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In chronic lymphocytic leukemia (CLL), natural killer (NK) cells show a dysfunctional phenotype that correlates with disease progression. Our aim was to restore NK cell functionality in CLL through a specifically targeted IL15-stimulating activity; IL15 targeting could, in fact, potentiate the activity of NK cells and reduce off-target effects. We designed and developed a cis-acting immunocytokine composed of an anti-CD56 single-chain Fragment variable (scFv) and IL15, labeled scFvB1IL15. scFvB1IL15 was tested in vitro on peripheral blood mononuclear cells (PBMCs) obtained from both different healthy donors (HDs) and CLL patients in order to evaluate its ability to target NK cells and enhance their activation and NK-mediated directed cytotoxicity. scFvB1IL15 specifically induced strong degranulation and cytokine and chemokine production in NK cells in both HD- and CLL patient-derived PBMC samples. Furthermore, compared to IL15 alone, it was able to induce higher levels of NKG2D- and NKp30-activating receptors and restore NK-mediated direct killing in the CLL patient-derived samples. The preliminary data presented in this work suggest that IL15’s targeting of NK cells via scFvB1 potentiates the effects of IL15 and that scFvB1IL15 can be a useful agent for overcoming NK functional gaps and contribute to NK-cell-based immunotherapies.

Intravascular large B-cell Lymphoma (IVLBCL) is a rare subtype of extranodal non-Hodgkin’s lymphoma that is challenging to diagnose and has a poor prognosis. Here, we describe two patients newly diagnosed with IVLBCL at our institution: an African man with hemophagocytic-syndrome-associated IVLBCL and an Italian woman with a cutaneous variant of IVLBCL. They presented with very different clinical manifestations. Both cases were diagnosed in a timely manner, successfully treated, and achieved long-lasting remissions.

Abstract The ubiquitination pathway is involved in the posttranslational modification of cellular proteins. However, the role of E3 ubiquitin ligase family proteins under abiotic stress conditions remains unclear, particularly in soybean. The core objective of the current study was to isolate and functionally characterize the GsPUB8 protein gene from wild soybean (Glycine soja) by using a homologous cloning method to investigate its abiotic stress responses. The GsPUB8 is a 40,562 Da molecular weight protein with 373 amino acid residues. The sequence alignment revealed the presence of U-box domain while the phylogenetic analysis showed an abundance of PUB8 proteins in both monocot and dicot plants. Analysis of gene structure predicted the absence of introns along with the presence of one exon. Furthermore, the activity of the GsPUB8 protein was anticipated in the plasma membrane and its expression was persuaded with NaCl, ABA, PEG6000, and NaHCO3 treatments with considerably higher manifestation in roots than leaves although, expressed in both vegetative and reproductive parts of G. soja. GsPUB8 protein showed 54% and 32% sequence identity to U-box domain containing 8 and 12 proteins from Arabidopsis thaliana and Oryza sativa subsp. japonica, respectively. GsPUB8 exhibited relatively higher expression under saline and drought stress particularly in roots. Whereas, the 3D model of GsPUB8 protein was generated using the SWISS-MODEL. This study can be used to manipulate the GsPUB8 protein or GsPUB8 gene for transformation purposes and its functional characterization under abiotic stress conditions.

In chronic lymphocytic leukemia (CLL), natural killer (NK) cells show a dysfunctional phenotype that correlates with disease progression. Our aim was to restore NK cell functionality in CLL through a specifically targeted IL15-stimulating activity; IL15 targeting could, in fact, potentiate the activity of NK cells and reduce off-target effects. We designed and developed a cis-acting immunocytokine composed of an anti-CD56 single-chain Fragment variable (scFv) and IL15, labeled scFvB1IL15. scFvB1IL15 was tested in vitro on peripheral blood mononuclear cells (PBMCs) obtained from both different healthy donors (HDs) and CLL patients in order to evaluate its ability to target NK cells and enhance their activation and NK-mediated directed cytotoxicity. scFvB1IL15 specifically induced strong degranulation and cytokine and chemokine production in NK cells in both HD- and CLL patient-derived PBMC samples. Furthermore, compared to IL15 alone, it was able to induce higher levels of NKG2D- and NKp30-activating receptors and restore NK-mediated direct killing in the CLL patient-derived samples. The preliminary data presented in this work suggest that IL15's targeting of NK cells via scFvB1 potentiates the effects of IL15 and that scFvB1IL15 can be a useful agent for overcoming NK functional gaps and contribute to NK-cell-based immunotherapies. [ABSTRACT FROM AUTHOR]

Tea is one of the most consumed and widely planted beverage plant worldwide, which contains many important economic, healthy, and cultural values. Low temperature inflicts serious damage to tea yields and quality. To cope with cold stress, tea plants have evolved a cascade of physiological and molecular mechanisms to rescue the metabolic disorders in plant cells caused by the cold stress; this includes physiological, biochemical changes and molecular regulation of genes and associated pathways. Understanding the physiological and molecular mechanisms underlying how tea plants perceive and respond to cold stress is of great significance to breed new varieties with improved quality and stress resistance. In this review, we summarized the putative cold signal sensors and molecular regulation of the CBF cascade pathway in cold acclimation. We also broadly reviewed the functions and potential regulation networks of 128 cold-responsive gene families of tea plants reported in the literature, including those particularly regulated by light, phytohormone, and glycometabolism. We discussed exogenous treatments, including ABA, MeJA, melatonin, GABA, spermidine and airborne nerolidol that have been reported as effective ways to improve cold resistance in tea plants. We also present perspectives and possible challenges for functional genomic studies on cold tolerance of tea plants in the future.

Using silicate fertilizer and bacterial inoculum as biofertilizer is significant for increasing soil silicon (Si) availability and rice agronomic performance. To use microbial technology for sustainable agriculture, it is crucial to have a deeper knowledge of how microbial populations shift among the plant hosts and related compartments, as well as how they respond to various fertilization models. In this study, the effects of silicate fertilizer, a single bacterial strain Bacillus mucilagniosis as biofertilizer, and their integrated application on soil physiochemical properties and soil microbiota structure, composition, and diversity in two eco-geographically diverse races (Indica and Japonica rice) were evaluated. Plant compartment, cultivar type, and fertilizer treatments contributed to microbiome variation. Indica and Japonica harbor different root microbiota; notably, taxa enriched in the rhizosphere soil were more diverse than in the root. Bacterial genera Leptonema, Azospira, Aquabacterium, Fluviicola, Aquabacterium, Leptonema, and fungal genera Metarhizium, Malassezia, and Cladosporium all were found in the rice core microbiome. Both silicate and biofertilizer applications increase the relative abundance of Betaproteobacteria, Deltaproteobacteria, and Actinobacteria, while suppressing fungal pathogens Alternaria and Fusarium. Silicate and bacterial inoculum applications increased the soil pH, available silicon content (ASi), available phosphorous (AP), available potassium (AK), and organic carbon (OC), while reduced the total nitrogen (N). These changes were also associated with major bacterial phyla Spirochaetes, Bacteroidetes, Actinobacteria, and Proteobacteria, except for Acidobacteria, and fungal phyla Ascomycota, Mortierellomycota and unassigned fungi. Several treatment-specific biomarkers were revealed through Linear discriminant analysis effect size (LEfSe) analysis. In conclusion, the change in the structure of root-associated communities driven by plant compartment and genetics suggests dynamic interactions in the host plant microbiome. Short-term silicate and biofertilizer amendments improved soil physiochemical status and altered bacterial and saprotrophic fungal communities, which have important implications for sustainable rice production.

BackgroundMacrophages are key targets of HIV-1 infection. We have previously described that the expression of CC chemokine ligand 2 (CCL2) increases during monocyte differentiation to macrophages and it is further up-modulated by HIV-1 exposure. Moreover, CCL2 acts as an autocrine factor that promotes viral replication in infected macrophages. In this study, we dissected the molecular mechanisms by which CCL2 neutralization inhibits HIV-1 replication in monocyte-derived macrophages (MDM), and the potential involvement of the innate restriction factors protein sterile alpha motif (SAM) histidine/aspartic acid (HD) domain containing 1 (SAMHD1) and apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) family members.ResultsCCL2 neutralization potently reduced the number of p24 Gag+ cells during the course of either productive or single cycle infection with HIV-1. In contrast, CCL2 blocking did not modify entry of HIV-1 based Virus Like Particles, thus demonstrating that the restriction involves post-entry steps of the viral life cycle. Notably, the accumulation of viral DNA, both total, integrated and 2-LTR circles, was strongly impaired by neutralization of CCL2. Looking for correlates of HIV-1 DNA accumulation inhibition, we found that the antiviral effect of CCL2 neutralization was independent of the modulation of SAMHD1 expression or function. Conversely, a strong and selective induction of APOBEC3A expression, to levels comparable to those of freshly isolated monocytes, was associated with the inhibition of HIV-1 replication mediated by CCL2 blocking. Interestingly, the CCL2 neutralization mediated increase of APOBEC3A expression was type I IFN independent. Moreover, the transcriptome analysis of the effect of CCL2 blocking on global gene expression revealed that the neutralization of this chemokine resulted in the upmodulation of additional genes involved in the defence response to viruses.ConclusionsNeutralization of endogenous CCL2 determines a profound restriction of HIV-1 replication in primary MDM affecting post-entry steps of the viral life cycle with a mechanism independent of SAMHD1. In addition, CCL2 blocking is associated with induction of APOBEC3A expression, thus unravelling a novel mechanism which might contribute to regulate the expression of innate intracellular viral antagonists in vivo. Thus, our study may potentially lead to the development of new therapeutic strategies for enhancing innate cellular defences against HIV-1 and protecting macrophages from infection.

Continuous cropping systems are the leading cause of decreased soil biological environments in terms of unstable microbial population and diversity index. Nonetheless, their responses to consecutive peanut monocropping cycles have not been thoroughly investigated. In this study, the structure and abundance of microbial communities were characterized using pyrosequencing-based approach in peanut monocropping cycles for three consecutive years. The results showed that continuous peanut cultivation led to a substantial decrease in soil microbial abundance and diversity from initial cropping cycle (T1) to later cropping cycle (T3). Peanut rhizosphere soil had Actinobacteria, Protobacteria, and Gemmatimonadetes as the major bacterial phyla. Ascomycota, Basidiomycota were the major fungal phylum, while Crenarchaeota and Euryarchaeota were the most dominant phyla of archaea. Several bacterial, fungal and archaeal taxa were significantly changed in abundance under continuous peanut cultivation. Bacterial orders, Actinomycetales, Rhodospirillales and Sphingomonadales showed decreasing trends from T1>T2>T3. While, pathogenic fungi Phoma was increased and beneficial fungal taxa Glomeraceae decreased under continuous monocropping. Moreover, Archaeal order Nitrososphaerales observed less abundant in first two cycles (T1&T2), however, it increased in third cycle (T3), whereas, Thermoplasmata exhibit decreased trends throughout consecutive monocropping. Taken together, we have shown the taxonomic profiles of peanut rhizosphere communities that were affected by continuous peanut monocropping. The results obtained from this study pave ways towards a better understanding of the peanut rhizosphere soil microbial communities in response to continuous cropping cycles, which could be used as bioindicator to monitor soil quality, plant health and land management practices.

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.

Abstract Background Lymphocyte-activation gene (LAG)3 is a 498 aa transmembrane type I protein acting as an immune inhibitory receptor. It is expressed on activated lymphocytes, natural killer cells and plasmacytoid dendritic cells. In activated lymphocytes, LAG3 expression is involved in negative control of cell activation/proliferation to ensure modulation and control of immune responses. In view of its deregulated expression in tumor-infiltrating lymphocytes, LAG3, together with the additional immune checkpoint inhibitors CTLA4 and PD1, is considered a major target in order to reverse the immunosuppression typically mounting in oncologic diseases. Since many patients still fail to respond to current immune checkpoints-based therapies, the identification of new effective immune inhibitors is a priority in the ongoing fight against cancer. Results We identified a novel human single-chain variable fragment (scFv) Ab against a conformational epitope of LAG3 by in vitro phage display technology using the recombinant antigen as a bait. This scFv (referred to as F7) was characterized in terms of binding specificity to both recombinant antigen and human LAG3-expressing cells. It was then rebuilt into an IgG format pre-optimized for clinical usage, and the resulting bivalent construct was shown to preserve its ability to bind LAG3 on human cells. Next, we analyzed the activity of the anti-LAG3 scFvF7 using two different antigen-specific CD8+ T lymphocyte clones as target cells. We proved that the reconstituted anti-LAG3 F7 Ab efficiently binds the cell membrane of both cell clones after peptide-activation. Still more significantly, we observed a striking increase in the peptide-dependent cell activation upon Ab treatment as measured in terms of IFN-γ release by both ELISA and ELISPOT assays. Conclusions Overall, the biotechnological strategy described herein represents a guiding development model for the search of novel useful immune checkpoint inhibitors. In addition, our functional data propose a novel candidate reagent for consideration as a cancer treatment.

Abstract 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.

STAT3 is a transcription factor that is activated in fibrotic diseases such as systemic sclerosis. Here the authors show that STAT3 is the converging point for multiple pro-fibrotic signalling pathways, and that its genetic ablation or inhibition ameliorate skin fibrosis in mouse models.

During HIV infection the severe depletion of intestinal CD4+ T-cells is associated with microbial translocation, systemic immune activation, and disease progression. This study examined intestinal and peripheral CD4+ T-cell subsets reconstitution under combined antiretroviral therapy (cART), and systemic immune activation markers.This longitudinal single-arm pilot study evaluates CD4+ T cells, including Th1 and Th17, in gut and blood and soluble markers for inflammation in HIV-infected individuals before (M0) and after eight (M8) months of cART. From January 2010 to December 2011, 10 HIV-1 naïve patients were screened and 9 enrolled. Blood and gut CD4+ T-cells subsets and cellular immune activation were determined by flow-cytometry and plasma soluble CD14 by ELISA. CD4+ Th17 cells were detected in gut biopsies by immunohistochemistry. Microbial translocation was measured by limulus-amebocyte-lysate assay to detect bacterial lipopolysaccharide (LPS) and PCR Real Time to detect plasma bacterial 16S rDNA.Eight months of cART increased intestinal CD4+ and Th17 cells and reduced levels of T-cell activation and proliferation. The magnitude of intestinal CD4+ T-cell reconstitution correlated with the reduction of plasma LPS. Importantly, the magnitude of Th17 cells reconstitution correlated directly with blood CD4+ T-cell recovery.Short-term antiretroviral therapy resulted in a significant increase in the levels of total and Th17 CD4+ T-cells in the gut mucosa and in decline of T-cell activation. The observation that pre-treatment levels of CD4+ and of CD8+ T-cell activation are predictors of the magnitude of Th17 cell reconstitution following cART provides further rationale for an early initiation of cART in HIV-infected individuals.ClinicalTrials.gov NCT02097381.