Role In Crosstalk Research Articles

49-OR: Synergistic Effect of ERAD and Autophagy in Preventing Proinsulin Aggregation and Promoting Beta-Cell Survival

Decline of proteostasis in the endoplasmic reticulum (ER) is thought to underlie molecular changes in β cell function and drive the pathogenesis of diabetes. ER-associated degradation (ERAD) and macroautophagy (hereafter “autophagy”) are two principal quality-control mechanisms responsible for the clearance of ER-resident proteins or protein aggregates, respectively, in the cell. However, the physiological significance of ERAD-autophagy crosstalk remains unexplored. Previously, using β cell-specific knockout mouse models, we reported that ERAD deficiency leads to β cell dedifferentiation, while autophagy deficiency leads to β cell loss by 4-5 months of age. Interestingly, we observed that loss of ERAD in β cells triggered activation of autophagy pathways, as monitored by LC3 flux. Furthermore, chloroquine-mediated inhibition of autophagy in ERAD-deficient islets led to an increased proinsulin aggregation. In mice, combined deletion of ERAD and autophagy in β cells caused marked hyperglycemia soon after weaning, with extensive loss of β cells and premature death at 2-3 months of age. Thus, our data demonstrate that ERAD and autophagy act synergistically to prevent proinsulin aggregation and promote β cell survival. This study reveals a critical role for crosstalk between the ERAD and autophagy pathways in preventing diabetes and provides the first evidence for synergism between ERAD and autophagy in vivo. Disclosure N. Shrestha: None. R.B. Reinert: None. P. Arvan: None. L. Qi: None. Funding JDRF (2-SRA-2018-539-A-B); National Institutes of Health (1R01DK117639-01, 1R01DK111174)

MON-635 FDXR Regulates Iron Metabolism and Glucose Metabolism in Liver

Iron is an essential cofactor for many proteins that function in electron transport or oxygen transport as heme or iron-sulfur cluster. On the contrary, iron also has the potential to cause oxidative damage if not carefully regulated and when in labial iron excess. Clinical studies show that elevated serum ferritin levels are observed in most patients with type 2 diabetes and non-alcoholic fatty liver disease (NAFLD). In this context, p53 is shown to induces some mitochondrial iron regulatory genes. The role of crosstalk between p53 and iron metabolism has not been sufficiently examined in the pathogenesis of diabetes and NAFLD.Here, we examined the role of ferredoxin reductase (FDXR), a key mitochondrial regulator for iron metabolism, as p53-inducible gene with focusing on the hepatocyte and liver. We confirmed that p53 induced FDXR expression in HepG2 cells and SKEHP1 cells. Biochemical analysis demonstrated that FDXR regulated ROS levels via iron metabolism. In vivo analysis, high-fat diet activated the p53-FDXR pathway in mice liver. We generated transgene expression in mice liver using adenovirus infection carrying shRNA or CRISPR Cas9 system. Treatment with the FDXR knockdown increased hepatic iron content and aggravated glucose intolerance. Besides, forkhead box protein O1 (FOXO1), a key transcriptional factor that induces phosphoenolpyruvate carboxylase and glucose-6-phosphatase increased ratio of nuclear localization, indicating hepatic gluconeogenesis activation. Consistently, biochemical analysis in HepG2 cells demonstrated that FDXR regulated insulin-dependent FOXO1 nuclear exclusion through oxidative stress.In conclusion, p53-inducible FDXR regulates iron metabolism and oxidative stress. FDXR inhibits iron accumulation and oxidative stress in liver and links to suppression of hepatic gluconeogenesis via insulin-dependent FOXO1 nuclear exclusion. The results of this study provide important new insights into relationship between iron metabolism and glucose metabolism as well as potentially identify novel therapeutic targets for the treatment of diabetes and NAFLD.

The Role of Crosstalk between AR3 and E2F1 in Drug Resistance in Prostate Cancer Cells.

Background: Drug resistance is one of the most prevalent causes of death in advanced prostate cancer patients. Combination therapies that target cancer cells via different mechanisms to overcome resistance have gained increased attention in recent years. However, the optimal drug combinations and the underlying mechanisms are yet to be fully explored. Aim and methods: The aim of this study is to investigate drug combinations that inhibit the growth of drug-resistant cells and determine the underlying mechanisms of their actions. In addition, we also established cell lines that are resistant to combination treatments and tested new compounds to overcome the phenomenon of double drug-resistance. Results: Our results show that the combination of enzalutamide (ENZ) and docetaxel (DTX) effectively inhibit the growth of prostate cancer cells that are resistant to either drug alone. The downregulation of transcription factor E2F1 plays a crucial role in cellular inhibition in response to the combined therapy. Notably, we found that the androgen receptor (AR) variant AR3 (a.k.a. AR-V7), but not AR full length (AR-FL), positively regulates E2F1 expression in these cells. E2F1 in turn regulates AR3 and forms a positive regulatory feedforward loop. We also established double drug-resistant cell lines that are resistant to ENZ+DTX combination therapy and found that the expression of both AR3 and E2F1 was restored in these cells. Furthermore, we identified that auranofin, an FDA-approved drug for the treatment of rheumatoid arthritis, overcame drug resistance and inhibited the growth of drug-resistant prostate cancer cells both in vitro and in vivo. Conclusion and significance: This proof-of-principle study demonstrates that targeting the E2F1/AR3 feedforward loop via a combination therapy or a multi-targeting drug could circumvent castration resistance in prostate cancer.

Dynorphins in Development and Disease: Implications for Cardiovascular Disease.

It is well-established that cardiovascular disease continues to represent a growing health problem and significant effort has been made to elucidate the underlying mechanisms. In this review, we report on past and recent high impact publications in the field of intracrine network signaling, focusing specifically on opioids and their interrelation with key modulators of the cardiovascular system and the onset of related disease. We present an overview of studies outlining the scope of cardiovascular and cerebrovascular processes that are affected by opioids, including heart function, ischemia, reperfusion, and blood flow. Specific emphasis is placed on the importance of dynorphin molecules in cerebrovascular and cardiovascular regulation. Evidence suggests that excessive or insufficient dynorphin could make an important contribution to cardiovascular physiology, yet numerous paradoxical observations frequently impede a clear understanding of the role of dynorphin. Thus, we argue that dynorphin-mediated signaling events for which an immediate regulatory effect is disputed should not be dismissed as unimportant, as they may play a role in cross-talk with other signaling networks. Finally, we consider the most recent evidence on the role of dynorphin during cardiovascular-related inflammation and on the potential value of endogenous and exogenous inhibitors of kappa-opioid receptor, a major dynorphin A receptor, to limit or prevent cardiovascular disease and its related sequelae.

Dissecting the role of crosstalk between glioblastoma subpopulations in tumor cell spreading

Glioblastoma (GBM) is a highly infiltrative brain cancer, which is thus difficult to operate. GBM cells frequently harbor Epidermal Growth Factor Receptor amplification (EGFRwt) and/or activating mutation (EGFRvIII), generating at least two different cellular subpopulations within the tumor. We examined the relationship between the diffusive architectures of GBM tumors and the paracrine interactions between those subpopulations. Our aim was to shed light on what drives GBM cells to reach large cell–cell distances, and whether this characteristic can be manipulated. We established a methodology that quantifies the infiltration abilities of cancer cells through computation of cell–cell separation distance distributions in 3D. We found that aggressive EGFRvIII cells modulate the migration and infiltrative properties of EGFRwt cells. EGFRvIII cells secrete HGF and IL6, leading to enhanced activity of Src protein in EGFRwt cells, and rendering EGFRwt cells higher velocity and augmented ability to spread. Src inhibitor, dasatinib, at low non-toxic concentrations, reduced the infiltrative properties of EGFRvIII/EGFRwt neurospheres. Furthermore, dasatinib treatment induced compact multicellular microstructure packing of EGFRvIII/EGFRwt cells, impairing their ability to spread. Prevention of cellular infiltration or induction of compact microstructures may assist the detection of GBM tumors and tumor remnants in the brains and improve their surgical removal.

Hydrogen alleviated organ injury and dysfunction in sepsis: The role of cross-talk between autophagy and endoplasmic reticulum stress: Experimental research

AimsSepsis is defined as a life-threatening organ dysfunction that is caused by a dysregulated host response to infection. Although much progress has been made in understanding the pathophysiology of sepsis, further discussion and study of the detailed therapeutic mechanisms are needed. Autophagy and endoplasmic reticulum stress are two pathways of the complicated regulatory network of sepsis. Herein, we focus on the cellular mechanism in which autophagy and endoplasmic reticulum stress participate in hydrogen (H2)-protected sepsis-induced organ injury. Materials and methodsMale C57BL/6 mice were randomly divided into the following groups: control group, cecal ligation puncture (CLP) group, CLP + tunicamycin(TM) group, CLP + 4-phenyl butyric acid (4-PBA) group, CLP + rapamycin (Rap) group, CLP + 3-methyladenine (3-MA) group, CLP + H2 group, CLP + H2 + 3-MA group, and CLP + H2 + TM group. After the experiment was completed, autophagosome was detected by transmission electron microscopy; protein PKR-like ER kinase (PERK), p-PERK, Eukaryotic translation initiation factor-2α (eIF2α), p-eIF2α, inositol-requiring enzyme1α(IRE1α), C/EBP homologous protein(CHOP), activating transcription factor(ATF), XBP-1, microtubule-associated protein 1 light(LC3), Beclin1, PTEN-induced putative kinase 1(PINK1), Parkin, and p65 subunit of Nuclear factor kappa B(NF-κb) were measured by Western blot; myeloperoxidase(MPO) activity in lung, bronchoalveolar lavage(BAL) total protein, lung wet-to-dry(W/D) ratio, serum biochemical indicators, 7-day survival rate, and pathological injury scores of lung, liver, and kidney were tested; and cytokines tumor necrosis factor-α(TNF-α), Interleukin(IL)-1β, and IL-6 and high mobility group box protein (HMGB)1 were detected by enzyme-linked immunosorbent assay(ELISA). ResultsWe demonstrated that sepsis induced endoplasmic reticulum stress. Moreover, it was found that an increase in endoplasmic reticulum impaired autophagy activity in sepsis, and the absence of endoplasmic reticulum stress attenuated tissue histological injury and dysfunction of lung, liver, and kidney in septic mice. Intriguingly, hydrogen alleviated the endoplasmic reticulum stress via the autophagy pathway and also mitigated inflammation and organ injury. ConclusionHydrogen provided protection from organ injury induced by sepsis via autophagy activation and endoplasmic reticulum stress pathway inactivation.

Diffuse Large B Cell Lymphoma (DLBCL)-Released NM23-H1 Promotes Monocyte Survival and Inflammatory Cytokine Release: A Mechanistic Link between the Dual Impacts of NM23-H1 Expression and Reduced Lymphocyte:Monocyte Ratio in DLBCL Prognosis

Elevated circulating levels of NM23-H1 have been associated with poor prognosis in haematological malignancies including AML and non-Hodgkin's lymphomas. In diffuse large B-cell lymphoma (DLBCL), several studies have demonstrated that both elevated circulating plasma levels and intra-tumoural levels of NM23-H1 correlate with poorer prognosis. These observations bring into question whether the mechanistic link of NM23-H1 expression with DLBCL prognosis depends an intrinsic intracellular mechanisms or is mediated by release of NM23-H1. We have shown that DLBCL lymphoma cell lines (Farage, HT, OCI-LY1, OCI-LY3, OCI-LY7, SUDHL4, SUDHL5 SUDHL6, U2932) express both NM23-H1 protein and the highly related protein NM23-H2. We also demonstrated that DLBCL cell lines release significant levels of NM23-H1 into their extracellular environment but not NM23-H2. Release of NM23-H1 was highly variable between cell lines with some releasing very high levels and some releasing much lower levels. Thus, DLBCL cell lines represent appropriate models to investigate the role of high and low levels of NM23-H1 on prognosis. The DLBCL cell lines HT and OCI-LY1 where selected for further study as representative of high and low level releasers of NM23-H1, respectively. CRISPR-Cas9 knockout of NM23-H1 (KO-NM23-H1) in HT and OCI-LY1 DLBCL cells had no impact on cell growth, viability or immuno-phenotype either in normoxic or hypoxic cultures. Thus NM23-H1 appears to not be required intrinsically by DLBCL cell lines. We therefore considered that the link between higher expression and release of NM23-H1 with prognosis is mediated via tumour-host environment interactions. We used an NSG mouse model transplanted subcutaneously with 1x106 CRISPR control (CTRL)-HT, CTRL-OCIL-Y1, KO-NM23-H1-HT or KO-NM23-H1-OCI-LY1 cell lines. We did not observe significant differences in tumour growth between CTRL and KO-NM23-H1 cells in the low NM23-H1 expressing OCI-LY1 DLBCL cell line. However, the high-expressing KO-NM23-H1-HT cells had significantly slower tumour progression and increased host survival when compared to CTRL-HT cells. We interpret to indicate that NM23-H1 release above a certain threshold provides a tumour growth advantage. Reduced lymphocyte:monocyte ratios (LMR) are also associated with poor prognosis in DLBCL. To investigate a potential functional link between NM23-H1 release and monocyte behaviour we first exposed peripheral blood leucocytes to Alexa 647 labelled fluorescent recombinant-NM23-H1 and found that monocytes but not neutrophils, T-cells or B-cells bound NM23-H1. We also observed that monocyte viability and survival were elevated in serum free cultures when supplemented with rNM23-H1, an observation that might indicate that elevated circulating NM23-H1 and reduced LMR in poor prognosis DLBCL may be mechanistically linked. We next co- cultured CTRL-HT or koNM23-H1-HT in a 1:1 ratios with purified monocytes from healthy human donors. Principle component analyses of 27 cytokines simultaneously measured by luminex assays identified that IL-1β, IL-6, IL-8, MIP-1α, MIP-1β and TNF-α were elevated when monocytes were co-cultured with CTRL-HT cells compared to co-culture with koNm23-H1-HT cells. In contrast, IP-10 was found elevated in the co-culture with koNm23-H1-HT cells. These observations indicate potentially important cross talk between the malignant DLBCL cells and innate immune cells of the host. Consistent with this, IL-6 and IL-8 serum levels have been shown to be elevated in pretreatment DLBCL patients compared to control subjects and MIP-1α, IL-6, and IL-8 serum levels have a greater association with DLBCL than follicular lymphoma. In conclusion ours is the first study to investigate potential mechanistic links between the associations of elevated NM23-H1 and poor prognosis in DLBCL and indicate a role for cross-talk between tumour cells and innate immune cells. Our data also indicate that NM23-H1 release from DLBCL cells may be a driving component in driving reduced LMRs that are also associated with poor prognosis. Whether reduced LMR and NM23-H1 release are casually related or not, the possibility that patients with both elevated monocytes and elevated circulating NM23-H1 levels are likely to represent a group with particularly poor prognosis requires urgent investigation. Disclosures Drayson: Abingdon Health: Consultancy, Equity Ownership. Rushworth:Abbvie: Research Funding; Janssen: Research Funding.

Mitochondria, Telomeres and Telomerase Subunits.

Mitochondrial functions and telomere functions have mostly been studied independently. In recent years, it, however, has become clear that there are intimate links between mitochondria, telomeres, and telomerase subunits. Mitochondrial dysfunctions cause telomere attrition, while telomere damage leads to reprogramming of mitochondrial biosynthesis and mitochondrial dysfunctions, which has important implications in aging and diseases. In addition, evidence has accumulated that telomere-independent functions of telomerase also exist and that the protein component of telomerase TERT shuttles between the nucleus and mitochondria under oxidative stress. Our previously published data show that the RNA component of telomerase TERC is also imported into mitochondria, processed, and exported back to the cytosol. These data show a complex regulation network where telomeres, nuclear genome, and mitochondria are co-regulated by multi-localization and multi-function proteins and RNAs. This review summarizes the connections between mitochondria and telomeres, the mitochondrion-related functions of telomerase subunits, and how they play a role in crosstalk between mitochondria and the nucleus.

Functional regulation of von Willebrand factor ameliorates acute ischemia-reperfusion kidney injury in mice

Acute kidney injury (AKI), an abrupt loss of renal function, is often seen in clinical settings and may become fatal. In addition to its hemostatic functions, von Willebrand factor (VWF) is known to play a role in cross-talk between inflammation and thrombosis. We hypothesized that VWF may be involved in the pathophysiology of AKI, major causes of which include insufficient renal circulation or inflammatory cell infiltration in the kidney. To test this hypothesis, we studied the role of VWF in AKI using a mouse model of acute ischemia-reperfusion (I/R) kidney injury. We analyzed renal function and blood flow in VWF-gene deleted (knock-out; KO) mice. The functional regulation of VWF by ADAMTS13 or a function-blocking anti-VWF antibody was also evaluated in this pathological condition. Greater renal blood flow and lower serum creatinine were observed after reperfusion in VWF-KO mice compared with wild-type (WT) mice. Histological analysis also revealed a significantly lower degree of tubular damage and neutrophil infiltration in kidney tissues of VWF-KO mice. Both human recombinant ADAMTS13 and a function-blocking anti-VWF antibody significantly improved renal blood flow, renal function and histological findings in WT mice. Our results indicate that VWF plays a role in the pathogenesis of AKI. Proper functional regulation of VWF may improve the microcirculation and vessel function in the kidney, suggesting a novel therapeutic option against AKI.

Pushing and pulling with no end in sight! The role of cross-talk between different forms of respiratory plasticity in modifying sleep apnoea.

Pushing and pulling with no end in sight! The role of cross-talk between different forms of respiratory plasticity in modifying sleep apnoea.

Subcellular Targets of Zinc Oxide Nanoparticles During the Aging Process: Role of Cross-talk Between Mitochondrial Dysfunction and Endoplasmic Reticulum Stress in the Genotoxic Response.

Zinc oxide nanoparticles (ZnO NPs) are being produced abundantly and applied increasingly in various fields. The special physicochemical characteristics of ZnO NPs make them incline to undergo physicochemical transformation over time (aging), which modify their bioavailability and toxicity. However, the subcellular targets and the underlying molecular mechanisms involved in the genotoxicity induced by ZnO NPs during aging process are still unknown. This study found that the acute cytotoxic effects of fresh ZnO NPs was largely regulated by mitochondria-dependent apoptosis, which the level of cleaved Caspase-3 and mitochondria damage were significantly higher than that of 60-day-aged ZnO NPs. In contrast, aged ZnO NPs induced more reactive oxygen species (ROS) production and endoplasmic reticulum (ER) stress marker protein (BIP/GRP78) expression and their genotoxicity could be dramatically suppressed by either ROS scavengers (dimethyl sulfoxide, catalase, and sodium azide) or ER stress inhibitor (4-phenylbutyrate). Using mitochondrial-DNA deficient (ρ0) AL cells, we further found that ER stress induced by aged ZnO NPs was triggered by ROS generated from mitochondria, which eventually mediated the genotoxicity of aged NPs. Our data provided novel information on better understanding the contribution of subcellular targets to the genotoxic response of ZnO NPs during the aging process.

Genome-wide identification and characterization of R2R3-MYB genes in Medicago truncatula.

MYB is a large family of plant transcription factors. Its function has been identified in several plants, while there are few reports in Medicago truncatula. In this study, we used RNA-seq data to analyze and identify R2R3-MYB genes in the genome of Medicago truncatula. Phylogenetic analysis classified 150 MtMYB genes into 21 subfamilies with homologs. Out of the 150 MtMYB genes, 139 were distributed among 8 chromosomes, with tandem duplications (TD) and segment duplications (SD). Microarray data were used for functional analysis of the MtMYB genes during growth and developmental processes providing evidence for a role in tissues differentiation, seed development processes, and especially the nodulation process. Furthermore, we investigated the expression of MtMYB genes in response to abiotic stresses using RNA-seq data, which confirmed the critical roles in signal transduction and regulation processes under abiotic stress. We used quantitative real-time PCR (qRT-PCR) to validate expression profiles. The expression pattern of M. truncatula MYB genes under different abiotic stress conditions suggest that some may play a major role in cross-talk among different signal transduction pathways in response to abiotic stresses. Our study will serve as a foundation for future research into the molecular function of M. truncatula R2R3-MYB genes.

Expression of TSLP and IL-33 receptors on sputum macrophages of asthma patients and healthy subjects

Objective: Local cytokine milieu (especially Th2 inflammatory type) secreted into the asthmatic airways affect the alternative activated macrophages polarization (M2). TSLP and IL-33 are important alarmins of allergic response associated with Th2 inflammation. The aim of the study was to investigate the expression of the receptors for epithelial derived cytokines: TSLP (TSLPR) and IL-33 (ST2) on induced sputum CD206 positive macrophages from asthma and healthy subjects and analyze the relationships between these receptors and clinical features of the disease. Methods: Immunofluorescence staining for CD206 and TSLPR or ST2 on sputum macrophages was performed in 20 adult patients with stable asthma – 75% with atopy (3 intermittent, 12 mild-to-moderate, 5 severe, of which 11 were on biological anty-IgE treatment) and 23 healthy adult controls – 48% with atopy. Results: Our study demonstrated an increased expression of TSLP and IL-33 receptors on bronchial CD206 positive macrophages in asthma group. TSLPR but not ST2 had also greater expression on CD206 negative macrophages in asthma patients. Increased expression of both investigated receptors was related to longer disease duration and impaired lung function. We observed increased count of CD206lowTSLPhigh macrophages as well as positive correlation of these cells with total serum IgE in patients with atopy. Conclusions: The macrophage response during allergic reaction is likely to be connected with TSLP but rather not with IL-33 action. Our study indicates an important role of crosstalk between macrophages, TSLP and IL-33 in asthma pathophysiology.

Converging Small Ubiquitin-like Modifier (SUMO) and Ubiquitin Signaling: Improved Methodology Identifies Co-modified Target Proteins

Post-translational protein modifications (PTMs) including small chemical groups and small proteins, belonging to the ubiquitin family, are essential for virtually all cellular processes. In addition to modification by a single PTM, proteins can be modified by a combination of different modifiers, which are able to influence each other. Because little is known about crosstalk among different ubiquitin family members, we developed an improved method enabling identification of co-modified proteins on a system-wide level using mass spectrometry. We focused on the role of crosstalk between SUMO and ubiquitin during proteasomal degradation. Using two complementary approaches, we identified 498 proteins to be significantly co-modified by SUMO and ubiquitin upon MG132 treatment. These targets included many enzymatic components of PTM machinery, involved in SUMOylation and ubiquitylation, but also phosphorylation, methylation and acetylation, revealing a highly complex interconnected network of crosstalk among different PTMs. In addition, various other biological processes were found to be significantly enriched within the group of co-modified proteins, including transcription, DNA repair and the cell cycle. Interestingly, the latter group mostly consisted of proteins involved in mitosis, including a subset of chromosome segregation regulators. We hypothesize that group modification by SUMO-targeted ubiquitin ligases regulates the stability of the identified subset of mitotic proteins, which ensures proper chromosome segregation. The mitotic regulators KIF23 and MIS18BP1 were verified to be co-modified by SUMO and ubiquitin on inhibition of the proteasome and subsequently identified as novel RNF4 targets. Both modifications on MIS18BP1 were observed to increase simultaneously during late mitosis, whereas the total protein level decreased immediately afterward. These results confirm the regulation of MIS18BP1 via SUMO-ubiquitin crosstalk during mitosis. Combined, our work highlights extensive crosstalk between SUMO and ubiquitin, providing a resource for further unraveling of SUMO-ubiquitin crosstalk.

Bacillus amyloliquefaciens Confers Tolerance to Various Abiotic Stresses and Modulates Plant Response to Phytohormones through Osmoprotection and Gene Expression Regulation in Rice.

Being sessile in nature, plants have to withstand various adverse environmental stress conditions including both biotic and abiotic stresses. Comparatively, abiotic stresses such as drought, salinity, high temperature, and cold pose major threat to agriculture by negatively impacting plant growth and yield worldwide. Rice is one of the most widely consumed staple cereals across the globe, the production and productivity of which is also severely affected by different abiotic stresses. Therefore, several crop improvement programs are directed toward developing stress tolerant rice cultivars either through marker assisted breeding or transgenic technology. Alternatively, some known rhizospheric competent bacteria are also known to improve plant growth during abiotic stresses. A plant growth promoting rhizobacteria (PGPR), Bacillus amyloliquefaciens NBRI-SN13 (SN13) was previously reported by our lab to confer salt stress tolerance to rice seedlings. However, the present study investigates the role of SN13 in ameliorating various abiotic stresses such as salt, drought, desiccation, heat, cold, and freezing on a popular rice cv. Saryu-52 under hydroponic growth conditions. Apart from this, seedlings were also exogenously supplied with abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA) and ethephon (ET) to study the role of SN13 in phytohormone-induced stress tolerance as well as its role in abiotic and biotic stress cross-talk. All abiotic stresses and phytohormone treatments significantly affected various physiological and biochemical parameters like membrane integrity and osmolyte accumulation. SN13 also positively modulated stress-responsive gene expressions under various abiotic stresses and phytohormone treatments suggesting its multifaceted role in cross-talk among stresses and phytohormones in response to PGPR. To the best of our knowledge, this is the first report on detailed analysis of plant growth promotion and stress alleviation by a PGPR in rice seedlings subjected to various abiotic stresses and phytohormone treatments for 0, 1, 3, 10, and 24 h.

Expression of lectin-like transcript-1 in human tissues

Background: Receptor-ligand pairs of C-type lectin-like proteins have been shown to play an important role in cross talk between lymphocytes, as well as in immune responses within concrete tissues and structures, such as the skin or the germinal centres. The CD161-Lectin-like Transcript 1 (LLT1) pair has gained particular attention in recent years, yet a detailed analysis of LLT1 distribution in human tissue is lacking. One reason for this is the limited availability and poor characterisation of anti-LLT1 antibodies. Methods: We assessed the staining capabilities of a novel anti-LLT1 antibody clone (2H7), both by immunohistochemistry and flow cytometry, showing its efficiency at LLT1 recognition in both settings. We then analysed LLT1 expression in a wide variety of human tissues. Results: We found LLT1 expression in circulating B cells and monocytes, but not in lung and liver-resident macrophages. We found strikingly high LLT1 expression in immune-privileged sites, such as the brain, placenta and testes, and confirmed the ability of LLT1 to inhibit NK cell function. Conclusions: Overall, this study contributes to the development of efficient tools for the study of LLT1. Moreover, its expression in different healthy human tissues and, particularly, in immune-privileged sites, establishes LLT1 as a good candidate as a regulator of immune responses.

Expression of lectin-like transcript-1 in human tissues.

Background: Receptor-ligand pairs of C-type lectin-like proteins have been shown to play an important role in cross talk between lymphocytes, as well as in immune responses within concrete tissues and structures, such as the skin or the germinal centres. The CD161-Lectin-like Transcript 1 (LLT1) pair has gained particular attention in recent years, yet a detailed analysis of LLT1 distribution in human tissue is lacking. One reason for this is the limited availability and poor characterisation of anti-LLT1 antibodies. Methods: We assessed the staining capabilities of a novel anti-LLT1 antibody clone (2H7), both by immunohistochemistry and flow cytometry, showing its efficiency at LLT1 recognition in both settings. We then analysed LLT1 expression in a wide variety of human tissues. Results: We found LLT1 expression in circulating B cells and monocytes, but not in lung and liver-resident macrophages. We found strikingly high LLT1 expression in immune-privileged sites, such as the brain, placenta and testes, and confirmed the ability of LLT1 to inhibit NK cell function. Conclusions: Overall, this study contributes to the development of efficient tools for the study of LLT1. Moreover, its expression in different healthy human tissues and, particularly, in immune-privileged sites, establishes LLT1 as a good candidate as a regulator of immune responses.

Adipose- and muscle-derived Wnts trigger pancreatic β-cell adaptation to systemic insulin resistance.

Wnt signaling molecules are associated with obesity, hyperlipidemia, and type 2 diabetes (T2D). Here, we show that two Wnt proteins, WNT3a and WNT4, are specifically secreted by skeletal muscle and adipose tissue during the development of insulin resistance and play an important role in cross-talk between insulin-resistant tissues and pancreatic beta cells. The activation of Frizzled receptor and Wnt signaling in pancreatic islets via circulating WNT3a in blood resulted in higher insulin secretion and an increase in beta cell proliferation, thus leading to islet adaptation in a pre-diabetic state. Interestingly, in fully developed T2D, the expression profiles of Wnt3a and Wnt4 in adipose tissue and muscle cells and blood plasma levels of these proteins were opposite to the pre-diabetic state, thus favoring the downregulation of Wnt signaling in beta cells and resulting in dysfunctional pancreatic islets. These results demonstrate that alterations in the secretion profile of a canonical Wnt activator (WNT3a) and inhibitor (WNT4) from insulin-resistant tissues during the development of T2D are responsible for triggering progression from a pre-diabetic to a diabetic state. We also show here that WNT3a and WNT4 are potent myokines, and their expression and secretion are regulated in response to nutritional and metabolic changes.

Development and characterization of Th17-related U-PLEX® assays

Abstract Th17-related cytokines mediate a host’s defense mechanisms against various infections and play a crucial role in crosstalk between the immune system and affected tissues. Here we describe the development and characterization of multiplexed Th17-related immunoassays on MSD’s flexible U-PLEX platform. Monoclonal and polyclonal antibodies were conjugated with biotin and/or SULFO-TAG™ and screened as capture and/or detection reagents. A number of analytical parameters were used to select antibody pairs, and assays were developed by optimizing antibody concentrations, calibrator curves, specificity, matrix tolerance, and assay robustness. Assay performance was verified to be compatible with other U-PLEX assays by running Th17-related multiplexes on the U-PLEX platform with controls and samples. Calibration curves showed expected signals, sensitivity, precision, and accuracy. Control samples for the assays had CVs < 10% within runs and < 25% between runs. Sensitivities were < 1 pg/mL for the majority of the assays. Dilution linearity and spike recovery studies demonstrated acceptable matrix tolerance and accurate quantification for most of the assays tested across all matrices (typically between 75% – 125%). Cross-reactivity between assays was shown to be typically < 0.5%. Results demonstrated a strong correlation between samples measured on U-PLEX multiplex and streptavidin singleplex panels with r2 values > 0.95 and slopes between 0.8–1.2. Over 20 Th17-related assays for human and mouse were developed for the U-PLEX platform. These assays enable the measurement of multiple proteins on biological matrices that are relevant to a wide range of life science applications and pre-clinical or clinical studies.

The role of shrimp miR-965 in virus infection.

RNAi, mediated by microRNAs (miRNAs), has attracted increasing attention for its important role in cross-talk between host and virus. However, the role of host miRNA in the virus infection invivo has not been intensively investigated. In this study, the effects of a shrimp miRNA (miR-965) on the white spot syndrome virus (WSSV) infection were characterized. The results indicated that the expression of miR-965 was significantly upregulated in shrimp in response to the WSSV challenge, suggesting its involvement in the virus infection. The miR-965 silencing led to significant increases of WSSV copies and virus-infected shrimp mortality, while the miR-965 overexpression resulted in the decreased WSSV copies and virus-infected shrimp mortality, indicating that miR-965 played a negative role in the WSSV infection. The further data revealed that miR-965 inhibited the virus infection by targeting the viral wsv240 gene, an important gene required for the WSSV infection in shrimp. The results demonstrated that miR-965 could promote the shrimp phagocytosis against virus infection by targeting the shrimp ATG5 (autophagy related 5) gene. Therefore, our findings presented novel evidence to better understand the anfractuous host-virus interactions invivo.