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Nuclear Factor Y (NF-Y) gene family regulates numbers of flowering processes. Two independent transgenic Arabidopsis lines overexpressing (OX) GmNFY-B1 and GmNFYB1-GR (GmNFYB1 fused with the glucocorticoid receptor) were used to investigate the function of NFY-B1 in flowering. Furthermore, GmNFYB1-GR lines were chemically treated with dexamethasone (Dex, synthetic steroid hormone), cycloheximide (Cyc, an inhibitor of protein biosynthesis), and ethanol to examine their effects on different flowering related marker genes. Our results indicated that the transgenic lines produced longer hypocotyl lengths and had fewer numbers of rosette leaves compared to the wild-type and nf-yb1 mutant plants under both long and short-day (LD and SD) conditions. The qRT-PCR assays revealed that transcript levels of all flowering time regulating genes, i.e. SOC, FLC, FT, TSF, LFY, GI2, AGL, and FCA showed higher transcript abundance in lines OX GmNFYB1-GR. However, FT and GI genes showed higher transcript levels under Dex and Dex/Cyc treatments compared to Cyc and ethanol. Additionally, 24 differentially expressed genes were identified and verified through RNA-seq and RT-qPCR in GmNF-YB1-GR lines under Cyc and Dex/Cyc treatments from which 14 genes were up-regulated and 10 were down-regulated. These genes are involved in regulatory functions of circadian rhythm, regulation of flower development in photoperiodic, and GA pathways. The overexpression of GmNF-YB1 and GmNF-YB1-GR promote flowering through the higher expression of flowering-related genes. Further GmNF-YB1 and its attachment with the GR receptor can regulate its target genes under Dex/Cyc treatment and might act as flowering inducer under LD and SD conditions.

Tea plant is an important economic crop, which is used to produce the world's oldest and most widely consumed tea beverages. Here, we present a high-quality reference genome assembly of the tea plant (Camellia sinensis var. sinensis) consisting of 15 pseudo-chromosomes. LTR retrotransposons (LTR-RTs) account for 70.38% of the genome, and we present evidence that LTR-RTs play critical roles in genome size expansion and the transcriptional diversification of tea plant genes through preferential insertion in promoter regions and introns. Genes, particularly those coding for terpene biosynthesis proteins, associated with tea aroma and stress resistance were significantly amplified through recent tandem duplications and exist as gene clusters in tea plant genome. Phylogenetic analysis of the sequences of 81 tea plant accessions with diverse origins revealed three well-differentiated tea plant populations, supporting the proposition for the southwest origin of the Chinese cultivated tea plant and its later spread to western Asia through introduction. Domestication and modern breeding left significant signatures on hundreds of genes in the tea plant genome, particularly those associated with tea quality and stress resistance. The genomic sequences of the reported reference and resequenced tea plant accessions provide valuable resources for future functional genomics study and molecular breeding of improved cultivars of tea plants.

Hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyl transferase (HCT) is mainly associated with monolignol biosynthesis, a central precursor to producing guaiacyl and syringyl lignins in plants. However, the explicit regulatory mechanism of HCT-mediated monolignol biosynthesis in plants still remained unclear. Here, the genome-wide analysis of the HCT gene family in Carthamus tinctorius as a target for understanding growth, development, and stress-responsive mechanisms was investigated. A total of 82 CtHCT genes were identified and characterized. Most of the CtHCTs proteins demonstrated the presence of two common conserved domains, including HXXXD and DFGWG. In addition, the conserved structure of protein motifs, PPI network, cis-regulatory units, and gene structure analysis demonstrated several genetic determinants reflecting the wide range of functional diversity of CtHCT-encoding genes. The observed expression analysis of CtHCT genes in different flowering stages under normal conditions partially highlighted their putative roles in plant growth and development pathways. Moreover, CtHCT genes appeared to be associated with abiotic stress responses as validated by the expression profiling in various flowering phases under light irradiation and MeJA treatment. Altogether, these findings provide new insights into identifying crucial molecular targets associated with plant growth and development and present practical information for understanding abiotic stress-responsive mechanisms in plants.

Background & Aims Premenopausal women who are HCV positive (HCV+) have failing ovarian function, which is likely to impact their fertility. Thus, we investigated the reproductive history, risk of infertility, and pregnancy outcomes in women of childbearing age who were HCV+. Methods Three different groups were studied: (1) Clinical cohort: 100 women who were HCV+ and also had chronic liver disease (CLD), age matched with 50 women who were HBV+ with CLD and with 100 healthy women; all women were consecutively observed in three gastroenterology units in hospitals in Italy; (2) 1,998 women who were HCV+ and enrolled in the Italian Platform for the Study of Viral Hepatitis Therapies (PITER); (3) 6,085 women, who were mono-infected with HCV, and 20,415 women, who were HCV−, from a large de-identified insurance database from the USA. Measurements: total fertility rate (TFR) defined as the average number of children that a woman would bear during her lifetime. To define the reproductive stage of each participant, levels of anti-Müllerian hormone (AMH) and 17β-estradiol were measured. Results Clinical cohort: women who were either HCV+ or HBV+ had similar CLD severity and age at first pregnancy. Based on a multivariate analysis, women who were HCV+ had a higher risk of miscarriage than those who were HBV+ (odds ratio [OR] 6,905; 95% CI 1.771–26.926). Among women who were HCV+, incidence of miscarriage was correlated with median AMH level (1.0 ng/ml). Achieving a sustained virologic response (SVR) after antiviral treatment reduced the risk of miscarriage (OR 0.255; 95% CI 0.090–0.723). In the PITER-HCV cohort, miscarriage occurred in 42.0% of women (44.6% had multiple miscarriages). TFR for women who were HCV+ and between 15 and 49 years of age was 0.7 vs. 1.37 of Italian population of the same age range. In the US cohort: compared with women who were HCV−, women who were HCV+ positive were significantly more likely to have infertility (OR 2.439; 95% CI 2.130–2.794), premature birth (OR 1.34; 95% CI 1.060–1.690), gestational diabetes (OR 1.24; 95% CI 1.020–1.510), and pre-eclampsia (OR 1.206; 95% CI 0.935–1.556), and were less likely to have a live birth (OR 0.754; 95% CI 0.622–0.913). Conclusions Ovarian senescence in women of childbearing age who are HCV+ is associated with a lower chance of live birth, greater risk of infertility, gestational diabetes, pre-eclampsia and miscarriage. Such risks could be positively influenced by successful HCV cure. Lay summary Most new cases of HCV infection are among people who inject drugs, many of whom are young women in their childbearing years. Women of reproductive age who are HCV+ display markers of ovarian senescence. This is associated with an increased burden in terms of infertility and adverse pregnancy outcomes, including stillbirth, miscarriage, fewer live births, and gestational diabetes. Early viral suppression with therapy is likely to mitigate these risks.

It has come to our attention that the PITER framework investigator, Alessandro Federico, was incorrectly listed as F. Alessandro in the original manuscript. Please note that the correct name of this author is Alessandro Federico (2nd University of Naples). The correct list of PITER investigators is in the footnote below.

The N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) superfamily, specifically the SNAP25-type proteins and t-SNAREs, have been proposed to regulate cellular processes and plant resistance mechanisms. However, little is known about the role of SNAP25-type proteins in combating abiotic stresses, specifically in wild soybean. In the current study, the isolation and functional characterization of the putative synaptosomal-associated SNAP25-type protein gene GsSNAP33 from wild soybean ( Glycine soja ) were performed. GsSNAP33 has a molecular weight of 33,311 Da and comprises 300 amino acid residues along with Qb-Qc SNARE domains. Multiple sequence alignment revealed the highest similarity of the GsSNAP33 protein to GmSNAP33 (91%), VrSNAP33 (89%), PvSNAP33 (86%) and AtSNAP33 (63%). Phylogenetic studies revealed the abundance of SNAP33 proteins mostly in dicotyledons. Quantitative real-time PCR assays confirmed that GsSNAP33 expression can be induced by salt, alkali, ABA and PEG treatments and that GsSNAP33 is highly expressed in the pods, seeds and roots of Glycine soja . Furthermore, the overexpression of the GsSNAP33 gene in WT Arabidopsis thaliana resulted in increased germination rates, greater root lengths, improved photosynthesis, lower electrolyte leakage, higher biomass production and up-regulated expression levels of various stress-responsive marker genes, including KINI , COR15A , P5Cs , RAB18 , RD29A and COR47 in transgenic lines compared with those in WT lines. Subcellular localization studies revealed that the GsSNAP33-eGFP fusion protein was localized to the plasma membrane, while eGFP was distributed throughout whole cytoplasm of onion epidermal cells. Collectively, our findings suggest that GsSNAP33 , a novel plasma membrane protein gene of Glycine soja , might be involved in improving plant responses to salt and drought stresses in Arabidopsis .

Signal transducer and activator of transcription 3 (STAT3) is phosphorylated by various kinases, several of which have been implicated in aberrant fibroblast activation in fibrotic diseases including systemic sclerosis (SSc). Here we show that profibrotic signals converge on STAT3 and that STAT3 may be an important molecular checkpoint for tissue fibrosis. STAT3 signaling is hyperactivated in SSc in a TGFβ-dependent manner. Expression profiling and functional studies in vitro and in vivo demonstrate that STAT3 activation is mediated by the combined action of JAK, SRC, c-ABL, and JNK kinases. STAT3-deficient fibroblasts are less sensitive to the pro-fibrotic effects of TGFβ. Fibroblast-specific knockout of STAT3, or its pharmacological inhibition, ameliorate skin fibrosis in experimental mouse models. STAT3 thus integrates several profibrotic signals and might be a core mediator of fibrosis. Considering that several STAT3 inhibitors are currently tested in clinical trials, STAT3 might be a candidate for molecular targeted therapies of SSc.

ObjectivesJanus kinase 2 (JAK2) has recently been described as a novel downstream mediator of the pro-fibrotic effects of transforming growth factor-β. Although JAK2 inhibitors are in clinical use for myelodysplastic syndromes, patients often rapidly develop resistance. Tumour cells can escape the therapeutic effects of selective JAK2 inhibitors by mutation-independent transactivation of JAK2 by JAK1. Here, we used selective JAK2 inhibition as a model to test the hypothesis that chronic treatment may provoke resistance by facilitating non-physiological signalling pathways in fibroblasts.MethodsThe antifibrotic effects of long-term treatment with selective JAK2 inhibitors and reactivation of JAK2 signalling by JAK1-dependent transphosphorylation was analysed in cultured fibroblasts and experimental dermal and pulmonary fibrosis. Combined JAK1/JAK2 inhibition and co-treatment with an HSP90 inhibitor were evaluated as strategies to overcome resistance.ResultsThe antifibrotic effects of selective JAK2 inhibitors on fibroblasts decreased with prolonged treatment as JAK2 signalling was reactivated by JAK1-dependent transphosphorylation of JAK2. This reactivation could be prevented by HSP90 inhibition, which destabilised JAK2 protein, or with combined JAK1/JAK2 inhibitors. Treatment with combined JAK1/JAK2 inhibitors or with JAK2 inhibitors in combination with HSP90 inhibitors was more effective than monotherapy with JAK2 inhibitors in bleomycin-induced pulmonary fibrosis and in adTBR-induced dermal fibrosis.ConclusionFibroblasts can develop resistance to chronic treatment with JAK2 inhibitors by induction of non-physiological JAK1-dependent transactivation of JAK2 and that inhibition of this compensatory signalling pathway, for example, by co-inhibition of JAK1 or HSP90 is important to maintain the antifibrotic effects of JAK2 inhibition with long-term treatment.

An experiment was conducted to determine the effect of different levels of zinc on the yield and growth of cotton in the field of Agronomy section ARI, Tandojam during the Kharif Season 2014. Seeds of cotton were sown in rows 75 x 30 cm in row and plant spacing in soil with four replications in Randomized Complete Block Design. Six zinc levels i.e. untreated 0.0, 5.0, 7.5, 10.0, 12.5 and 15.0 kg ha -1 were evaluated. The results reveals that plant height, number of sympodia plant -1 , number of productive bolls plant -1 , fibre length, G.O.T (%) and seed cotton yield kg ha -1 affected significantly by the zinc levels, while plant population and number of monopodial branches were not affected. Application of zinc from 5.00 to 15.00 showed similar effect. However, control resulted different in taller plants (130.55 in), while application of 15.00 kg zn ha -1 produced maximum sympodia (16.35 plant -1 ) however productive bolls were more at 10.00 kg zn ha -1 (50.30 plant -1 ). The staple length was maximum (27.00 mm) at 7.5 kg zn ha -1 , while G.O.T% was greater (38.28%) at 5.00 kg zn ha -1 , whereas maximum seed cotton yield was recorded at 7.5 kg zn ha -1 (2556.70 kg ha -1 ). For the trait of seed cotton yield plant 1 , there was no any difference between applications of zinc sulphates 5.00 to 7.5kg ha 1 .

The core objective of instant study was to check the effectiveness of Aloe vera edible coating on postharvest life and physicochemical characteristics of eggplants under different storage temperatures. For this purpose, different formulations of Aloe vera based coating (non-poisonous) was applied at concentration of 0, 0.1%, 0.5%, and 1.0%, respectively on eggplants. The coated eggplants were stored at two different temperatures [10°C and (30±2)°C] and examined for weight loss, firmness, stem color, shriveling, total soluble solids, pH, acidity, vitamin C, sugar (total sugar, reducing sugar and non-reducing sugar) and N, P, K, Ca and Na for two weeks. The obtained results showed that weight loss, shriveling, total soluble solids, pH, sugar (total sugar and non-reducing sugar) increased and firmness, stem color, acidity, reducing sugar, vitamin C minimized during the storage period. The 0.5% Aloe coating at 10°C showed significant effect and delayed the changes in above parameters. Aloe vera coating remained almost ineffective in altering nutrient homeostasis (N, P, K, Ca and Na) of eggplants. The optimistic results gained in the current study could additionally investigate in larger market experiments and also could extensive to other tropical/subtropical fruits and vegetables.

Background Ebola hemorrhagic fever is caused by the Ebola filovirus (EBOV), which is one of the most aggressive infectious agents known worldwide. The EBOV pathogenesis starts with uncontrolled viral replication and subversion of both the innate and adaptive host immune response. The multifunctional viral VP35 protein is involved in this process by exerting an antagonistic action against the early antiviral alpha/beta interferon (IFN-α/β) response, and represents a suitable target for the development of strategies to control EBOV infection. Phage display technology permits to select antibodies as single chain Fragment variable (scFv) from an artificial immune system, due to their ability to specifically recognize the antigen of interest. ScFv is ideal for genetic manipulation and to obtain antibody constructs useful for targeting either antigens expressed on cell surface or intracellular antigens if the scFv is expressed as intracellular antibody (intrabody) or delivered into the cells. Results Monoclonal antibodies (mAb) in scFv format specific for the EBOV VP35 were isolated from the ETH-2 library of human recombinant antibodies by phage display technology. Five different clones were identified by sequencing, produced in E.coli and expressed in CHO mammalian cells to be characterized in vitro. All the selected scFvs were able to react with recombinant VP35 protein in ELISA, one of the scFvs being also able to react in Western Blot assay (WB). In addition, all scFvs were expressed in cell cytoplasm as intrabodies; a luciferase reporter gene inhibition assay performed in A549 cells showed that two of the scFvs can significantly hamper the inhibition of the IFN-β-induced RIG-I signaling cascade mediated by EBOV VP35. Conclusion Five antibodies in scFv format recognize an active form of EBOV VP35 in ELISA, while one antibody also recognizes VP35 in WB. Two of these scFvs were also able to interfere with the intracellular activity of VP35 in a cell system in vitro. These findings suggest that such antibodies in scFv format might be employed to develop therapeutic molecules able to hamper EBOV infections.

Additional file 3: Figure S7AIII. The treatment with the divalent scFvF7-Fc Ab increases the activation of peptide-stimulated Mart1-specific CD8+ T lymphocytes in terms of IFN-Îł secretion (Exp. II and Exp. III). For details see Legend of Fig. 7a. (PPTX 41 kb)

ObjectivesHedgehog signalling plays a critical role during the pathogenesis of fibrosis in systemic sclerosis (SSc). Besides canonical hedgehog signalling with smoothened (SMO)-dependent activation of GLI transcription factors, GLI can be activated independently of classical hedgehog ligands and receptors (so-called non-canonical pathways). Here, we aimed to evaluate the role of non-canonical hedgehog signalling in SSc and to test the efficacy of direct GLI inhibitors that target simultaneously canonical and non-canonical hedgehog pathways.MethodsThe GLI inhibitor GANT-61 was used to inhibit canonical as well as non-canonical hedgehog signalling, while the SMO inhibitor vismodegib was used to selectively target canonical hedgehog signalling. Furthermore, GLI2 was selectively depleted in fibroblasts using the Cre-LoxP system. The effects of pharmacological or genetic of GLI2 on transforming growth factor-β (TGF-β) signalling were analysed in cultured fibroblasts, in bleomycin-induced pulmonary fibrosis and in mice with overexpression of a constitutively active TGF-β receptor I.ResultsTGF-β upregulated GLI2 in a Smad3-dependent manner and induced nuclear accumulation and DNA binding of GLI2. Fibroblast-specific knockout of GLI2 protected mice from TBRact-induced fibrosis. Combined targeting of canonical and non-canonical hedgehog signalling with direct GLI inhibitors exerted more potent antifibrotic effects than selective targeting of canonical hedgehog signalling with SMO inhibitors in experimental dermal and pulmonary fibrosis.ConclusionsOur data demonstrate that hedgehog pathways and TGF-β signalling both converge to GLI2 and that GLI2 integrates those signalling to promote tissue fibrosis. These findings may have translational implications as non-selective inhibitors of GLI2 are in clinical use and selective molecules are currently in development.

Rice (Oryza sativa L.) is one of the most important cereals grown worldwide, mostly in warm regions. However, cold temperature is a major environmental factor that limits rice cultivation in temperate climates. γ-Aminobutyric acid (GABA), glutamic acid (Glu), and calcium chloride (CaCl2) play significant roles in key regulatory pathways throughout plant development. Here, we investigated the effects of Glu, CaCl2, and GABA in the culture medium on the cold tolerance of rice at the seedling stage. The medium components for cold tolerance of rice were optimized using response surface methodology (RSM). Plants treated with cold stress alone (without Glu, CaCl2, or GABA applications) displayed decreased weight, lower relative water content (RWC), maximum photochemical efficiency of PSII (F v/F m) and PSII efficiency, and higher relative electrolyte leakage (REL). However, after application of Glu, CaCl2, and GABA, the opposite results were documented, which could be due to an alleviation of cold-induced effects by restoration of membrane integrity. However, the levels of REL were considerably decreased due to Glu, CaCl2, and GABA treatment. RWC, F v/F m, PSII efficiency, and the average of subordinate functional values (ASFV) increased with the addition of Glu, CaCl2 and GABA. A central composite design indicated that the optimal concentrations of culture components for the cold tolerance of rice were 2.21 mg/mL, 2.94 mM and 2.68 mM of Glu, CaCl2, and GABA, respectively, and the maximal ASFV (0.987) was obtained under optimal conditions. Analysis of variance for the regression model suggested that the model can predict exactly the cold tolerance of rice seedlings, and the optimal culture components can be used to enhance the cold tolerance of rice seedlings.

The enzyme myo-inositol-1-phosphate synthase (MIPS EC 5.5.1.4) catalyzes the first step of myo-inositol biosynthesis, a product that plays crucial roles in plants as an osmoprotectant, transduction molecule, cell wall constituent and production of stress related molecule. Previous reports highlighted an important role of MIPS family genes in abiotic stresses particularly under salt stress tolerance in several plant species; however, little is known about the cellular and physiological functions of MIPS2 genes under abiotic conditions. In this study, a novel salt stress responsive gene designated GsMIPS2 from wild soybean Glycine soja 07256 was functionally characterized contained an open reading frame (ORF) of 1 533 bp coding a peptide sequence of 510 amino acids along with mass of 56 445 ku. Multiple sequence alignment analysis revealed its 92%-99% similarity with other MIPS family members in legume proteins. Quantitative real-time PCR results demonstrated that GsMIPS2 was induced by salt stress and expressed in roots of soybean. The positive function of GsMIPS2 under salt response at different growth stages of transgenic Arabidopsis was also elucidated. The results showed that GsMIPS2 transgenic lines displayed increased tolerance as compared to WT and atmips2 mutant lines under salt stress. Furthermore, the expression levels of some salt stress responsive marker genes, including KIN1, RD29A, RD29B, P5Cs and COR47 were significantly up-regulated in GsMIPS2 overexpression lines than wild type and atmips2 mutant. Collectively, these results suggested that GsMIPS2 gene was a positive regulator of plant tolerance to salt stress. This was the first report to demonstrate that overexpression of GsMIPS2 gene from wild soybean improved salt tolerance in transgenic Arabidopsis.

Germin-like proteins (GLPs) are ubiquitous plant glycoproteins (belonging to the cupin super family) that play diverse roles, including abiotic stress resistance in many plant species. To identify the molecular functions underlying abiotic stress responses, the expression of germin-like protein encoding genes of soybean GmGLPs was analyzed. qRT-PCR analyses of 21 GmGLPs transcripts abundances were conducted in soybean leave tissues. The results showed that GmGLPs transcripts were highly abundant upon treatments with high salinity, PEG6000, abscisic acid (ABA) and methyl viologen (MV). The peaks of transcript copiousness induced by PEG6000 and NaCl were mostly observed after 18 h, while some genes expressed earlier than 4 h after abiotic stress treatment. A specific GmGLP7 gene, that was highly abundant under salinity, drought, ABA and MV conditions, was further characterized. The ectopic overexpression of GmGLP7 (Glyma.08G226800.1) in transgenic Arabidopsis enhanced drought, salt, and oxidative tolerance and resulted in hypersensitive phenotypes toward ABA-mediated seed germination and primary root elongation, compared to the wild-type. Taken together, these results suggest that GmGLP7 positively confers abiotic tolerance in plants.

Human monoclonal antibodies are a powerful tool with increasingly successful exploitations and the single chain fragment variable format can be considered the building block for the implementation of more complex and effective antibody-based constructs. Phage display is one of the best and most efficient methods to isolate human antibodies selected from an efficient and variable phage display library. We report a method for the construction of a human naïve single-chain variable fragment library, termed IORISS1. Many different sets of oligonucleotide primers as well as optimized electroporation and ligation reactions were used to generate this library of 1.2×10(9) individual clones. The key difference is the diversity of variable gene templates, which was derived from only 15 non-immunized human donors. The method described here, was used to make a new human naïve single-chain fragment variable phage display library that represents a valuable source of diverse antibodies that can be used as research reagents or as a starting point for the development of therapeutics. Using biopanning, we determined the ability of IORISS1 to yield antibodies. The results we obtained suggest that, by using an optimized protocol, an efficient phage antibody library can be generated.

Size and variability often represent an obstacle in generating an effective antibody gene library for the detection of an abundant repertoire of antigens. Therefore, optimizing the construction of a large library is essential for the selection of high-affinity reactive fragments. Here, we report a highly efficient method for the construction of a human naïve antibody gene library for the selection of antibodies as single-chain variable fragments. This protocol is based on many different sets of oligonucleotide primers and multistep electroporation and ligation reactions.This advanced method can be adopted by any molecular biology laboratory to generate a naïve library for use in isolating single-chain fragment variables against specific targets.

ObjectivesSystemic sclerosis (SSc) fibroblasts remain activated even in the absence of exogenous stimuli. Epigenetic alterations are thought to play a role for this endogenous activation. Trimethylation of histone H3 on lysine 27 (H3K27me3) is regulated by Jumonji domain-containing protein 3 (JMJD3) and ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX) in a therapeutically targetable manner. The aim of this study was to explore H3K27me3 demethylases as potential targets for the treatment of fibrosis.MethodsJMJD3 was inactivated by small interfering RNA-mediated knockdown and by pharmacological inhibition with GSKJ4. The effects of targeted inactivation of JMJD3 were analysed in cultured fibroblasts and in the murine models of bleomycin-induced and topoisomerase-I (topoI)-induced fibrosis. H3K27me3 at the FRA2 promoter was analysed by ChIP.ResultsThe expression of JMJD3, but not of UTX, was increased in fibroblasts in SSc skin and in experimental fibrosis in a transforming growth factor beta (TGFβ)-dependent manner. Inactivation of JMJD3 reversed the activated fibroblast phenotype in SSc fibroblasts and prevented the activation of healthy dermal fibroblasts by TGFβ. Pharmacological inhibition of JMJD3 ameliorated bleomycin-induced and topoI-induced fibrosis in well-tolerated doses. JMJD3 regulated fibroblast activation in a FRA2-dependent manner: Inactivation of JMJD3 reduced the expression of FRA2 by inducing accumulation of H3K27me3 at the FRA2 promoter. Moreover, the antifibrotic effects of JMJD3 inhibition were reduced on knockdown of FRA2.ConclusionWe present first evidence for a deregulation of JMJD3 in SSc. JMJD3 modulates fibroblast activation by regulating the levels of H3K27me3 at the promoter of FRA2. Targeted inhibition of JMJD3 limits the aberrant activation of SSc fibroblasts and exerts antifibrotic effects in two murine models.

Size and variability often represent an obstacle in generating an effective antibody gene library for the detection of an abundant repertoire of antigens. Therefore, optimizing the construction of a large library is essential for the selection of high-affinity reactive fragments. Here, we report a highly efficient method for the construction of a human naive antibody gene library for the selection of antibodies as single-chain variable fragments. This protocol is based on many different sets of oligonucleotide primers and multistep electroporation and ligation reactions.This advanced method can be adopted by any molecular biology laboratory to generate a naive library for use in isolating single-chain fragment variables against specific targets.

The present experiment was laid out in randomized complete block design with three replications in order to estimate the genetic divergence, correlation and path analysis in ten upland cotton genotypes. The mean squares revealed highly significant differences (P

BackgroundActivating transcription factor 3 (ATF3), a member of the ATF/cAMP-responsive element binding (CREB) family of transcription factors, regulates cellular response to stress including oxidative stress. The aim of this study was to analyse the role of ATF3 in fibroblast activation in systemic sclerosis (SSc).MethodsATF3 was analysed by reverse transcription quantitative PCR, western blot and immunohistochemistry. ATF3 knockout fibroblasts and mice were used to study the functional role of ATF3. Knockdown experiments, reporter assays and coimmunoprecipitation were performed to study the effects of ATF3 on Smad and activation protein 1 (AP-1) signalling. The role of c-Jun was analysed by costaining, specific inactivation and coimmunoprecipitation.ResultsTransforming growth factor-β (TGFβ) upregulates the expression of ATF3 in SSc fibroblasts. ATF3-deficient fibroblasts were less sensitive to TGFβ, whereas ectopic expression of ATF3 enhanced the profibrotic effects of TGFβ. Mechanistically, ATF3 interacts with Smad3 directly on stimulation with TGFβ and regulates Smad activity in a c-Jun-dependent manner. Knockout of ATF3 protected mice from bleomycin-induced fibrosis and fibrosis induced by overexpression of a constitutively active TGFβ receptor I. Reporter assays and analyses of the expression of Smad target genes demonstrated that binding of ATF3 regulates the transcriptional activity of Smad3.ConclusionsWe demonstrate for the first time a key role for ATF3 in fibrosis. Knockout of the ATF3 gene reduced the stimulatory effect of TGFβ on fibroblasts by interfering with canonical Smad signalling and protected the mice from experimental fibrosis in two different models. ATF3 might thus be a candidate for molecular targeted therapies for SSc.

Light is one of the most important environmental factors for the growth and development of plants. To adapt to changes in day length, the photoreception and transmission of the light signals in plants mainly depend on the various light receptor proteins. The PAS/LOV protein (PLP) has a PAS domain in the N-terminal and LOV domain in the C-terminal and has been confirmed as a new type of blue light receptor in Arabidopsis thaliana. However, the role of its counterpart in soybean remains largely unclear. In this study, the expression pattern of the GmPLP1 under different light qualities was determined by real-time RT-PCR analysis using the cultivar 'DongNong 42', a photosensitive soybean cultivar, suggesting that GmPLP1 was affected by the circadian clock and was a dark-induced gene. Moreover, the mRNA abundance increased significantly under blue light. Further analysis revealed that overexpression of GmPLP1 displayed the inhibition of hypocotyl elongation under blue light, and the expression of CRY1, CRY2, CKL3, CKL4, BIT1, and HY5 were simultaneously increased in GmPLP1-transgenic Arabidopsis, suggesting that the shortened hypocotyl was associated with the up-regulation of these genes. Taken together, our results suggest that GmPLP1, which is a new possible type of blue light photoreceptor in soybean, plays an important role in the blue light signaling pathway.

Background Chronic and exaggerated fibroblast activation is a central hallmark of Systemic Sclerosis (SSc) fibrotic disease and results in a high morbidity and mortality. Epigenetic changes might play important roles in mediating chronic fibroblast activation. Trimethylation of H3 at lysine residue K27 (H3K27me3) is a repressive epigenetic mark that was recently identified as an important negative regulator of fibroblast activation [1]. Jumonji domain containing protein 3 (JMJD3) mediates H3K27me3–demethylation. JMJD3 inhibitors are being tested as therapeutic strategies in malignant diseases. Objectives The aim of this study was to characterize the role of JMJD3 in fibrotic disease and to explore JMJD3 as a potential drug target in SSc. Methods Expression analyses of JMJD3 were performed using qPCR, IF and Western blot. siRNA mediated knockdown and the pharmacologic H3K27me3-demethylase inhibitor GSKJ4 were used to target JMJD3. In vivo, we analyzed the effects of GSKJ4 in bleomycin-induced dermal fibrosis and in Topoisomerase-I-induced (TopoI) fibrosis. H3K27me3 levels at the Fra2 promotor were analyzed by CHIP. Results We observed increased expression of JMJD3 in SSc skin compared to healthy controls. Fibroblast-specific overexpression of JMJD3 was also reflected in experimental fibrosis models. TGFβ upregulated JMJD3. Inhibition of JMJD3 increased H3K27me3 in vitro and in vivo . Inhibition of JMJD3 reverted the activated fibroblast phenotype in SSc fibroblasts and decreased the expression of contractile fibers and of α-smooth muscle actin. In addition, JMJD3 inhibition reduced the basal and TGFβ induced collagen secretion of SSc fibroblasts. JMJD3 regulated the TGFβ induced expression of Fra2. GSKJ4 reverted the TGFβ induced reduction of H3K27me3 at the Fra2 promotor. Moreover, the anti-fibrotic effects of JMJD3 inhibition were evened in Fra2 knockout fibroblasts. Overexpression of Fra2 in JMJD3-knockdown fibroblasts restored the profibrotic effect of JMJD3. In vivo , inhibition of JMJD3 ameliorated fibrosis in bleomycin- and TopoI- induced experimental fibrosis and reduced dermal thickening, hydroxyproline content and myofibroblast differentiation. Conclusions We present first evidence that JMJD3 contributes to the activated phenotype of SSc fibroblasts. TGFβ upregulated JMJD3. Inhibition of JMJD3 prevented the aberrant activation of fibroblasts in vitro and ameliorated dermal fibrosis in several mouse models in vivo . The profibrotic effects of JMJD3 might be mediated by reducing the H3K27me3 at the Fra2 promotor and consecutive overexpression of Fra2. References Kramer, M., et al., Inhibition of H3K27 histone trimethylation activates fibroblasts and induces fibrosis. Ann Rheum Dis, 2013. 72(4): p. 614–20. Disclosure of Interest None declared

In the present study, the nuclear factor-Y subunit B (GmNFYB1) gene, induced by abscisic acid (ABA), 8% polyethylene-glycol (PEG) 6000, and NaCl, was cloned from soybean. GmNFYB1 is located in the ...

Carcinoembryonic antigen–related cell adhesion molecule 1 (CEACAM1) is a cell surface glycoprotein involved in intercellular binding, belonging to the immunoglobulin superfamily. It is involved in cell-cell recognition and modulates cellular processes that range from vascular angiogenesis to the regulation of insulin homeostasis and T-cell proliferation. Aberrant expression of CEACAM1 is often associated with progression and metastatic potential in melanoma, lung carcinoma and other types of tumor. Tumor-specific antigens such as CEACAM1 are ideal targets for cancer immunotherapy because they are over-expressed by the cancer cell and not on non-malignant tissues, minimizing the risk of autoimmune destruction. Many of the limitations of therapeutic use of rodent monoclonal antibodies (mAbs) can now be overcome by exploiting the use of recombinant antibody fragments and the advances in antibody engineering methods to improve tumor retention, reduce immunogenicity and modulate pharmacokinetics. In addition, a novel effective model of immunotherapeutic treatments of tumors includes antibody drug conjugates (ADCs) that combine specific mAbs and antibody fragments with cytotoxic drugs, proteins, enzymes, radionuclides and nanoparticles. This review aims to describe how these antibody engineering approaches can meet the challenges for generating new and effective antibody constructs for diagnosis and therapy of CEACAM1 expressing malignancies.

Systemic sclerosis (SSc) is a complex heterogeneous autoimmune disease with unknown etiology. It has three major pathologic hallmarks: vasculopathy, immune activation and extensive fibrosis that is an excessive accumulation of extracellular matrix proteins released from activated fibroblasts and destroys the physiological tissue architecture and function of the involved organs. Particularly, diffuse cutaneous SSc (dcSSc) has a highly increased risk of morbidity and mortality. Unfortunately, no targeted treatment is approved for fibrosis in SSc and there is a great medical need for new anti-fibrotic therapies for SSc. In this thesis, we evaluated the anti-fibrotic effects of targeting ATF3 and EN1 transcription factors. Both molecules have a distinct contribution to the disease’s pathogenesis and each one could serve as a potential target for the treatment of fibrosis in SSc. In the first part of this thesis, we evaluated the role of ATF3 in skin fibrosis as the most commonly affected tissue. Furthermore, we showed that ATF3 is a crucial regulator of TGF-β signaling in SSc. ATF3-deficient fibroblasts are less responsive to TGF-β with impaired production of extracellular matrix components such as collagens and activation of Smad-dependent transcription, reduced activity in reporter assays and decreased levels of TGF-β target genes. On the contrary, overexpression of ATF3 amplified the effect of TGF-β on fibroblast activation. Given that TGF-β stimulates the expression of ATF3 in a Smad3-dependent manner, ATF3 may represent an endogenous enhancer of canonical TGF-β signaling and its overexpression may thus contribute to the aberrant activation of TGF-β signaling in SSc. Furthermore, ATF3 deletion ameliorated fibrosis in several complementary mouse models of SSc. In the second part of this thesis, we investigated the role of EN1 in the pathogenesis of SSc. We demonstrated that EN1 regulates TGF-β signaling which is the core pathway in fibrotic diseases. Inactivation of EN1 signaling reduced the TGF-β mediated activation of fibroblasts and production of collagens and target genes. Moreover, fibroblast-specific knockout of EN1 ameliorated fibrosis in several mouse models. The results of both parts of the thesis might have clinical implications. Targeted inactivation of ATF3 and EN1 may thus offer potential to interfere with persistent TGF-β signaling and aberrant fibroblast activation in fibrotic diseases.

Systemic sclerosis (SSc) is a complex heterogeneous autoimmune disease with unknown etiology. It has three major pathologic hallmarks: vasculopathy, immune activation and extensive fibrosis that is an excessive accumulation of extracellular matrix proteins released from activated fibroblasts and destroys the physiological tissue architecture and function of the involved organs. Particularly, diffuse cutaneous SSc (dcSSc) has a highly increased risk of morbidity and mortality. Unfortunately, no targeted treatment is approved for fibrosis in SSc and there is a great medical need for new anti-fibrotic therapies for SSc. In this thesis, we evaluated the anti-fibrotic effects of targeting ATF3 and EN1 transcription factors. Both molecules have a distinct contribution to the disease’s pathogenesis and each one could serve as a potential target for the treatment of fibrosis in SSc. In the first part of this thesis, we evaluated the role of ATF3 in skin fibrosis as the most commonly affected tissue. Furthermore, we showed that ATF3 is a crucial regulator of TGF-β signaling in SSc. ATF3-deficient fibroblasts are less responsive to TGF-β with impaired production of extracellular matrix components such as collagens and activation of Smad-dependent transcription, reduced activity in reporter assays and decreased levels of TGF-β target genes. On the contrary, overexpression of ATF3 amplified the effect of TGF-β on fibroblast activation. Given that TGF-β stimulates the expression of ATF3 in a Smad3-dependent manner, ATF3 may represent an endogenous enhancer of canonical TGF-β signaling and its overexpression may thus contribute to the aberrant activation of TGF-β signaling in SSc. Furthermore, ATF3 deletion ameliorated fibrosis in several complementary mouse models of SSc. In the second part of this thesis, we investigated the role of EN1 in the pathogenesis of SSc. We demonstrated that EN1 regulates TGF-β signaling which is the core pathway in fibrotic diseases. Inactivation of EN1 signaling reduced the TGF-β mediated activation of fibroblasts and production of collagens and target genes. Moreover, fibroblast-specific knockout of EN1 ameliorated fibrosis in several mouse models.

Objective To determine the functions of in vitro primed Natural Killer (NK) cells in Human Immunodeficiency Virus (HIV-1) infection and the role of IL-2, IL-12 and IL-15 in enhancing the NK survival and activity in terms of viral suppression and of purging of HIV provirus. Methods Peripheral Blood Mononuclear Cells (PBMCs) and CD4+ T lymphocytes cells obtained from eight healthy donors were infected in vitro with HIV-1 and p24 was measured with and without IL-2, IL-12 and IL-15. We studied the effect of NK pulsed in vitro with IL-2, IL-12 and IL-15 on HIV replication by measurement of p-24 and DNA-provirus load when added into the culture of PBMCs and CD4+ T lymphocytes cells infected in vitro. We evaluated the effect of NK cells pulsed with IL-2, IL-12 and IL-15 on HIV replication and DNA-load into the culture of CD4+ T lymphocytes cells and PBMCs by trans-well chamber. Results We found high levels of p24 in the supernatants of PBMCs and CD4+ T lymphocytes cells cultured with IL-2, IL-12, and IL-15. We observed a significant reduction of p24 in the culture both of infected PBMCs and CD4+ T lymphocytes cells in which was added NK pulsed with IL-15. We did not obtain the some results with NK pulsed with IL-2 and IL-12. We observed a power effect of NK pulsed with IL-15 on HIV-DNA. The trans-well chamber experiments showed that the effect of NK is both direct and both mediated by realizing of soluble factors. Conclusions This study highlights some important effects of IL 15 on NK in HIV patients anyway our results are preliminary and descriptive and others studies will be needed to provide rationale for immune therapies.

The study was conducted to estimate the general combining ability (GCA) and specific combining ability (SCA) of wheat genotypes crossed in a line ´ tester fashion. The mean squares due to F1 hybrids, female lines, male testers/pollinators and lines ´ tester interaction were significant for majority of the characters studied. The significance of GCA and SCA variances thus suggested that both additive and non- additive genes were controlling majority of the characters, yet additive genes were more prominent because variances due to GCA by and large were higher than due to SCA. Among the three female lines evaluated, Khirman displayed maximum positive GCA effects for spike length (0.08) and seeds/spike (0.67), while other female lines which showed maximum positive GCA effects were Mehran for plant height (3.05), number of tillers/plant (1.00), spikelets/spike (1.92) and seed index (3.42) and Kiran for seeds/spike (0.67) and yield/plant (1.86). From the male testers, TD-1 exhibited greater GCA effects for number of tillers/plant (2.96), spikelets/spike (0.25), seed index (0.61) and yield/plant (2.22), whereas, Marvi displayed highest positive GCA effects for plant height (2.88), spike length (0.37) and seeds/spike (6.41). The specific combining ability estimates indicated, if hybrid crop development is feasible then, crosses Mehran ´ TD-1 for spike length; Kiran ´ TD-1 for plant height and seeds/spike and Khirman ´ Marvi for number of tillers/plant, spikelets/spike, seed index and yield/plant may be the hybrids of choice.