate concentration on P3HB accumulation. As shown in Additional file We next cultivated co-4-hydroxybutyrate) (P3HB4HB) exhibits favorable biodegradability and a wide range of physical properties ranging from highly crystalline plastic to elastic rubber, thus it is considered to be one of the most promising PHA materials. The synthesis of P3HB4HB copolymer from structural unrelated carbon sources such as glucose in E. coli has been reported [E. coli that can produce P3HB4HB from acetate as carbon source.Poly when grown in mineral medium supplemented with 10 g/L yeast extract and 5 g/L acetate  yielded 2.96 g/L CDW, containing 58.01 wt% P(3HB-co-5.79 mol% 4HB) . Similarly, the addition of 1 g/L citrate as joint carbon source resulted in 3.62 g/L CDW, containing 59.48 wt% P(3HB-co-3.43 mol% 4HB). The addition of α-ketoglutarate and citrate as additional carbon sources increased the cell growth and P3HB4HB production, but had no obvious effect on improving 4HB monomer content.Previously, the addition of TCA cycle intermediates including α-ketoglutarate and citrate were found to be effective for increasing metabolic flux into 4HB precursor when glucose was employed as carbon source . TherefoE. coli metabolizes acetate as a sole carbon and energy source, the genes involved in acetate uptake, glyoxylate cycle, TCA cycle, and gluconeogenesis were all up-regulated [When egulated . The addco-3-hydroxyvalerate) (PHBV) is more flexible and tougher than P3HB homopolymer and has been commercially produced for many years [Megasphaera elsdenii propionyl-CoA transferase and P3HB synthesis operon phaCAB, was co-transformed with pMCS-pta-ackA into E. coli JM109. The resulting recombinant reached 2.64 g/L CDW, containing 12.52 wt% P(3HB-co-6.58 mol% 3HV)  under the same culture condition. PHBV production titer was increased from 0.15 to 0.33 g/L with pta-ackA overexpression, further demonstrating that pta-ackA overexpression was an effective strategy for improving acetate-dependent biopolymer synthesis.Poly(3-hydroxybutyrate-ny years . The norny years . The conE. coli harboring phaCAB operon and propionate permease (or propionyl-CoA synthase) failed to incorporate 3HV into the biopolymer in M9 medium containing 2 g/L yeast extract, 1 g/L sodium propionate, and 20 g/L sodium acetate [Previously, recombinant  acetate . HoweverprpP gene encoding propionate permease of R. eutropha H16 was overexpressed to study its effect on PHBV synthesis. E. coli JM109 (p68-pct-CAB + pMCS-pta-ackA-prpP) resulted in 3.15 g/L CDW, containing 34.77 wt% P(3HB-co-10.37 mol% 3HV)  increased gradually with the increase of corn steep liquor concentration, yet the P3HB production exhibit a trend of first increasing then decreasing. The addition of 4 g/L and 6 g/L of corn steep liquor to MM medium containing 5 g/L acetate yielded 0.91 and 0.96 g/L P3HB, respectively. Although 10 g/L corn steep liquor addition led to highest CDW among the different corn steep liquor concentration condition, the P3HB concentration was only 0.56 g/L. Therefore, the favorable corn steep liquor concentration for P3HB production was 4–6 g/L under our culture condition. These results indicated that P3HB can be synthesized when corn steep liquor is employed as a nitrogen source, albeit with somewhat lower efficiency in comparison with yeast extract. The use of corn steep liquor for PHA production from acetate therefore merits further research.Shake flask cultivations using various concentration  of corn steep liquor instead of yeast extract were performed Fig. . The CDWE. coli JM109 (pBHR68 + pMCS-pta-ackA) showed the highest P3HB accumulation of 0.04 g/L among all strains studied when cultivated in MM medium supplemented with 6 g/L CSL. The P3HB titer obtained without acetate was much lower than that achieved by acetate supplementation. These results further demonstrated that acetate was served as the main carbon source to support effective P3HB synthesis.In addition, we performed shake flask studies without acetate addition to evaluate the contribution of CSL to P3HB production. As shown in Additional file E. coli was engineered to synthesize P3HB, P3HB4HB, and PHBV using acetate as a main carbon source. The production of biopolymers was significantly improved by the overexpression of phosphotransacetylase and acetate kinase. In shake flask cultures, the engineered E. coli produced 1.27 g/L of P3HB and 1.71 g/L P(3HB-co-5.79 mol% 4HB), respectively, with minimal medium supplemented 10 g/L yeast extract and 5 g/L acetate. When 1 g/L citrate was added as assistant carbon source, P3HB4HB production titer was increased to 2.15 g/L. The overexpression of propionyl-CoA transferase and propionate permease lead to a production titer of 1.09 g/L P(3HB-co-10.37 mol% 3HV) when 5 g/L acetate and 1.5 g/L propionate were simultaneously supplied. To our knowledge, this is the first study reporting the efficient production of P3HB, P3HB4HB, and PHBV using acetate as a main carbon source by engineered E. coli.In this study, Additional file 1: Table S1. Oligonucleotides used in this study. Table S2. P3HB production by E. coli strains cultivated in MM medium supplemented with CSL. Figure S1. Effect of acetate concentration on cell growth and P3HB production.


Apart from active allergen avoidance, immunotherapy is regarded as the most effective form of treatment available for type I allergies. Such treatments involve the administration of allergen preparations in various forms and by various routes. Virus-like particles (VLPs) offer a very effective platform for immunization with the allergen and are characterized by high immunogenicity, low allergenicity and high clinical efficacy. Formulations that include Toll-like receptor ligands, T cell stimulatory epitopes and/or depot-forming adjuvants appear to enhance activation of the relevant immune cells. Short nucleotide sequences including CpG motifs have also been intensively explored as potent stimulators of dendritic cells and B cells.The present paper is based on a systematic literature search in PubMed and MEDLINE, and focuses on the pertinent immunological processes and on clinical data relating to use of VLPs and CpG motifs for the treatment of allergic rhinitis (AR).Many published studies have reported positive clinical results following administration of VLPs, either alone or in combination with CpG motifs and, in some cases, even in the absence of the allergen-specific allergen.These results indicate that VLPs modulate immune responses in ways which underline their exceptional promise as a platform for the immunotherapy of allergic disorders. However, clinical evaluations remain limited, and further large-scale and longer-term studies will be necessary to substantiate the efficacy and safety of these novel therapies. There has been a significant increase in the incidence of allergic disorders of the upper respiratory tract in recent decades, and these syndromes are now among the most common of all chronic illnesses in Europe –3. AccorSIT generally involves the repeated sublingual or subcutaneous application of an allergen extract in progressively increasing doses during the initial phase, followed by the regular administration of a sustaining dose, if necessary –7. This Since its inception, allergen-specific immunotherapy has developed in parallel with vaccination. In the early years of the 20th century, the effect of SIT was explicitly equated with that of vaccination against the toxins postulated to be present in pollen. Thus, Dunbar (1903) developed a hyperimmune serum in animals that had been vaccinated with pollen extracts and used this preparation (“Pollantin”), administered as nasal drops, to treat patients suffering from hay fever. Although these efforts might appear rather outlandish to us today, they were in fact well thought out and can be said to have anticipated the concept of blocking antibodies by over a century. Indeed, the analogy between SIT and vaccination has been revived in recent years . AgainstVirus-like particles (VLPs) are of particular interest as a possible basis for the development of an immunotherapy for the treatment of inhalation allergies –24.Pathogen-specific VLP-based vaccines have been shown to be well tolerated and immunologically effective for prophylactic vaccination against papillomavirus and hepatitis B virus and, most recently, against malaria, and are now in use in clinical practice –27.VLPs based on the coat protein of the bacteriophage Qbeta have been used as carriers of diverse target structures to raise antibodies directed against tumors and infectious agents . MoreoveThe recent review by Mohsen et al. provides an excellent summary of the rationale for the use of VLPs as antigen carriers in vaccines, discusses the optimal structural characteristics of VLPs as a platform to achieve the desired immunological effects, and provides an overview of the agonists of the Toll-like receptor family (TLT) members, which can be included in VLPs to enhance the immune response .Fig. Bacteriophage-based VLPs—packed with oligonucleotides enriched in CpG motifs (CpGs)—and displaying tumor antigens on their surfaces, have been shown to activate specific T cells in melanoma patients . SimilarIn this article, we therefore review clinical data on the use of VLPs and CpG motifs in the treatment of allergic rhinitis.The misregulation of the immune response to environmental antigens that triggers the development of allergies is characterized by the excessive (relative to the Th1-type) development of a Th2-type pattern of cytokines dominated by interleukins (IL)-4, -5, and -13, which results in the generation and activation of IgE-producing B lymphocytes and effector cells that generate an allergic inflammatory reaction . IL-17-pThe regulatory cytokines IL-10 and TGF-β suppress both the proliferation of effector T cells and the production of cytokines . They arThe normal immune response differs from its allergic counterpart in the relatively high numbers of allergen-specific Tregs with regard to the Th1 and Th2 cells . The proThe corresponding transcription factors therefore serve as reliable markers for each of the various T cell subpopulations . It is gIt is therefore conceivable that IL-4 reduces tolerance to antigens generally, and conversely that the counteracting Th1 or Treg population acts to promote tolerance to allergens .These immunological mechanisms also account for the actions of VLPs and CpG motifs in alleviating allergic disorders.Non-allergic immune responses can be induced either by the use of high concentrations of the appropriate antigen or another immunological trigger, such as an adjuvant or VLPs. For the induction of T cell responses, only the antigen’s binding site for the major histocompatibility complex (MHC) II is essential. Therefore, activation of immunity and tolerance by SIT at the level of the T cell could in principle be achieved using a mixture of peptides bearing T cell-specific epitopes . AllergyAs with conventional vaccinations, the efficacy of the intervention is strongly dependent on the ability of the inducing agent to trigger a potent and long-lasting induction of antibody production and T‑helper cells, immunological memory cells and a potent B cell response. This is where VLPs offer a potential advantage as immunogens in allergy vaccines , 38.Virus-like particles (VLPs) are nanoparticles that contain no viral RNA or DNA and are therefore incapable of replication. As a rule, VLPs are made up of proteins derived from capsids or coat proteins of either viruses or bacteriophages , 22, 27.Qb-VLPs are each made up of 180 identical copies of the capsid protein of the bacteriophage, and are of the same size and form as the native bacterial virus. VLPs spontaneously incorporate bacterial RNA during their assembly. The protein coat stabilizes the nucleic acids and protects them from enzymatic degradation. Moreover, these Qb-VLPs can be disassembled and reassembled in the presence of oligodeoxynucleotides (ODNs).+ T cells. Moreover, the size of VLPs, which is of the order of 30 nm, also permits their antigenic peptides to be presented by MHC Class I molecules, which leads to the induction of a CD8+ T cell response [The uptake of antigens by antigen-presenting cells (APCs) is strongly dependent on features such as the size, form, surface structure, hydrophobicity, hydrophilicity, and charge distribution, which all contribute to optimal interaction with the MHC-II receptor , 39. Theresponse , 44.Many APCs are dendritic cells (DCs), which can in turn be divided into several populations. Myeloid DCs (mDCs) mainly secrete IL-12, plasmacytoid DCs (pDCs) produce IFN-α, and CD8+ lymphoid DCs can induce the production of cytotoxic T lymphocytes –47. TLR Particular molecular structures found on the surfaces or in internal components of viruses and other microorganisms are among the most potent activators of immune cells in humans. Among these structures are unmethylated CpG oligodinucleotide motifs, which are commonly found in bacterial genomes but are relatively rare in the human genome, in which most CpG dinucleotides are methylated , 38.CpG oligodeoxynucleotides (CpG-ODN) constitute a class of synthetically produced single-stranded oligonucleotides, which contain a relatively high proportion of CG motifs. CG sequence motifs  rarely occur in the human genome: CG accounts for less than 2% of all dinucleotides in the human genome, and in many cases, the C in this context occurs in methylated form .In the genomes of bacteria and viruses, on the other hand, the CG dinucleotide is not only far more common, the cytosine is also predominantly found in the unmethylated state. In humans, the adaptive immune system recognizes unmethylated CG motifs in bacterial and viral nucleic acids by means of TLR9 , 38. Sin+ and CD4+ T cells in melanoma patients [CpG-ODNs have been used together with tumor antigens to stimulate the induction of specific CD8patients . CpGs hapatients , 31 and patients .Unmethylated CpG motifs that occur in oligonucleotides or longer DNA molecules bind to TLR9, thus, activating intracellular signal pathways that lead to the secretion of cytokines and chemokines, and upregulation of the expression of cell-surface receptors . The CpGThe CpG sequence motifs used as adjuvants normally consists of six bases, with the central CG sequence being flanked on either side by two outer bases.In type A CpG-ODNs, the CpG motifs form part of a palindromic sequence, which is bracketed by guanosines. Palindromes are sequences found within the same strand that can pair with one another via complementary bases. The nucleotides are typically linked by phosphodiester bonds and can therefore be degraded rapidly by DNases in vivo.Type B CpG-ODNs contain one or more CpG dinucleotides which are covalently linked exclusively by phosphorothioate bridges.Type C CpG-ODNs combine the features of A and B. They contain phosphorothioate bonds, as well as CpG motifs within palindromic sequences that are held together by phosphodiester linkages.Three types of CpG-ODN, termed A, B and C, have been described , 38:In tThe various types of CpG-ODNs differ in their patterns of activation. Type A primarily induces the synthesis of α‑interferon (IFN-α) in pDCs, while type B is a strong activator of B lymphocytes and triggers virtually no IFN-α response. Type C possesses both of these properties to some degree.In humans, TLR9 is found primarily in the endosomes of plasmacytoid DCs and B lymphocytes. Stimulation of TLR9 results in the production and secretion of diverse Th1-targeting cytokines and chemokines and induces an IgG class switch in B cells . TLR9 isa local Th1-mediated immune response which is associated with suppression of the allergic reaction driven by the Th2 response,production of IFN-α, which has the potential to convert the Th2 response into a Th1/Treg response in human PBMCs in vitro ,down-regulation of IgE synthesis, anda reduction in the activities of effector cells such as mast cells, basophils and eosinophils as a consequence of the stimulation of TLR9 .Based on the available experimental data, CpGs can be postulated to have several effects on the immune systems of allergy patients . They caOver the course of evolution, the human immune system has developed highly effective mechanisms that enable it to recognize and respond to highly repetitive molecular structures which are characteristic of pathogenic microorganisms such as viruses and bacteria. This is one reason why viruses and viral proteins serve as strong activators of the human immune system. VLPs mimic the repetitive character of the structure of virus coats, and they form spontaneously during the synthesis of specific viral capsid proteins. However, unlike true viruses, VLPs do not carry a viral genome and hence are not infectious. Normally they are very well tolerated and highly immunogenic , 56.Antigenic determinants displayed on the surface of VLPs are recognized by B cell receptors, and the particles themselves are efficiently taken up by antigen-presenting cells. Following intracellular processing, T cell epitopes present on the VLP coats are transported to the plasma membrane of the APC in association with MHC molecules and presented to interacting immune cells.One other property of VLPs makes them particularly suitable for use in allergy sufferers: Allergens that have been coupled to VLPs are unable to provoke anaphylactic reactions in allergenic individuals. This feature has been demonstrated experimentally for several VLP platforms recently , 57, 58 VLPs can play a dual role in allergy vaccination. First, they act as an adjuvant to facilitate antigen presentation and, second, they can help to dampen the Th2 response by enhancing Th1 polarization, which is important for sustained immunity to the allergen.VLPs are supramolecular particles with a diameter of between 25 and 100 nm . A key cThe lack of genetic information that would enable them to replicate and integrate into the genome of host cells increases the safety.They can be produced economically under standard GMP (Good Manufacturing Procedures) conditions.Owing to their form and size, they are quickly drained from the lymph nodes and can induce strong humoral immune responses.VLPs can transport and display virtually any antigen on their surfaces. Such antigens can be produced either by genetic fusion with the VLP coat protein (as chimeras) or by chemically attaching them to the surface of the particles (as coupled epitopes).Immunostimulatory agents can be packaged inside VLPs.Depot-forming adjuvants can be included to mimic the kinetics of natural viral infections , 22, 60.VLPs possess essentially the same key immunological features as real viruses and are just as immunogenic as pathogenic viruses without inducing any of the latter’s deleterious effects. This confers several important advantages on VLPs as immunotherapeutic agents.Over the past several years, CpG-containing oligonucleotides have been successfully employed to treat a range of clinically important conditions , 61–66. In the following, we discuss the results of four phase I clinical studies designed to assess the impact of VLPs and TLR ligands on allergic rhinitis.Kündig et al. conducted a randomized phase I study using RNA-loaded Qβ-VLPs coupled to a synthetic 16-amino-acid fragment of the house dust mite (HDM) allergen Der p 1 (Qβ-Der p 1). Like CpG motifs, RNA is recognized by TLRs, albeit by TLR7/8, not TLR9. The agent was administered in three doses to a group of 24 healthy subjects, and the antibody titer was monitored over the course of the following 18 months . The onlAmbrosia) [In another study, Creticos et al. tested a conjugate comprising the Amb a1 antigen linked to CpG-ODN on 25 patients who were allergic to ragweed (mbrosia) . In thisSenti et al. assessed the safety, tolerability and clinical efficacy of an intervention based on the use of type A CpGs delivered by Qβ-VLPs (QbG10) . The vacKlimek et al. have published the results of a randomized, double-blind, multicentric, placebo-controlled phase II study, in which they made use of the QbG10 VLPs loaded with type A CpG (CYT003-QbG10) that were employed in the trial conducted by Senti et al. . HoweverThe mechanism responsible for the immunological effect observed here is, however, not yet understood, and further investigations will be necessary to confirm the results of this proof-of-concept study. Based on the results of in vitro studies and various experiments in animals, it is thought that the induction of a potent Th1 immune response and the production of IFN-α described above blunts the Th2 response, which is overactive in allergy sufferers Fig. . FurtherThe significance of the VLP-specific B cell response for the positive clinical effects is still unclear. In mouse models, as well as in human subjects, VLPs can induce a B cell response in the absence of an additional adjuvant , 91. In Other VLP platforms make use of effectively universal T cell epitopes instead of CpGs. These novel constructs are now being developed for use against diseases such as Alzheimer’s and psoriasis, but also to treat persons who are allergic to cats .A vaccine designed for the treatment of peanut allergy is now undergoing preclinical development .VLPs, in general and in particular, combined with CpGs as adjuvants have been successfully employed for the treatment of infectious diseases and malignancies. Thanks to the immunomodulatory characteristics associated with their viral structure, they have also been used as therapeutic agents against allergic disorders—especially for treatment of allergic rhinitis and asthma. Indeed, VLPs loaded with CpGs appear to promote the reconstitution of a physiological immune response in allergy patients, although the underlying immunological mechanisms are not yet fully understood.Early clinical trials with various forms of VLP- and/or CpG-based vaccines have not only demonstrated that these agents are safe, but they have also been shown to have promising immunological and clinical effects . Large-scale controlled studies will, however, be necessary in order to learn more about the broader clinical properties of this new technology. Nevertheless, theoretically, the use of VLPs in combination with CpG motifs has great potential in the treatment of allergic patients—but also as a prophylactic measure in individuals who are at risk of developing allergies.


Dcaf17) is a member of DCAF family genes that encode substrate receptor proteins for Cullin-RING E3 ubiquitin ligases, which play critical roles in many cellular processes. To unravel the function of DCAF17, we performed expression profiling of Dcaf17 in different tissues of wild type mouse by qRT-PCR and generated Dcaf17 knockout mice by gene targeting. Expression profiling of Dcaf17 showed highest expression in testis. Analyses of Dcaf17 transcripts during post-natal development of testis at different ages displayed gradual increase in Dcaf17 mRNA levels with the age. Although Dcaf17 disruption did not have any effect on female fertility, Dcaf17 deletion led to male infertility due to abnormal sperm development. The Dcaf17−/− mice produced low number of sperm with abnormal shape and significantly low motility. Histological examination of the Dcaf17−/− testis revealed impaired spermatogenesis with presence of vacuoles and sloughed cells in the seminiferous tubules. Disruption of Dcaf17 caused asymmetric acrosome capping, impaired nuclear compaction and abnormal round spermatid to elongated spermatid transition. For the first time, these data indicate that DCAF17 is essential for spermiogenesis.DDB1– and CUL4–associated factor 17 ( Almost half of these infertility cases are attributed to male infertility, where about 75% of all the male factor infertility attributed to defective spermatogenesis5. Despite of advancement in the fields of biomedicine and genetics, the underlying causes of male infertility remain largely unknown in about 50% cases and they are categorized as idiopathic infertility cases1.Infertility is a global health problem that affects 15% of couples worldwide7. During spermiogenesis, many biochemical and structural changes necessary for the formation of acrosome, nucleus elongation, chromatin condensation, formation of flagellum and removal of cytoplasmic residual body are carried out in an intricate manner10. Normal spermatogenesis requires regulated protein homeostasis and disruption of such balance results in male infertility13.Mammalian spermatogenesis is a complex and highly regulated process that occurs within the seminiferous tubules, where the pluripotent spermatogonial stem cells proliferate through mitotic cell divisions and differentiate into primary spermatocytes. Primary spermatocytes then undergo two meiotic divisions to produce haploid round spermatids, which subsequently differentiate into highly specialized spermatozoa by a unique metamorphosis process called spermiogenesis16. In UPS, ubiquitin is covalently transferred to one or more lysine residues of the target protein through a series of enzymatic reactions involving ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligase enzyme (E3)18. E3 ubiquitin ligases (E3s) are diverse enzymes with modular, single protein or multi-protein complex structures that provide specificity toward a variety of substrates for the ubiquitination21. Cullin (CUL) 4 A and CUL4B, members of cullin-RING finger E3 ligase (CRL) sub-family, have been shown to play distinct and essential roles during spermatogenesis25. Both the proteins have displayed complementary expression patterns in adult mouse testis, where CUL4A is mainly present in meiosis-stage spermatocytes, while CUL4B is predominantly expressed in Sertoli cells, spermatogonia and spermatids24. Disruption of Cul4A caused male infertility because of the defective meiotic progression25. Whereas, the male infertility phenotype resulted from Cul4B deletion was due to abnormal post-meiotic sperm development23.The ubiquitin-proteasome system (UPS) plays an important role in numerous developmental processes, including spermatogenesis, by selective and timely protein turnover for proper cellular functions in eukaryotesDNA damage-binding protein 1), as an adaptor protein, which in turn recruits different substrate receptor proteins known as DDB1- and CUL4-associated factors (DCAFs)28. These DCAFs have been suggested to determine the substrate specificity in different CUL4-DDB1-based E3 ligase complexes27. Mutations in DCAF17 gene caused an extremely rare autosomal recessive disorder known as Woodhouse-Sakati syndrome (WSS) in human29. Patients with WSS display hypogonadism, alopecia, diabetes, mental retardation, deafness and extrapyramidal symptoms31. Male patients with DCAF17 mutations showed azoospermia with very less germ cell mass and large number of Sertoli cells in testicular biopsies31. However, the role of DCAF17 in the male reproductive system is not clear. To investigate the role of DCAF17 protein in mammalian spermatogenesis, we generated and characterized global Dcaf17 mutant mice. Our data show that Dcaf17 mutant male mice are infertile and the DCAF17 protein is required for normal spermiogenesis process and sperm functionality.CUL4 proteins interact with the E2 enzyme via the RING finger protein Hrt1/ROC1/Rbx1 at their C terminus. Whereas at their N terminus, they employ DDB1 . The Dcaf17 mRNA level at Day 5 testis was chosen as a calibrator and standardized to one. The 18S rRNA levels were used as endogenous control. The fold change in the Dcaf17 gene expression, normalized to 18S rRNA and relative to 5 dpp testis, is reported  mice were created by deleting exon 4 of the Dcaf17 using gene targeting approach  with concomitant premature stop codon after 59 residues  were viable and showed no gross abnormalities. Due to male infertility, the colony was maintained through heterozygous mating system and continuous pups’ genotyping.To investigate the role of the ach Fig. . The Dcaing Fig. . The Dcaice Fig. . DeletioPCR Fig.  and cDNAPCR Fig. . The cDNues Fig. . All theDcaf17 KO mice, controlled breeding experiments were carried out where adult WT and Dcaf17−/− males were individually mated with WT or Dcaf17−/− adult females. Mating behavior of WT and Dcaf17−/− was monitored by checking the ability to produce vaginal (copulatory) plugs in the female mice. All the females with WT or Dcaf17−/− genotype produced vaginal plugs after mating either with WT or Dcaf17−/− male mice, suggesting normal mating behavior among both the genotypes , suggesting that there is no effect of Dcaf17 deletion on female fertility in the mice. All the Dcaf17+/− heterozygous KO mice were fertile and produced comparable number of pups with that of WT mice (Data not shown). These data show that Dcaf17 homozygous KO male mice are infertile and the infertility phenotype did not result from abnormal sexual behavior but resulted from defective testicular function.Breeding three WT females and three WT males produced 46 pups in 13 litters with an average litter size of 3.54 pups  with 68% motility as compared to the WT mice of the same age that produced 10 ± 6.3 × 106 sperm with more than 90% motility  compared to that of 8 weeks Dcaf17 KO mice and 35 fold less sperm than their age-matched WT control. Eight months old WT showed about 1.4 fold sperm count reduction (7 ± 2.57 × 106) in comparison to their genotype counterpart at 8 weeks of age , which labels mitochondria; FITC-labeled lectin PNA (green), which labels acrosome; and DAPI (blue), which labels the nucleus  was observed  shaped head   Fig. . Interesent Fig. . In content Fig. . TogetheDcaf17 mutant spermatocytes progress properly through meiosis, we analyzed the expression and localization patterns of meiotic markers by immunolabeling of squashed preparation from WT and Dcaf17−/− adult mice testes. We used antibodies against axial element, SYCP3  and transverse filaments, SYCP1  to examine the formation of synaptonemal complex and phosphorylated histone H2AFX (γH2AX) antibody to detect DNA double strand breaks (DSBs) before the synaptonemal complex is fully established and the unsynapsed X and Y chromosomes during the process of meiosis36. The labeling patterns for SYCP3 and SYCP1 proteins, in Dcaf17−/− spermatocytes were indistinguishable from that in WT littermate spermatocytes . In WT testis, the manchette formation begins at step 8 of spermiogenesis, when elongation of the round spermatid starts by nucleus polarization and change of nucleus shape from spherical to slightly elongated  and cap phase (steps 4–7) of spermiogenesis between two genotypes, which is consistent with PAS staining of testes sections  were observed between WT and Dcaf17 mutant mice  of DCAF17 proteins during normal sperm development.In several studies, it was shown that the CUL4 ubiquitin ligases (CUL4A and CUL4B) play essential roles in male germ cell development. Both 27. Ubiquitination plays very important roles in regulation of different aspects of normal sperm development and testicular functions50. The CUL4A and CUL4B function as a scaffold protein for Cullin4-based E3 ligase complex assembly26. Due to the comparable similarities in the testicular phenotypes of Cul4A, Cul4B and Dcaf17 KO mice, we speculate that the function(s) of CUL4A, CUL4B and DCAF17 proteins may be interlinked for the regulation of normal spermatogenesis via ubiquitin mediated protein homeostasis. However, it is needed to be confirmed by appropriate experimental analyses.DCAF17 is a member of a DCAF family proteins that function as receptor protein for different Cullin4-based E3 ligase complexes and provides substrate specificity to the E3 ligase complex for protein ubiquitinationDcaf17 KO mice successfully characterized the male infertility phenotype caused due to Dcaf17 deletion and demonstrated the importance of DCAF17 protein in maintaining normal spermiogenesis. It has also provided an ideal model to further investigate the molecular mechanisms of DCAF17, E3 ligase complex components and their target proteins in sperm development and male fertility.In conclusion, our studies on Dcaf17 knockout (KO) mice were generated by inGenious Targeting Laboratory, Inc. . The CMV-Cre mice (B6.C-Tg(CMV-cre)1Cgn/J) were obtained from the Jackson Laboratory . Mice were housed in the animal facility at King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia, under controlled conditions with 12 hours light/dark cycles and free access to feed and water. All the experiments and animal procedures were approved by the Animal Care and Use Committee of the King Faisal Specialist Hospital and Research Centre (Protocol RAC# 210-0006), and executed according to institutional and international guidelines for the care of experimental animals.Conditional Dcaf17 gene in the mouse genome was carried out using gene targeting approach. Briefly, a 10.46 kb fragment containing exon 4 of Dcaf17 gene from Dcaf17-containing C57BL/6 BAC clone (RP23: 359E23) was subcloned to construct the targeting vector (pSP72 backbone). The FRT/loxP flanked Neo cassette was inserted upstream of exon 4 in intron 3–4 in an opposite direction in relation to the target gene. The single loxP site was inserted downstream of the exon 4 in intron 4–5 sequence. The target region was 930 bp long and included exon 4 of Dcaf17 gene. The region was designed to have a short homology arm (SA) extending 1.4 kb upstream of the LoxP/FRT-flanked pGK-gb2 Neo cassette and a long homology arm (LA) extending 8.06 kb to the 3′-end of the single lox P located downstream of the exon 4. The targeting construct was linearized by NotI restriction digestion and transfected into the BA1 (C57BL/6 × 129/SvEv) mouse embryonic stem cells (mESCs). The targeted mESC clones were selected for G418 antibiotic resistance. Positive mESC clones were screened by PCR using A1 or A2 forward primers and UNI reverse primer to confirm the short homology arm integration ). The Dcaf17wt/fl mice were back-crossed to generate homozygous mice for Dcaf17fl/fl, on a C57BL/6 background. The breeding was continued to establish the founder line. Genomic DNA from tail biopsies of offspring was genotyped for the presence of the Dcaf17fl/fl allele by PCR using LOX1 and LAN1 primers, and sequenced by SDL2 and LOX1 primers to confirm LoxP site retention 1Cgn/J) to remove the neomycin cassette and Dcaf17 exon 4. Cre-mediated deletion of Dcaf17 exon 4 was confirmed by performing PCR using WSS-F and WSS-R primers on tail DNA samples collected from the F1 pups as shown in Fig. Dcaf17+/− mice were crossed to generate Dcaf17−/− homozygous mutants.Disruption of ion Figs  and S2B.ion Figs  using thion Figs  and S2B.Dcaf17 exon 4, was performed by routine PCR. The primers used for genotyping are listed in Table Genomic DNA was isolated from tail biopsies using Gentra Puregene Mouse Tail Kit , according to manufacturer’s instructions. Genotype analysis, to confirm excision of Dcaf17−/−, were mated individually with sexually mature (8–10 weeks old) female mice of WT or Dcaf17−/− genotype (n = 3 to 5) in a controlled breeding experiment for the period of 2–6 months. Females were observed for the presence of vaginal plugs and signs of pregnancy. Litter numbers and pups produced by contorlled breeding were recorded.Sexually mature (8–10 weeks old) male mice (n = 3 to 5) of each genotypes, WT and Dcaf17−/− mice (n = 3) were dissected and collected in 1 ml of EmbryoMax Human Tubal Fluid (HTF) medium  pre warmed at 37 °C. The collected cauda epididymides were incised using fine scissors and sperms were allowed to swim out by incubating for 15–30 minutes at 37 °C under 5% CO2. Ten μl of sperm suspension medium was transferred to the hemocytometer and the total number of sperm was determined using a brightfield microscope . The total numbers of sperm were counted and the average was calculated from 5 microscopic fields per caudal epididymis. Sperm motility was evaluated immediately by placing a 10 μl drop of sperm suspension on hemocytometer and counting the sperm under a brightfield at 400× magnification. Sperm motility presented as percentage of motile sperm of total sperm number.The cauda epididymides from 8 weeks and 8 months old WT and To study the sperm morphology, sperm smears were prepared by pipetting 20 µl of cauda epididymides sperm suspension on glass slides. After drying the slide, the sperm smear was fixed with 2% paraformaldehyde (PFA) by incubating at room temperature for 10–15 minutes, washed three times with 1X PBS and stained with Diff-Quick stain  according to the manufacturer’s protocol. Diff-Quick stained slides were observed under optical microscope . Digital images were captured using OLYMPUS DP72 microscope camera  and OLYMPUS cellSens Entry 1.6 imaging software . Adobe Photoshop software  and Microsoft PowerPoint software were used to assemble images into figures. No post-acquisition modifications were made to the original images.Dcaf17 KO mice at different postnatal ages  as well as on epididymides of sexually mature (8 weeks old) mice (n = 3). The tissues were collected and directly fixed in 4% paraformaldehyde (PFA) or in Bouin’s fixative for overnight at 4 °C. Fixed tissues were washed with distilled water and dehydrated in a series of 70–100% ethanol solutions. Dehydrated tissues were embedded in paraffin and 5 µm thick sections were prepared on glass slides using microtome . Paraffin embedded tissue sections were deparaffinized, rehydrated and stained with hematoxylin and eosin (H&E) or periodic acid-Schiff (PAS) staining according to standard protocols52. Stained testis and epididymis sections were observed at room temperature using Olympus BX53  optical microscope. Digital images were captured using OLYMPUS DP72 microscope camera  and OLYMPUS cellSens Entry 1.6 imaging software . Adobe Photoshop software  and Microsoft PowerPoint software were used to assemble images into figures. No post-acquisition modifications were made to the original images.For the evaluation of spermatogenesis, histological analyses were performed on the testes of WT and 53. Briefly, the testes of adult WT and Dcaf17−/− mice were dissected and decapsulated by removing tunica albuginea. Seminiferous tubules of decapsulated testis were dispersed and fixed in freshly prepared 2% formaldehyde in phosphate-buffered saline (PBS) containing 0.05% Triton X-100  for 10 minutes at room temperature. Pieces (~1 mm) of seminiferous tubules were placed in a drop (~15 µl) of fixing solution on a clean glass slide. The pieces were gently minced with fine tip tweezers and were covered with glass cover slip. The cells were squashed by applying external pressure on coverslip. The slides were frozen by immersing in liquid nitrogen. The slides were immediately used for immunostaining or stored at −80 °C until required.Squash preparation of testicular samples was performed as previously described24. Briefly, the cauda epididymides were dissected from sexually mature (8–10 weeks old) WT and Dcaf17−/− mice (n = 3 for each genotype) and sperm were allowed to swim out as described before. Aliquots of sperm suspension were transferred to 1.5 ml centrifuge tubes and centrifuged at 500 × g for 10 minutes at room temperature. The HTF medium was removed and the sperm were incubated with MitoTracker solution  at 37 °C for 30 minutes. Sperm were collected by centrifugation and then fixed with 4% PFA, permeabilized in 0.2% Triton X-100  and a smear was prepared. Slides were then incubated with PNA-FITCI  (1:1000 dilution in PBS) at room temperature for 45 minutes. Slides were washed and mounted with VECTASHIELD mounting medium containing DAPI (1.5 µg/ml) .Evaluation of defects in sperm head and mid piece was carried out using MitoTracker, Peanut agglutinin (PNA) and 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) staining as described previouslyFor immunostaining of testes squash preparations, the slides were hydrated and washed in PBS. After washing in PBS, the slides were boiled for 20 min in 1× DakoCytomation Target Retrival Solution (pH 6.0)  using a microwave oven for antigen unmasking. The slides were then washed three times in PBS for 5 min each and blocked for non-specific antibody binding by incubating with 10% normal goat serum  in PBS for 1 h at room temperature. After blocking, the slides were washed with PBS and incubated overnight at 4 °C with appropriate primary antibodies diluted in 0.5% BSA and 0.1% Triton X100 in PBS. The following antibodies were used: mouse monoclonal anti-SYCP3  and rabbit polyclonal anti-SYCP1  at 1:200 dilution; rabbit polyclonal phospho-Histon H2A.x (ser139) antibody  at 1:500 dilution or mouse monoclonal alpha-tubulin (DM1A)  at 1:1000 dilution. Following the primary antibody incubation, the slides were washed with PBS (3 times 5 minutes each) and were further incubated in dark with appropriate secondary antibodies at room temperature for 1 hour. All secondary antibodies; FITC conjugated goat anti-rabbit-IgG (SC-2012), rodamin conjugated goat anti-rabbit-IgG (SC-2091), FITC conjugated goat anti-mouse-IgG (SC-2010), or rodamin conjugated goat anti-mouse-IgG (SC-2092) were purchased from Santa Cruz Biotechnology  and used at 1:300 dilution. After secondary antibody incubation, slides were washed in PBS, and mounted in VECTASHIELD mounting medium with DAPI (1.5 µg/ml) . For sperm and testes immunofluorescence analyses, three mice of each genotype were used. From each mouse, three slides were prepared. All the samples were observed at room temperature using a Zeiss Axio imager Z2 epifluorescence microscope  and digital images were captured with an Axiocam MRm microscope camera  and AxioVision imaging software ZEN2011. Adobe Photoshop software  and Microsoft PowerPoint software were used to assemble images into figures. No post-acquisition modifications were made to the original images.In Situ Cell Death Detection kit  to detect DNA fragmentation in apoptotic cells according to manufacturer’s recommended protocol. Briefly, testis paraffin sections from 8 weeks and 8 months old WT and Dcaf17−/− mice testes were deparaffinized with xylene, rehydrated in dilution series of ethanol, and permeabilized by incubating the sections with proteinase K  solution  for 15–30 minutes at room temperature. Following permeabilization, the tissue sections were washed twice in PBS and incubated with TUNEL reaction mixture for 60 minutes at 37 °C in the dark. Positive controls were treated with 3 U/ml Dnase I  solution  for 10 minutes before TUNEL labeling, while negative controls were treated with labeling solution without TdT enzyme. After TUNEL mixture incubation, sections were washed three times (5 minutes each) with PBS, and mounted in VECTASHIELD mounting medium . Three serial testicular sections (5 µm) each from three different mice of each WT and Dcaf17−/− genotypes were used for TUNEL assay. All the samples were observed under 20× objective at room temperature using a Zeiss Axio imager Z2 epifluorescence microscope  and digital images were captured with an Axiocam MRm microscope camera  and AxioVision imaging software ZEN2011. All the fluorescence images were acquired under the same exposure time (190.5 ms). The average number of TUNEL-positive cells per tubule was determined by analyzing 100 seminiferous tubules per animal. Adobe Photoshop software  and Microsoft PowerPoint software were used to assemble images into figures. No post-acquisition modifications were made to the original images.Terminal deoxynucleotidyl transferase (TdT) mediated dUTP-biotin nicked end-labeling (TUNEL) assay was performed by using Dcaf17 mRNA levels in different tissues and during testis development, adult WT mouse tissues  and 5, 14, 23, 32, 42 and 56 days postpartum (dpp) testes were collected and snap frozen immediately by immersing in liquid nitrogen. Total RNA was extracted using QIAGEN RNeasy mini kit and then treated with DNase I according to the manufacturer’s protocol . The RNA quality was checked by visualizing intact 28S and 18S RNA bands on agarose gel-electrophoresis using ethidium bromide staining. Quantification of total RNA was carried out using NanoDrop 2000c spectrophotometer . Reverse transcription was performed on 500 ng total RNA from each tissue using Superscript III Frist Strand Synthesis system  as per manufacturer’s protocol.To determine −ΔCt method and reported as fold change. Primers used for this experiment are listed in Table The qRT-PCR was performed using 5 µl of 1:5 diluted cDNA products as templates from different tissues in a 20 µl of total reaction mixture for each cDNA sample. The qPCR reaction mixture contained 10ul, SYBER Green Supermix  and 1 µl of 100 nM of each forward and reverse primer. For each sample, a parallel reaction was done using 18 s rRNA primers as endogenous control. Quantitative RT-PCR was carried out using C1000 Touch thermal cycler . Reaction steps were 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 30 sec, primer annealing at 58 °C for 30 sec, and primer extension at 72 °C for 30 sec, followed by melting curve analysis  to verify specificity of amplicons. All samples were analyzed in triplicate and 18 s rRNA was used as internal control for data normalization, which were analyzed by the 2Dcaf17−/− adult mice were collected, cut roughly into 1 mm cubes and immediately fixed in 2.5% glutaraldehyde solution prepared in 0.1 M PBS (pH 7.4) for 1 hour at room temperature. After glutaraldehyde fixation, tissues were rinsed twice 5 minutes each with 0.1 M PBS and post-fixed in 1% Osmium Tetroxide (OsO4) for 1 hour at room temperature. After the OsO4 treatment, tissues were washed with distilled water twice for 5 minutes each and pre-stained in 2% uranyl acetate for 30 minutes at room temperature. Tissues were then washed with distilled water for 5 minutes, dehydrated in a series of gradient acetone and embedded in Epon resin (Poly/BED 812). Semi-thin sections (0.5–0.7 µm) were cut using LEICA EM UC6 ultra microtome , stained with toluidine blue and inspected under the bright field microscope. The relevant areas were chosen and ultrathin (50–70 nm) sections were prepared with a diamond knife using LEICA EM UC6 ultra microtome. Sections were picked up on Gilder hexagonal mesh copper grids  and stained with uranyl acetate followed by lead citrate. Ultrathin sections were examined with JEM-1230 JEOL transmission electron microscope  operated at 60 kV. Digital images were acquired using KeenView Soft Imaging System camera  and iTEM imaging software .For the ultra-structural analysis, testes and epididymides of WT and P-value < 0.05 was considered statistically significant.All data from sperm count, motility, TUNEL assay and qRT-PCR were analyzed using Graphpad prism 5 software and presented as the mean ± standard error of the mean (SEM). All the experiments were performed in triplicate and were repeated three times. For each experiment, 3 to 5 animals were used. A one-way ANOVA or two-Ways ANOVA followed by post hoc Bonferroni’s multiple or Tukey’s multiple comparisons tests were performed to compare the data. A All data generated or analyzed during this study are included in this published article and its supplementary information files.Dataset 1


Listeria monocytogenes, Shigella flexneri, Rickettsia spp., and Burkholderia spp. disseminate within nonphagocytic cells, such as epithelial and endothelial cells, through a process referred to as cell-to-cell spread [A subset of intracellular pathogens, including l spread . These pPathogenic bacteria gain access to the host cell actin assembly machinery through bacterial engulfment in membrane-bound compartments termed primary vacuoles from which the bacteria escape through secretion of bacterial factors that challenge the integrity of the vacuole membrane . This inL. monocytogenes primary vacuole escape in human epithelial cells is facilitated by the production of the pore-forming toxin Listeriolysin O (LLO) [ O (LLO)  and phos O (LLO) .S. flexneri vacuole escape depends on the type-three secretion system (T3SS). Similar to L. monocytogenes LLO, the T3SS translocases, primarily IpaC, contribute to vacuole escape by forming pores in the vacuole membrane [S. flexneri vacuole escape [S. flexneri T3SS effector protein IpgD [Rickettsia spp. produce phospholipases that are implicated in vacuole escape; however, this mechanism is still unclear [Burkholderia spp. escape vacuoles through the activity of the T3SS [membrane ,6. The he escape . The recein IpgD , suggest unclear –11. Burkthe T3SS .Once in the cytosol, intracellular bacteria spread from cell to cell by first acquiring actin-based motility (ABM). In uninfected cells, actin polymerization relies on actin nucleators and their cognate regulators . In infeS. flexneri and L. monocytogenes, the actin-related protein ARP2/3 complex—a critical host cell actin nucleator—is recruited to the bacterial pole. S. flexneri secretes a bacterial autotransporter protein IcsA , whose activity recruits the ARP2/3 nucleation-promoting factor Wiskott−Aldrich Syndrome protein (N-WASP), and consequently ARP2/3, to the bacterial pole [In ial pole ,16.L. monocytogenes expresses a bacterial factor, ActA [By contrast, or, ActA , that reor, ActA  through or, ActA –21 and aRickettsia spp. encode RickA, an N-WASP mimic that recruits ARP2/3 to the bacterial surface and induces ABM [R. rickettsii produce Sca2, which is required for cell-to-cell spread and resembles actin nucleators of the formin family [uces ABM ,23. In an family . While Rn family . RickA in family .Listeria and Rickettsia, B. thailandensis BimA acts as a nucleator that activates Arp2/3. However, orthologs of BimA from pathogenic counterparts B. pseudomallei and B. mallei mimic actin nucleators of the host Ena/VASP family [Similarly to P family . SimilarP family .Cytosolic ABM allows intracellular pathogens to reach the plasma membrane at sites of cell−cell contacts, where they form membrane protrusions that project into adjacent cells . This prL. monocytogenes releases Tuba/N-WASP-mediated tension at cell−cell contacts by secreting Internalin C (InlC), which binds Tuba, thereby displacing N-WASP [R. parkeri effector protein Sca4 has been proposed to release tension by interfering with vinculin−α-catenin interactions, potentially creating unequal actomyosin tension at cell junctions and promoting bacterial spread. This mechanism has been shown to contribute to protrusion resolution [S. flexneri and Burkholderia spp. overcome membrane tension is unknown.g N-WASP . The R. solution . How S. L. monocytogenes cytosolic ABM, membrane protrusion formation relies on the AIP1/CFL1-dependent disassembly machinery [In addition to the ARP2/3-dependent actin assembly machinery required for achinery . The disachinery . Efficieachinery  and formachinery , whose fS. flexneri protrusions. In addition to ARP2/3, the host formins mDia1/2 localize to protrusions and are required for their proper formation [It is presumed that, similar to cytosolic ABM, IcsA and N-WASP/Arp2-3 are responsible for actin polymerization in ormation . Myosin-ormation .Rickettsia spp. ABM relies on the actin cytoskeleton, it was recently observed that R. parkeri protrusions uniquely lack actin tails, suggesting that the R. parkeri protrusion formation may not rely on the forces generated by actin assembly [Although assembly . This poB. pseudomallei and B. thailandensis infection [Burkholderia dissemination [Actin-containing membrane protrusions are formed during nfection ,35, but mination ,35. HoweDuring bacterial spread from cell to cell, membrane protrusions resolve into double membrane vacuoles (DMVs), whose inner and outer membranes are contributed by the primary infected cell and the adjacent cell, respectively . The forL. monocytogenes, the host AIP1/CFL1-dependent disassembly machinery is critical not only for the formation but also for the resolution of protrusions [L. monocytogenes spread in epithelial cells is unclear.In trusions . It was trusions . The bactrusions . An additrusions ; howeverS. flexneri, protrusions are resolved in a two-step process. The collapse of the protrusion neck, presumably due to the disassembly of the actin cytoskeleton network, results in the formation of intermediate structures termed vacuole-like protrusions (VLPs) [In s (VLPs) . Formatis (VLPs) ,39. The s (VLPs) , but theRickettsia effector protein Sca4 contributes to Rickettsia spp. protrusion resolution [Burkholderia spp. have been observed in protrusions, they have not been observed in DMVs [As mentioned above, the solution . Althoug in DMVs ; therefoIn order to resume ABM in the cytosol of adjacent cells, spreading bacterial pathogens must escape from the DMVs formed as a result of protrusion resolution. In contrast with primary vacuole escape, DMV escape is a complex process that requires the destabilization of two membranes .L. monocytogenes accomplishes DMV escape by using pore-forming toxins and enzymes that challenge the integrity of the vacuole membranes [embranes . Similarembranes .S. flexneri mutants lacking functional T3SS are trapped in DMVs, it was proposed that, similar to primary vacuole escape, the T3SS translocases may mediate DMV escape through pore formation [Because ormation . In addiormation . IcsB isormation , althougRickettsia spp. DMV escape is poorly understood, and whether Burkholderia spp. forms DMVs altogether is unknown.The mechanism supporting L. monocytogenes and S. flexneri and are now fairly well understood for Rickettsia spp. and Burkholderia spp. By contrast, further investigation will be required to uncover the mechanisms supporting the formation and resolution of protrusions resolution and DMV escape for most pathogens that spread from cell to cell.In conclusion and as shown in