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•HIV DRM widespread in youths.•Dual-class resistance common.•Strengthen monitoring of drug resistance.

Novel bismuth selenite iodate oxide BiSeIO6was synthesized in a mild hydrothermal condition. BiSeIO6was crystallized in the polar space group Pna21of an orthorhombic system. The crystal structure features a three-dimensional framework composed of three types of lone pair cations with distorted BiO7polyhedra, SeO3pyramids, and IO3pyramids in one structure. Interestingly, BiSeIO6exhibits a strong and phase-matchable second-harmonic generation (SHG) of ∼6 times that of KH2PO4(KDP). Dipole moment analysis shows that all three local acentric groups of BiO7, SeO3, and IO3cooperatively contribute to the large macroscopic polarization and thereby strong SHG efficiency of BiSeIO6. In addition, BiSeIO6has a broad transparency range from 0.35 to 11 μm, indicating its promising nonlinear optical applications from visible to mid-infrared bands.

A lithology identification method based on stacking multi-model fusion was studied, which solved the problem of poor recognition performance of traditional single machine learning models. In the experiment, logging data underwent preprocessing using outlier and linear analysis. Nine data features were filtered to identify valid features. Classification and regression tree, K-nearest neighbour algorithm, random forest, and extreme gradient boosting were used as base models. Principal component analysis calculated the weights of each model and applied them to the light gradient boosting machine metamodel in the second layer, constructing a multi-layer ensemble learning model. The fusion model improved the F1-score by 1.63 percentage points compared to random forest. In the siltstone with the best average recognition performance, the improvement was 9.24 percentage points over the K-nearest neighbour algorithm. These results verify the higher accuracy and F1-score of the fusion model as compared to traditional single algorithms, demonstrating the effectiveness of the fusion model method. [Received: April 28, 2023; Accepted: July 28, 2023]

We construct a numerical model for multipulse laser drilling. It is found that the previous laser-pulse-induced temperature accumulation, thermal stress occurrence, and crater morphology change promote subsequent pulse laser drilling. Among them, previous laser-pulse-induced temperature accumulation contributes significantly to the drilled crater depth when the workpiece temperature is higher than its melting point just before the subsequent laser pulse irradiation, especially in a short pulse interval condition. The crater morphology change becomes the main contributor when the workpiece temperature decreases below the melting point, often in a long pulse interval condition. Besides, the previous occurrence of laser-pulse-induced thermal stress always has had little influence on the drilled crater. This work can be a theoretical reference, especially for multipulse laser manufacturing.

Mitophagy has a neuroprotective effect on reactive oxygen species (ROS)-induced neurodegenerative diseases. The walnut-derived polypeptide (TW-7) has antioxidant activity and protects nerves by promoting autophagy. However, its action mechanism against oxidative stress through mitophagy remains obscure. Therefore, we aimed to assess the effects of TW-7 on HT-22 cells under oxidative stress. Mitochondrial ultrastructure and cristae number were observed by transmission electron microscopy. The results showed that TW-7 (100 μM) restored the fluorescence intensity of the mitochondrial membrane potential to 0.99 ± 0.04 (P< 0.05), decreased H2O2-induced opening of mitochondrial permeability transition pores, and inhibited mitochondrial bioenergetic deficits. Moreover, it significantly increased activities of antioxidant enzymes to 186.88 ± 5.40 U/mgprot, 40.08 ± 0.87 mU/mgprot, and 23.57 ± 0.77 U/mgprot (P< 0.05), based on superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) assay results, respectively. Consistently, it decreased cellular and mitochondrial ROS levels by 51.71 ± 0.81 and 49.75 ± 0.69% (P< 0.05). TW-7 also downregulated C-Jun N-terminal kinase (JNK) phosphorylation and activated PTEN-induced putative kinase 1 (PINK1)-mediated mitophagy in H2O2-induced HT-22 cells treated with JNK activator (anisomycin) and inhibitor (SP600125). Furthermore, TW-7 inhibited the mitochondrial apoptosis pathway by downregulation of the cytoplasmic cytochrome C, caspase-9, and cleaved-caspase-3 expression. Additionally, BDNF and SNAP-25 levels significantly increased to protect the synaptic function. Collectively, TW-7 improved oxidative stress-mediated nerve cell injury via JNK-regulated PINK1-mediated mitophagy.

Mitophagy has a pivotal protective function in the pathogenesis of neurological disorders. However, the mechanism of its modulation remains elusive, especially in PINK1-mediated mitophagy. Here, we investigated the neuroprotective effects of a walnut-derived peptide, YVLLPSPK, against scopolamine-induced cognitive deficits in mice and explored the underlying PINK1-mediated mitophagy mechanisms in H2O2-treated HT-22 cells. Using the Morris water maze, we showed that YVLLPSPK relieved the cognitive deficiency by alleviating oxidative stress. Mitochondrial morphology was observed in mice hippocampal tissues using transmission electron microscopy (TEM). Both Western blot and immunofluorescence analysis illustrated YVLLPSPK promoted the expression of mitophagy-related proteins and activated the NRF2/KEAP1/HO-1 pathway. Subsequently, an NRF2 inhibitor (ML385) was used to verify the contribution of the YVLLPSPK-regulated NRF2/KEAP1/HO-1 pathway in PINK1-mediated mitophagy in H2O2-treated HT-22 cells. These data suggested that YVLLPSPK improved learning and memory in scopolamine-induced cognitive-impaired mice through a mechanism associated with PINK1-mediated mitophagy via the NRF2/KEAP1/HO-1 pathway.

Natural-derived peptides are effective substances in attenuating oxidative stress. However, their specific mechanisms have not been fully elucidated, especially in peptide-mediated autophagy. In the present study, TWLPLPR, YVLLPSPK, and KVPPLLY, novel peptides from Juglans mandshuricaMaxim, prevented reactive oxygen species (ROS) production, elevated glutathione peroxidase (GSH-Px) activity and adenosine 5′-triphosphate (ATP) levels, and ameliorated apoptosis in Aβ25–35(at a concentration of 50 μM for 24 h)-induced PC12 cells (P< 0.01). Both western blot and immunofluorescence analysis illustrated that the peptides regulated Akt/mTOR signaling through p-Akt (Ser473) and p-mTOR (S2481) and promoted autophagy by increasing the levels of LC3-II/LC3-I and Beclin-1 while lowering p62 expression (P< 0.01). The autophagy inhibitor (3-methyladenine, 3-MA) and inducer (rapamycin, RAPA) were combined used to confirm the contribution of peptide-regulated autophagy in antioxidative effects. Moreover, the peptides increased the levels of LAMP1, LAMP2, and Cathepsin D (P< 0.05) and promoted the fusion with lysosomes to form autolysosomes, accelerating ROS removal. These data suggested that walnut-derived peptides regulated oxidative stress by promoting autophagy in the Aβ25–35-induced PC12 cells.

We aimed to explore the impact of blood pressure (BP) levels on atherosclerosis in a rural Chinese population with a low-education level, low income, high incidence of stroke, and high prevalence of hypertension. B-mode ultrasonography was used to measure carotid intima-media thickness (CIMT) in adults aged ≥ 45 years with no history of stroke or cardiovascular disease. A total of 5403 eligible subjects were included in this study. The mean CIMT was 0.57 mm overall, 0.58 mm for men and 0.56 mm for women. Systolic blood pressure (SBP) and hypertension were significantly associated with increased CIMT. CIMT increased by 0.42 μm for every 1 mm Hg-increase in SBP (P< 0.001). The mean CIMT in participants with a history of hypertension was 17.42 μm greater than that in participants with no history of hypertension (P< 0.001). Diastolic blood pressure (DBP) was a protective factor, as CIMT decreased by 0.44 μm with every 1 mm Hg-increase in DBP (P= 0.011).

Influenza A virus (IAV) remains a major worldwide health threat, especially to high-risk populations, including the young and elderly. There is an unmet clinical need for therapy that will protect the lungs from damage caused by lower respiratory infection. Here, we analyzed the role of EMAPII, a stress- and virus-induced pro-inflammatory and pro-apoptotic factor, in IAV-induced lung injury. First, we demonstrated that IAV induces EMAPII surface translocation, release, and apoptosis in cultured endothelial and epithelial cells. Next, we showed that IAV induces EMAPII surface translocation and release to bronchoalveolar lavage fluid (BALF) in mouse lungs, concomitant with increases in caspase 3 activity. Injection of monoclonal antibody (mAb) against EMAPII attenuated IAV-induced EMAPII levels, weight loss, reduction of blood oxygenation, lung edema, and increase of the pro-inflammatory cytokine TNF alpha. In accordance with the pro-apoptotic properties of EMAPII, levels of caspase 3 activity in BALF were also decreased by mAb treatment. Moreover, we detected EMAPII mAb-induced increase in lung levels of M2-like macrophage markers YM1 and CD206. All together, these data strongly suggest that EMAPII mAb ameliorates IAV-induced lung injury by limiting lung cell apoptosis and shifting the host inflammatory setting toward resolution of inflammation.

Cigarette smoking (CS) adversely affects the physiologic function of endothelial progenitor, hematopoietic stem and progenitor cells. However, the effect of CS on the ability of adipose stem/stromal cells (ASC) to promote vasculogenesis and rescue perfusion in the context of ischemia is unknown. To evaluate this, ASC from nonsmokers (nCS‐ASC) and smokers (CS‐ASC), and their activity to promote perfusion in hindlimb ischemia models, as well as endothelial cell (EC) survival and vascular morphogenesis in vitro were assessed. While nCS‐ASC improved perfusion in ischemic limbs, CS‐ASC completely lost this therapeutic effect. In vitro vasculogenesis assays revealed that human CS‐ASC and ASC from CS–exposed mice showed compromised support of EC morphogenesis into vascular tubes, and the CS‐ASC secretome was less potent in supporting EC survival/proliferation. Comparative secretome analysis revealed that CS‐ASC produced lower amounts of hepatocyte growth factor (HGF) and stromal cell‐derived growth factor 1 (SDF‐1). Conversely, CS‐ASC secreted the angiostatic/pro‐inflammatory factor Activin A, which was not detected in nCS‐ASC conditioned media (CM). Furthermore, higher Activin A levels were measured in EC/CS‐ASC cocultures than in EC/nCS‐ASC cocultures. CS‐ASC also responded to inflammatory cytokines with 5.2‐fold increase in Activin A secretion, whereas nCS‐ASC showed minimal Activin A induction. Supplementation of EC/CS‐ASC cocultures with nCS‐ASC CM or with recombinant vascular endothelial growth factor, HGF, or SDF‐1 did not rescue vasculogenesis, whereas inhibition of Activin A expression or activity improved network formation up to the level found in EC/nCS‐ASC cocultures. In conclusion, ASC of CS individuals manifest compromised in vitro vasculogenic activity as well as in vivo therapeutic activity. StemCells2018;36:856–867 The detrimental effect of smoking on bioactivity of mesenchymal stem/stromal cells is expected, but has not been studied. This study has shown that smoking dramatically decreases the ability of adipose stromal cells to promote recovery of blood perfusion in the mouse ischemic tissue and support endothelial cell vasculogenic activity, which was in part due to increased secretion of angiostatic factor Activin A.

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Transplantation of mesenchymal stromal cells (MSCs) has been shown to effectively prevent lung injury in several preclinical models of acute respiratory distress syndrome (ARDS). Since MSC therapy is tested in clinical trials for ARDS, there is an increased need to define the dynamics of cell trafficking and organ-specific accumulation. We examined how the presence of ARDS changes retention and organ-specific distribution of intravenously delivered MSCs isolated from subcutaneous adipose tissue [adipose-derived stem cells (ADSCs)]. This type of cell therapy was previously shown to ameliorate ARDS pathology. ARDS was triggered by lipopolysaccharide (LPS) aspiration, 4 h after which 300,000 murine CRE+ADSCs were delivered intravenously. The distribution of ADSCs in the lungs and other organs was assessed by real-time polymerase chain reaction (PCR) of genomic DNA. As anticipated, the majority of delivered ADSCs accumulated in the lungs of both control and LPS-challenged mice, with minor amounts distributed to the liver, kidney, spleen, heart, and brain. Interestingly, within 2 h following ADSC administration, LPS-challenged lungs retained significantly lower levels of ADSCs compared to control lungs (~7% vs. 15% of the original dose, respectively), whereas the liver, kidney, spleen, and brain of ARDS-affected animals retained significantly higher numbers of ADSCs compared to control animals. In contrast, 48 h later, only LPS-challenged lungs continued to retain ADSCs (~3% of the original dose), whereas the lungs of control animals and nonpulmonary organs in either control or ARDS mice had no detectable levels of ADSCs. Our data suggest that the pulmonary microenvironment during ARDS may lessen the pulmonary capillary occlusion by MSCs immediately following cell delivery while facilitating pulmonary retention of the cells.

Camelina sativais a self-pollinating and facultative outcrossing oilseed crop. Genetic engineering has been used to improve camelina yield potential for altered fatty acid composition, modified protein profiles, improved seed and oil yield, and enhanced drought resistance. The deployment of transgenic camelina in the field posits high risks related to the introgression of transgenes into non-transgenic camelina and wild relatives. Thus, effective bioconfinement strategies need to be developed to prevent pollen-mediated gene flow (PMGF) from transgenic camelina. In the present study, we overexpressed the cleistogamy (i.e. floral petal non-openness)-inducing PpJAZ1gene from peach in transgenic camelina. Transgenic camelina overexpressing PpJAZ1showed three levels of cleistogamy, affected pollen germination rates after anthesis but not during anthesis, and caused a minor silicle abortion only on the main branches. We also conducted field trials to examine the effects of the overexpressed PpJAZ1on PMGF in the field, and found that the overexpressed PpJAZ1dramatically inhibited PMGF from transgenic camelina to non-transgenic camelina under the field conditions. Thus, the engineered cleistogamy using the overexpressed PpJAZ1is a highly effective bioconfinement strategy to limit PMGF from transgenic camelina, and could be used for bioconfinement in other dicot species.

Adipose stem/stromal cells (ASCs) after isolation produce numerous angiogenic growth factors. This justifies their use to promote angiogenesis per transplantation. In parallel, local coimplantation of ASC with endothelial cells (ECs) leading to formation of functional vessels by the donor cells suggests the existence of a mechanism responsible for fine‐tuning ASC paracrine activity essential for vasculogenesis. As expected, conditioned media (CM) from ASC promoted ECs survival, proliferation, migration, and vasculogenesis. In contrast, media from EC‐ASC cocultures had neutral effects upon EC responses. Media from cocultures exhibited lower levels of vascular endothelial growth factor (VEGF), hepatic growth factor, angiopoietin‐1, and stromal cell‐derived factor‐1 compared with those in ASC CM. Activin A was induced in ASC in response to EC exposure and was responsible for overall antivasculogenic activity of EC‐ASC CM. Except for VEGF, activin A diminished secretion of all tested factors by ASC. Activin A mediated induction of VEGF expression in ASC, but also upregulated expression of VEGF scavenger receptor FLT‐1 in EC in EC‐ASC cocultures. Blocking the FLT‐1 expression in EC led to an increase in VEGF concentration in CM. In vitro pre‐exposure of ASC to low number of EC before subcutaneous coimplantation with EC resulted in decrease in vessel density in the implants. In vitro tests suggested that activin A was partially responsible for this diminished ASC activity. This study shows that neovessel formation is associated with induction of activin A expression in ASC; this factor, by affecting the bioactivity of both ASC and EC, directs the crosstalk between these complementary cell types to establish stable vessels. StemCells2015;33:3039–3051

The Po2within the kidney changes dramatically from cortex to medulla. The present experiments examined the effect of changing Po2on epithelial Na channel (ENaC)-mediated Na transport in the collecting duct using the mpkCCD-c14 cell line. Decreasing ambient O2concentration from 20 to 8% decreased ENaC activity by 40%; increasing O2content to 40% increased ENaC activity by 50%. The O2effect required several hours to develop and was not mimicked by the acid pH that developed in monolayers incubated in low-O2medium. Corticosteroids increased ENaC activity at each O2concentration; there was no interaction. The pathways for O2and steroid regulation of ENaC are different since O2did not substantially affect Sgk1, α-ENaC, Gilz, or Usp2–45 mRNA levels, genes involved in steroid-mediated ENaC regulation. The regulation of ENaC activity by these levels of O2appears not to be mediated by changes in hypoxia-inducible factor-1α or -2α activity or a change in AMP kinase activity. Changes in O2concentration had minimal effect on α- or γ-ENaC mRNA and protein levels; there were moderate effects on β-ENaC levels. However, 40% O2induced substantially greater total β- and γ-ENaC on the apical surface compared with 8% O2; both subunits demonstrated a greater increase in the mature forms. The α-ENaC subunit was difficult to detect on the apical surface, perhaps because our antibodies do not recognize the major mature form. These results identify a mechanism of ENaC regulation that may be important in different regions of the kidney and in responses to changes in dietary NaCl.

A polymorphism at position 460(G ←W) of the α-adducin gene was found to be associated with essential hypertension in some but not all studies. The aim of the present study was to further investigate the association of the α-adducin 460W allele with essential hypertension in Chinese population.

The prevalence rate of allergic diseases, such as asthma, atopic rhinitis (AR), and atopic dermatitis (AD), has been significantly increasing over the years because of environmental changes. Type 2 immunity is mediated by allergic inflammation initiated by an innate immune response. This response is orchestrated by type 2 cytokines (interleukin [IL]-4, IL-5, IL-9, and IL-13) together with other cells. The dendritic cell [DC]-T helper 2 (Th2) cell axis is the conventional type 2 immune pathway, and is currently known as the group 2 innate lymphoid cell (ILC2)-DC-Th2 axis that mediates type 2 inflammation. ILC2s strongly mediate type 2 inflammation in allergic diseases. ILC2s are activated by epithelial cell-derived cytokines, such as IL-25 and IL-33, and thymic stromal lymphopoietin. Additionally, ILC2s are activated by mast cell lipid inflammatory mediators, such as cysteinyl leukotrienes and prostaglandin D2. ILC2s produce a large amount of type 2 cytokines. The important role of ILC2s in the pathogenesis of type 2-mediated disease has resulted in ILC2-derived cytokines being a target for therapeutic development. In this review, we discuss type 2 immunity, mainly the ILC2-DC-Th2 axis, and other immune cells, the dominant role of ILC2s in asthma, AR, and AD, and therapeutic targets against type 2 cytokines.

Computational tool-assisted primer design for real-time reverse transcription (RT) PCR (qPCR) analysis largely ignores the sequence similarities between sequences of homologous genes in a plant genome. It can lead to false confidence in the quality of the designed primers, which sometimes results in skipping the optimization steps for qPCR. However, the optimization of qPCR parameters plays an essential role in the efficiency, specificity, and sensitivity of each gene’s primers. Here, we proposed an optimized approach to sequentially optimizing primer sequences, annealing temperatures, primer concentrations, and cDNA concentration range for each reference (and target) gene. Our approach started with a sequence-specific primer design that should be based on the single-nucleotide polymorphisms (SNPs) present in all the homologous sequences for each of the reference (and target) genes under study. By combining the efficiency calibrated and standard curve methods with the 2−ΔΔCtmethod, the standard cDNA concentration curve with a logarithmic scale was obtained for each primer pair for each gene. As a result, an R2≥ 0.9999 and the efficiency (E) = 100 ± 5% should be achieved for the best primer pair of each gene, which serve as the prerequisite for using the 2−ΔΔCtmethod for data analysis. We applied our newly developed approach to identify the best reference genes in different tissues and at various inflorescence developmental stages of Tripidium ravennae, an ornamental and biomass grass, and validated their utility under varying abiotic stress conditions. We also applied this approach to test the expression stability of six reference genes in soybean under biotic stress treatment with Xanthomonas axonopodispv. glycines(Xag). Thus, these case studies demonstrated the effectiveness of our optimized protocol for qPCR analysis.

The increasing use of zinc oxide nanoparticles (ZnO NPs) as feed additives has raised huge environmental concerns in the anaerobic digestion (AD) of livestock wastes. In this study the 30-day effect of ZnO NPs on AD performance of cattle manure was investigated under optimal conditions (temperature=55 °C, initial pH=10, total solids contents=10%), which were obtained from response surface methodology. Results showed that ZnO NPs (5–100 mg/g TS) promoted the accumulation of soluble chemical oxygen demand, soluble protein and polysaccharide, but inhibited their following fermentation and methane production processes. This inhibition became stronger as ZnO NPs concentration increased, leading to the methane production declined by 84.55% (5 mg/ g TS), 92.39% (30 mg/g TS), and 93.72% (100 mg/g TS), respectively. High-throughput sequencing results demonstrated that the decrease in the abundances of functional bacteria from 72.11% to 11.24% (in families Ruminococcaceaeand Lachnospiraceae), and of methanogens from 96.82% to < 1% (in genus Methanothermobacter)led to poor fermentation and methanogenesis, respectively. Predictive functional analysis indicated that ZnO NPs can enhance abundances of AD-friendly functional genes instead of possible genetic expression, resulting in the low methane production. Our findings highlight a deep understanding of the role of ZnO NPs in cattle manure AD and are critical for guiding feed additive application.

The aim of this article was to assess whether and how BARX1 affects the progression of malignant phenotype of endometrial carcinoma (EC) cells. BARX1 levels and its prognostic value were evaluated using the EC-related RNA sequence dataset from The Cancer Genome Atlas (TCGA) database. Functional experiments were performed to evaluate the biological roles of BARX1 in EC HEC-1-A and KLE cells by silencing BARX1. BARX1 was upregulated in EC tissues according to the public database and in EC cells. High expression of BARX1 led to a poor prognosis and significantly related to clinical stage, pathological grade, death, histological subtypes, and menopause status in patients with EC. Silencing BARX1 notably suppressed the aggressive phenotypes of EC cells, as evidenced by inhibiting cells viability, growth, invasion and migration. Furthermore, depletion of BARX1 decreased the phosphorylation (p) levels of ERK and MEK, also reinforced the suppressive effects of ERK/MEK pathway blocker PD98059 on the p-ERK and p-MEK levels. Together, our results demonstrated that BARX1 functions as a carcinogen by regulating the cell viability, invasion, and migration at least partly through the ERK/MEK pathway.

Rechargeable aluminum-ion batteries (AIBs) are promising candidates for energy storage because of their low cost, high safety, and high power density due to the three-electron redox reactions. However, the applications of AIBs are hindered by their limited lifetime and practically achievable energy density. Herein, we present a novel Fe-doped NiSe nanoflake cathode material, derived from a Ni-Fe layered double hydroxide (LDH), affording a long cycle life and high energy density. Synergism between the ultrafine nanostructure and Fe doping provided shorter ion diffusion pathways and created multiple active sites in the cathode. As a result, the prepared Fe-NiSe battery achieved an outstanding energy density of 402.5 W h kg−1, and an ultra-long lifespan of 13500 cycles with a capacity decay of only 0.0026% per cycle. Furthermore, based on the mass of the entire pouch-type cell, the fabricated AIB delivered an energy density of 101.2 W h kg−1. This work paves the way for serviceable high-energy-density AIBs.

A greater understanding of the mechanisms behind HSPC trafficking is vital to increase the efficacy of HSPC therapy. The composition of adult blood plasma (ABP) is well documented, in particular proteins and metabolites, but very little is known about umbilical cord blood plasma (CBP) which may contain a host of bioactive proteins and lipids released as a stress response during birth as opposed to ABP which is generally regarded as homeostatic. Physiologic stress response may result in altered concentration gradients of these factors thereby creating differing gradients between the marrow and peripheral blood compartments. We sought to investigate factors in CBP and ABP and their effects on HSPC migration.Both ABP (Research Blood Components, Boston, MA) and CBP was filtered through 0.22 μm filters to remove debris and any cells remaining in the plasma. ABP contained 14% Citrate Phosphate Dextrose Anticoagulant (CPDA) by volume while CBP varied between 28–40% CPDA by volume. ABP samples used in migration assays were diluted to match the CBP concentration with PBS. We screened for the concentrations of 115 known proteins using a multiplexed ELISA assay and compared a pool of 10 CBPs against a pool of 10 ABPs. Umbilical cord blood (UCB) from research units not meeting clinical cell dose threshold was provided by the New York Blood Center (P. Rubinstein, MD). We found 43 proteins were elevated at least two-fold in CBP versus ABP, 16 of which were elevated 10-fold relative to ABP. Out of these 43 proteins, 6 have potential implications on HSPC mobilization: IL8, GCSF (CSF-2), VCAM, MCP1, MIP3, and CXCL10. The concentrations of the proteins in CBP are in pg/mL: IL8 546.85; GCSF 609.91; MCP1 1142.02; MIP3 80.80; VCAM 4, 016, 017.46; and CXCL10 218.27. The fold increase in CBP for these proteins are: IL8 19.39; GCSF 6.39; MCP1 4.12; MIP3 3.48; VCAM 4.20; and CCL10 2.00. The relative contribution of each protein to migration was measured by preparing aliquots of CBP and treating the aliquots with neutralizing antibodies toward each protein (Abcam, Cambridge, MA). Antibodies were incubated at a concentration of 1μg/mL in accordance with recommended concentrations from Abcam. In addition to these 6 proteins, S1P and C3a concentrations were also investigated due to their potential effect in HSPC mobilization. Migration experiments were conducted using Transwell plates (Corning Life Sciences, Lowell, MA). UCB was obtained 24–48 hrs following delivery, and CBP and mononuclear cells were isolated by centrifugation through a Ficoll-Paque density gradient. UCB CD34+ cells were selected by magnetic labeling and sorting using AutoMACS magnetic cell sorter (Miltenyi Biotec, Auburn, CA). UCB HSPCs were placed in upper transwells (8.0 μm pores; 1.5 × 105 cells/well) and the lower well contained CBP, ABP, or fresh RPMI basal media as a control. The cells were allowed to migrate towards the various solutions for 3 h. Cells that migrated were counted and immunophenotyped via hemocytometer and flow cytometry (BD FACS Calibur). In a comparison of migration towards CBP vs. ABP (n = 11 and 10), CBP exhibited an average increase in migration by 157.8 ± 44.1%. Migrations towards CBP depleted of one of the 6 proteins exhibited the following HSPC migrations compared to untreated CBP (100%) were: 48.9 ± 17% for IL8-neutralized (n=6); 90.2 ± 20.4% for GCSF-neutralized (n=5); 102 ± 18.0% for MCP-neutralized (n=5); 71.7 ± 19.8% for MIP3-neutralized (n=6); 35.4 ± 14.7% for VCAM-neutralized (n=4); and 51.7 ± 9.5% for CXCL10-neutralized CBP (n=4).All samples of CBP and ABP used in the migration studies were analyzed for S1P concentration by LC-MS/MS. S1P concentrations in CBP samples ranged from 0.95 to 2.27 (n = 6) times the concentration of S1P in ABP. Additionally, C3a in these CBPs and ABP was analyzed by an ELISA (BD Biosciences, San Jose CA). In CBP samples, C3a varied from 111–297 ng/mL (n = 13) compared to 661 ng/mL in ABP.An improved knowledge of the factors that influence mobilization may provide us with a better approach towards stem cell priming and graft HSPC engineering prior to transplantation. The proteins examined here and the effects of S1P and C3a on HSPC migration may provide novel insights into the factors that influence HSPC trafficking. Further understanding of HSPC migration to proteins released in stress response may be exploited to direct HSPC trafficking in the autologous and allogeneic setting.Lynch: SphynKx Therapeutics LLC: Equity Ownership.

Umbilical cord blood cells (UCB) from allogeneic donors have been established as an alternative source for HSC transplantation in patients who lack suitably HLA matched bone marrow or peripheral blood stem cells from adult donors. Transplantation using 2 unit UCB has been shown to compensate the low engraftment and slow hematopoietic recovery resulting from 1 unit UCB transplantation in full stature adult patients. At present, there are no unit specific factors that reliably predicts for the “winning unit” in 2 unit UCB transplantation, e.g. cell viability, number of infused total nucleated cells, CD34+ or CD3+ cells, sex mismatch, ABO blood group, and degree of HLA mismatch. In vivo mouse models suggest that CD34 negative subsets play an important role. Among CD34 negative subsets, CD8 T subset accounts for approximately 34.0+/-23.3% of T lymphocytes from UCB. In bone marrow transplantation CD8 T cells have been found to facilitate donor hematopoietic cell engraftment. Moreover, it has been reported that 1 dominant unit coincides with a specific CD8 T cell response against the non-engrafted unit which was not observed from CD4 or NK cells.In this study, we used volunteer donated UCB research units (kindly provided by P. Rubinstein, MD, New York Blood Center). Mononuclear cells (MNC) were purified by Ficoll gradient centrifugation, and CD3 T cells were isolated with CD3 MicroBeads (Miltenyi Biotec; autoMACS). The purified CD3 (confirmed by FACS >95% purity) cells were labeled with CFSE and DDAO-SE. After labeling, the cells from two different donors were mixed in 96-well U-bottom plates for continued culture in 37 °C 5% CO2. The expansion from each labeled donor cells was evaluated using flow cytometry; the dead cells were gated out using propidium iodide, and the data was analyzed using FlowJo software. For proper T cells activation, we also compared different activation conditions using i.) anti-CD3/CD28 Beads, ii.) anti-CD3 antibody plus anti-CD28 antibody, and iii.) cytokine IL-2. The schematic illustration of methods is shown in Figure 1.We noted that T cells from UCB are primarily at nai¨ve stage as determined by CD45RA (93.8 +/- 7.11%) and CCR7 (84.9 +/- 12.0%) expression. We also determined the optimal activation condition using a modified mixed lymphocyte reaction from 2 UCB units. Four days after incubation, the proliferation from 2 units labeled with CFSE and DDAO-SE could be reproducibly distinguished using FL1 channel for CFSE and FL4 channel for DDAO-SE (Figure 1). The optimal concentration for labeling using CFSE (1 mM) and DDAO (1 µM or 3 mM) was determined by titration. To avoid cell toxicity resulting from CFSE and DDAO-SE labeling, as well as self-crossing from each donor using two dyes, we examined additional mixed lymphocyte analyses in which each donor was labeled with CFSE or DDAO-SE respectively and vice versa. As shown in Figure 1, we found consistently that the predicated dominant unit accounted for the majority of culture (73.2% stained with DDAO; 63.5% stained with CFSE) after 4 days co-culture. The dominance was not correlated with cell proliferation indicated by the proliferation index (1.12 for dominant and 1.48 for another unit). After confirmation of this in vitro assay, further studies were conducted to evaluate the IFN-? release of 2 UCB units in this optimized mixed lymphocyte assay in the condition using cytokine IL-2. Interestingly, we could only detect IFN-? by intracellular staining in one unit when co-culture was set-up using CD3 T cells from each unit; the expression of IFN-? was not detected when we used CD3 T cells from 1 unit. The correlation between dominance and the expression of IFN-? is currently under investigation.UCB Transplantation is an important alternative for patients lacking bone marrow or peripheral blood stem cell donors. With the establishment of this novel modified mixed lymphocyte in vitro assay for prediction of the “winning” immune dominant unit, routine analyses can be performed to guide unit selection. Further interventions can be exploited to preferentially treat the expected dominant unit with glycosylation, cytokines, prostaglandins, or C3a compliments to further enhance hematopoietic stem cells trafficking and engraftment to the marrow.No relevant conflicts of interest to declare.

T-cell transcription factor NFAT1 (NFATc2) has been reported to regulate cell cycle regulatory proteins including cyclins, cyclin-dependent kinases (CDKs), and p21. MicroRNA-184 (miR-184) has been noted to have close sequence homology to the 3’ UTR of NFAT1 and to regulate NFAT1 mRNA translation in human adult and umbilical cord blood (UCB) CD3+CD4+ T-cells. In this study, we are characterizing the functional role of NFAT1 in T-ALL cells to determine whether its dysregulation may alter normal cell cycle regulation and thereby may contribute to T-ALL leukemogenesis.In this study we used MOLT-4 cell line to investigate the role of NFAT1 in T-ALL, since MOLT-4 represents early stage of human T-ALL (CD4+CD8+ double positive) compared with other T-cell lines including Jurkat. We developed two distinct approaches to explore NFAT1 signalling, either by manipulating the expression level of NFAT1 protein by blocking miR-184, or by overexpressing a constitutively active form of NFAT1 (caNFAT1) holding a HA-tag (Addgene plasmid 11792, A. Rao, PhD, Harvard University). caNFAT1 was introduced into gateway system expression vector pEF-DEST51. MOLT-4 cells were maintained in complete RPMI 1640 medium according to ATCC guidelines. Electroporation transfection was performed on Amaxa Nucleofector I using program C-05. 200 nM of anti-miR-184 miRNA inhibitor (Dharmacon) or 2 μ g of plasmid containing caNFAT1 was transfected into 2×10e6 MOLT-4 cells in each sample. Transfection efficiency was tested using pmaxGFP (Lonza). Proteins were extracted at various time points after transfection using RIPA buffer (PIERCE) with protease inhibitor cocktail and tested for the expression of NFAT1 (BD Biosciences), phosphorylated NFAT1 (Santa Cruz Biotechnology), HA (Roche), and beta-actin (Sigma-Aldrich). Cell cycle was measured at various time points by pelleting down 0.5–1 X10e6 cells, followed by ethanol fixation, and incubation in propidium iodide (PI) (10 μ g/ml) staining solution and flow cytometric measurement for PI incorporation. Cell apoptosis was measured by PE-conjugated annexin V staining (BD Biosciences) in transfected MOLT-4 cells including empty plasmid controls.The over-expression of caNFAT1 in MOLT-4 led to increased proportion of cells in G2 phase than in controls (10.8% vs. 7.3% on day5, and 11.8% vs. 8.1% on day6). In addition to these cell cycle changes in MOLT-4 treated with caNFAT1 plasmid, higher rates of apoptosis were noted on day 2 (28.3% vs. 15.5%), which notably leveled off from day 3 to day 6 potentially attributed to compensatory cellular responses. Previous work by this group has shown higher expression of miR-184 in MOLT-4 by 56 fold compared with normal human CD4/8 double positive T-cells. Here, miR-184 knockdown was also notable for a larger proportion of MOLT-4 cells in G2 cell cycle phase and higher apoptosis, albeit modest changes compared with that exerted by caNFAT1.In MOLT-4 cell line, this preliminary data suggests that increasing active non-phosphorylated NFAT1 is associated with enhanced proportion of cells in G2 phase as well as enhanced resultant apoptosis. Taken together, regulation of NFAT1 may be exploited as potential T-ALL targeted therapy.No relevant conflicts of interest to declare.

Abstract 4699