TARDBP drives T-cell acute lymphoblastic leukemia progression by binding MDM2 mRNA, involving ß-catenin pathway.

T-cell acute lymphoblastic leukemia (T-ALL) is a dangerous hematological malignancy. The trans-activation response DNA binding protein (TARDBP), an RNA/DNA binding protein, is involved in the growth and metastasis of multiple cancers. However, TARDBP has not been reported in T-ALL. It was found that TARDBP was highly expressed in pediatric T-ALL samples by microarray GSE26713 (log2 fold change >1, p < .05). Herein, TARDBP was silenced and overexpressed by lentivirus transduction in T-ALL cell lines, including Jurkat and Molt4 cells. In vitro, silencing TARDBP inhibited T-ALL cell proliferation and cycle progression and accelerated cell apoptosis, while overexpressing TARDBP induced the opposite effects. In addition, we investigated whether the ß-catenin pathway could be activated by TARDBP in T-ALL cells. Moreover, XAV-939, a ß-catenin inhibitor, was capable of suppressing the malignant phenotypes in TARDBP-overexpressed T-ALL cells. In vivo, TARDBP-silenced or TARDBP-overexpressed T-ALL cells were injected into mice. We found that TARDBP promoted T-ALL cell growth in the spleens and bone marrows of mice. On the basis of GSE26713, there was a significant correlation between TARDBP and mouse double minute 2 (MDM2). The RIP-PCR assay demonstrated that TARDBP bound MDM2 mRNA in T-ALL cells. The rescue experiments further revealed the roles of the TARDBP/MDM2 axis in T-ALL cell phenotypes, which was also reflected by mRNA-seq. In aggregate, we explored a promising biomarker, TARDBP, for T-ALL treatment. The underlying mechanisms might involve the interaction with MDM2 mRNA and the regulation of the ß-catenin pathway.

Antibody-Free Glycogen Nanoparticles Engage Human Immune T Cells for Intracellular Delivery of Small Drugs or mRNA.

T cells play a major role in immune defense against viral infections and diseases such as cancer. Accordingly, developing nanoparticle (NP) systems to effectively deliver therapeutics to T cells is of interest. However, NP-mediated delivery of drugs to T cells is challenging because of the nonphagocytic nature of T cells. To engage T cells and induce cellular internalization, NPs are typically decorated with specific receptor-targeting antibodies, often using laborious and costly procedures. Herein, we report that natural glycogen NPs (i.e., nanosugars) with different sizes (20-80 nm) and surface charges (neutral and positively charged) engage Jurkat T cells, undergo intracellular trafficking, and release encapsulated drug without the use of receptor-targeting antibodies. Specifically, glycogen-resveratrol constructs are employed to reactivate HIV-1 latently infected Jurkat T cells (J-Lat A2) and trigger proviral expression. Both neutral and positively charged glycogen NPs engage with J-Lat A2 cells. Large (84 ± 29 nm) and positively charged (23 ± 5 mV) NPs, denoted phytoglycogen-ethylenediamine (PGEDA) NPs, readily associate with the cell membrane and are internalized (60%) in J-Lat A2 cells but remain confined in the endocytic vesicles, with moderate reactivation of latent HIV-1 (4.7 ± 0.5%). Conversely, small (21 ± 5 nm) and positively charged (10 ± 6 mV) NPs, bovine glycogen-EDA (BGEDA) NPs, associate slowly with T cells but show nearly 100% internalization and efficient endosomal escape properties, resulting in 1.5-fold higher reactivation of latent HIV-1 in T cells. PGEDA NPs and BGEDA NPs are also internalized by primary human T cells (>90% cell association) and enable the transfection of mRNA, with BGEDA NPs showing a 2-fold higher transfection than PGEDA NPs. This work highlights the potential of BGEDA NPs for the effective intracellular delivery of small-molecule drugs and mRNA in T cells.

A Customizable Platform to Integrate CAR and Conditional Expression of Immunotherapeutics in T Cells.

The potential of chimeric antigen receptor (CAR)-based immunotherapy as a promising therapeutic approach is often hindered by the presence of highly immunosuppressive tumor microenvironments (TME). Combination therapies with either co-administration or built-in expression of additional TME-modulating therapeutic molecules to potentiate the functions of CAR-T cells can cause systemic toxicities due to the lack of control over the delivery of biologics. Here, we present a proof-of-concept engineered platform in human Jurkat T cells that combines CAR with a therapeutic gene circuit capable of sensing ß-galactosidase (a reported cancer-associated signal) and subsequently activate the production of customized therapeutic gene products. We have demonstrated the integration of the chemically induced proximity (CIP) and associated signal sensing technologies with CAR in this study. A ß-galactosidase-activatable prodrug was designed by conjugating a galactose moiety with a CIP inducer abscisic acid (ABA). We showed that Jurkat T cells engineered with CAR and the ABA-inducible genetic circuits can respond to recombinant ß-galactosidase to drive the production and secretion of various immunotherapeutics including an anti-cancer agent, an immunomodulatory cytokine, and immune checkpoint inhibitors. Our design is highly modular and could be adapted to sense different cancer-related signals to locally produce antitumor therapeutics that can potentially boost CAR-T efficacy and persistence.

Numerical assessments of geometry, proximity and multi-electrode effects on electroporation in mitochondria and the endoplasmic reticulum to nanosecond electric pulses.

Most simulations of electric field driven bioeffects have considered spherical cellular geometries or probed symmetrical structures for simplicity. This work assesses cellular transmembrane potential build-up and electroporation in a Jurkat cell that includes the endoplasmic reticulum (ER) and mitochondria, both of which have complex shapes, in response to external nanosecond electric pulses. The simulations are based on a time-domain nodal analysis that incorporates membrane poration utilizing the Smoluchowski model with angular-dependent changes in membrane conductivity. Consistent with prior experimental reports, the simulations show that the ER requires the largest electric field for electroporation, while the inner mitochondrial membrane (IMM) is the easiest membrane to porate. Our results suggest that the experimentally observed increase in intracellular calcium could be due to a calcium induced calcium release (CICR) process that is initiated by outer cell membrane breakdown. Repeated pulsing and/or using multiple electrodes are shown to create a stronger poration. The role of mutual coupling, screening, and proximity effects in bringing about electric field modifications is also probed. Finally, while including greater geometric details might refine predictions, the qualitative trends are expected to remain.

Targeting telomerase with MST-312 leads to downregulation of CCND1, MDM2, MYC, and HSP90AA1 and induce apoptosis in Jurkat cell line.

Acute lymphoblastic leukemia is a challenging disease to treat, especially in older adults who are most commonly diagnosed and have a high risk of relapse, even with current treatment options. MST-312, targets the RNA component of telomerase, inhibiting its activity and leading to growth arrest and telomere shortening in cancer cells. This study aimed to investigate the effects of MST-312 on apoptosis rates and the expression of telomerase target genes, CCND1, MDM2, MYC, and HSP90AA1, in Jurkat cell line. Jurkat cell line was cultured and treated with various concentrations of MST-312(0 µM, 0.5 µM, 1 µM, 2 µM, and 4 µM). The optimal concentration of MST-312 was determined using MTT assay. Flow cytometry was employed to evaluate the apoptosis induced by MST-312 treatment. The expression levels of the target genes were measured using real-time polymerase chain reaction before and after the treatment with MST-312. P-value < 0.05 was considered statistically significant. The percentages of apoptotic cells after 48 h, as determined by flow cytometry analysis, were 30.32%, 52.35%, 57.60%, and 68.82%, respectively, compared to the control group which was 4.6%. The expression levels of all genes, including CCND1, MDM2, MYC, and HSP90AA1, were decreased compared to the control group. The results showed that MST-312 induced dose- and time-dependent apoptosis and downregulated the expression of CCND1, MDM2, MYC, and HSP90AA1in Jurkat cell line.

Physiologically Achievable Concentration of 2-Deoxy-D-Glucose Stimulates IFN-γ Secretion in Activated T Cells In Vitro.

2-deoxy-D-glucose (2DG) is a glycolysis and protein N-glycosylation inhibitor with promising anti-tumor and immunomodulatory effects. However, 2DG can also suppress T cell function, including IFN-γ secretion. Few human T cell studies have studied low-dose 2DG, which can increase IFN-γ in a Jurkat clone. We therefore investigated 2DG's effect on IFN-γ in activated human T cells from PBMCs, with 2DG treatment commenced either concurrently with activation or 48 h after activation. Concurrent 2DG treatment decreased IFN-γ secretion in a dose-dependent manner. However, 2DG treatment of pre-activated T cells had a hormetic effect on IFN-γ, with 0.15-0.6 mM 2DG (achievable in vivo) increasing and >2.4 mM 2DG reducing its secretion. In contrast, IL-2 levels declined monotonously with increasing 2DG concentration. Lower 2DG concentrations reduced PD-1 and increased CD69 expression regardless of treatment timing. The absence of increased T-bet or Eomes expression or IFNG transcription suggests another downstream mechanism. 2DG dose-dependently induced the unfolded protein response, suggesting a possible role in increased IFN-γ secretion, possibly by increasing the ER folding capacity for IFN-γ via increased chaperone expression. Overall, low-dose, short-term 2DG exposure could potentially improve the T cell anti-tumor response.

Glycogen nanoparticles for efficient mRNA transduction to T lymphocytes.

T lymphocyte therapies demonstrate significant promise in the treatment of cancer and infectious diseases. An efficient gene delivery system is essential for the safe and reliable introduction of exogenous genes, especially mRNA, into cells to achieve therapeutic purposes. Commercial transfection reagents are suitable for the transduction of plasmids to adherent cells, whereas they are ineffective for suspension cells such as T lymphocytes and for unstable mRNA. Moreover, the cytotoxicity of transfection reagents themselves constitutes an impediment to their application. The challenge of mRNA transduction to T lymphocytes with high efficiency is notably formidable. An innovative transfection strategy is urgently needed. In this study, we synthesized aminated glycogen (AGly) nanoparticles as gene vectors, encapsulating mRNA to facilitate the efficient transfection of T lymphocytes. Compared to commercial transfection reagent polyethylenimine (PEI), the AGly demonstrated favorable biocompatibility. The positive charge provided AGly with pH buffering ability and mRNA-binding capacity. AGly formed stable nanoparticles with mRNA, which were readily internalized by suspension cells and enhanced the cellular uptake of mRNA. In the T lymphocyte model cell lines (Jurkat cells and HuT 78 cells), AGly demonstrated superior transfection efficiency than that of PEI. Consequently, AGly can emerge as a viable mRNA vector for the efficient transfection of T lymphocytes whilst circumventing the issue of cytotoxicity. The AGly designed in this study provides a novel concept for the exploitation of transfection reagents and proposes a promising methodology for the proficient transfection of T lymphocytes which may significantly contribute to the treatment of cancer and other complex diseases.

IL23R mutations associated with decreased risk of psoriasis lead to the differential expression of genes implicated in the disease.

Psoriasis is an incurable immune-mediated skin disease, affecting around 1%-3% of the population. Various lines of evidence implicate IL23 as being pivotal in disease. Genetic variants within the IL23 receptor (IL23R) increase the risk of developing psoriasis, and biologic therapies specifically targeting IL23 demonstrated high efficacy in treating disease. IL23 acts via the IL23R, signalling through the STAT3 pathway, mediating the cascade of events that ultimately results in the clinical presentation of psoriasis. Given the essential role of IL23R in disease, it is important to understand the impact of genetic variants on receptor function with respect to downstream gene regulation. Here we developed model systems in CD4+ (Jurkat) and CD8+ (MyLa) T cells to express either the wild type risk or mutant (R381Q) protective form of IL23R. After confirmation that the model system expressed the genes/proteins and had a differential effect on the phosphorylation of STAT3, we used RNAseq to explore differential gene regulation, in particular for genes implicated with risk to psoriasis, at a single time point for both cell types, and in a time course experiment for Jurkat CD4+ T cells. These experiments discovered differentially regulated genes in the cells expressing wild type and mutant IL23R, including HLA-B, SOCS1, RUNX3, CCR5, CXCR3, CCR9, KLF3, CD28, IRF, SOCS6, TNFAIP and ICAM5, that have been implicated in both the IL23 pathway and psoriasis. These genes have the potential to define a IL23/psoriasis pathway in disease, advancing our understanding of the biology behind the disease.

Influence of Anticoagulants and Heparin Contaminants on the Suitability of MMP-9 as a Blood-Derived Biomarker.

In contrast to other common anticoagulants such as citrate and low-molecular-weight heparin (LMWH), high-molecular-weight heparin (HMWH) induces the expression of matrix metalloproteinase (MMP)-9, which is also measured as a biomarker for stroke in blood samples. Mechanistically, HMWH-stimulated T cells produce cytokines that induce monocytic MMP-9 expression. Here, the influence of further anticoagulants (Fondaparinux, Hirudin, and Alteplase) and the heparin-contaminating glycosaminoglycans (GAG) hyaluronic acid (HA), dermatan sulfate (DS), chondroitin sulfate (CS), and over-sulfated CS (OSCS) on MMP-9 was analyzed to assess its suitability as a biomarker under various conditions. Therefore, starved Jurkat T cells were stimulated with anticoagulants/contaminants. Subsequently, starved monocytic THP-1 cells were incubated with the conditioned Jurkat supernatant, and MMP-9 mRNA levels were monitored (quantitative (q)PCR). Jurkat-derived mediators secreted in response to anticoagulants/contaminants were also assessed (proteome profiler array). The supernatants of HMWH-, Hirudin-, CS-, and OSCS-treated Jurkat cells comprised combinations of activating mediators and led to a significant (in the case of OSCS, dramatic) MMP-9 induction in THP-1. HA induced MMP-9 only in high concentrations, while LMWH, Fondaparinux, Alteplase, and DS had no effect. This indicates that depending on molecular weight and charge (but independent of anticoagulant activity), anticoagulants/contaminants provoke the expression of T-cell-derived cytokines/chemokines that induce monocytic MMP-9 expression, thus potentially impairing the diagnostic validity of MMP-9.

Label-free high-throughput impedance-activated cell sorting.

Cell sorting holds broad applications in fields such as early cancer diagnosis, cell differentiation studies, drug screening, and single-cell sequencing. However, achieving high-throughput and high-purity in label-free single-cell sorting is challenging. To overcome this issue, we propose a label-free, high-throughput, and high-accuracy impedance-activated cell sorting system based on impedance detection and dual membrane pumps. Leveraging the low-latency characteristics of FPGA, the system facilitates real-time dual-frequency single-cell impedance detection with high-throughput (5 × 104 cells per s) for HeLa, MDA-MB-231, and Jurkat cells. Furthermore, the system accomplishes low-latency (less than 0.3 ms), label-free, high-throughput (1000 particles per s) and high-accuracy (almost 99%) single-particle sorting using FPGA-based high-precision sort-timing prediction. In experiments with Jurkat and MDA-MB-231 cells, the system achieved a throughput of up to 1000 cells per s, maintaining a pre-sorting purity of 28.57% and increasing post-sorting purity to 97.09%. These findings indicate that our system holds significant potential for applications in label-free, high-throughput cell sorting.

Impact of controlled ice nucleation on intracellular dehydration, ice formation and their implications on T cell freeze-thaw viability.

Cryopreservation is important in manufacturing of cell therapy products, influencing their safety and effectiveness. During freezing and thawing, intracellular events such as dehydration and ice formation can impact cell viability. In this study, the impact of controlling the ice nucleation temperature on intracellular events and viability were investigated. A model T cell line, Jurkat cells, were evaluated in commercially relevant cryoformulations (2.5 and 5 % v/v DMSO in Plasma-Lyte A) using a cryomicroscopic setup to monitor the dynamic changes cells go through during freeze-thaw as well as a controlled rate freezer to study bulk freeze-thaw. The equilibrium freezing temperatures of the studied formulations and a DMSO/Plasma-Lyte A liquidus curve were determined using DSC. The cryomicroscopic studies revealed that an ice nucleation temperature of -6°C, close to the equilibrium freezing temperatures of cryoformulations, led to more intracellular dehydration and less intracellular ice formation during freezing compared to either a lower ice nucleation temperature (-10 °C) or uncontrolled ice nucleation. The cell membrane integrity and post thaw viability in bulk cryopreservation consistently demonstrated the advantage of the higher ice nucleation temperature, and the correlation between the cellular events and cell viability.

CD38 in SLE CD4 T cells promotes Ca2+ flux and suppresses interleukin-2 production by enhancing the expression of GM2 on the surface membrane.

CD38 has emerged as a potential therapeutic target for patients with systemic lupus erythematosus (SLE) but it is not known whether CD38 alters CD4+ T cell function. Using primary human T cells and CD38-sufficient and CD38-deficient Jurkat T cells, we demonstrate that CD38 shifts the T cell lipid profile of gangliosides from GM3 to GM2 by upregulating B4GALNT1 in a Sirtuin 1-dependent manner. Enhanced expression of GM2 causes ER stress by enhancing Ca2+ flux through the PLCγ1-IP3 pathway. Interestingly, correction of the calcium overload by an IP3 receptor inhibitor, but not by a store-operated calcium entry (SOCE) inhibitor, improves IL-2 production by CD4+ T cells in SLE. This study demonstrates that CD38 affects calcium homeostasis in CD4+ T cells by controlling cell membrane lipid composition that results in suppressed IL-2 production. CD38 inhibition with biologics or small drugs should be expected to benefit patients with SLE.

An Integrated Strategy of UHPLC-ESI-MS/MS Combined with Bioactivity-Based Molecular Networking for Identification of Antitumoral Withanolides from Athenaea fasciculata (Vell.) I.M.C. Rodrigues & Stehmann.

BACKGROUND: Athenaea fasciculata, a Brazilian native species from the Solanaceae family, is recognized as a promising source of bioactive withanolides, particularly Aurelianolide A and B, which exhibit significant antitumoral activities. Despite its potential, research on the chemical constituents of this species remains limited. This study aimed to dereplicate extracts and partitions of A. fasciculata to streamline the discovery of bioactive withanolides. METHODS: Using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), various extracts-including n-hexane, methanol, and ethanol-were analyzed, and their mass spectrometry data were processed through the GNPS platform for the generation of molecular networking. The results indicated that crude extracts displayed comparable cytotoxicity against Jurkat cells, by treatment at 150 µg/mL, while alcoholic extracts achieved approximately 80% inhibition of K562 cells and K562-Lucena 1 at the same concentration. Notably, the dichloromethane partition exhibited the highest cytotoxicity across leukemia cell lines, particularly against Jurkat cells (IC50 = 14.34 µg/mL). A total of 22 compounds were annotated by manual inspection and different libraries, with six of them demonstrating significant cytotoxic effects. CONCLUSIONS: This research underscores the therapeutic potential of A. fasciculata and highlights the effectiveness of integrating advanced analytical methods in drug discovery, paving the way for further exploration of its bioactive compounds.

Phytochemical Characterization of Bilberries and Their Potential as a Functional Ingredient to Mitigate Ochratoxin A Toxicity in Cereal-Based Products.

Mycotoxin contamination of cereals and cereal-based products is a serious problem for food safety. Antioxidant-rich ingredients such as bilberries (Vaccinium myrtillus L., VM) may mitigate their harmful effects. Firstly, total phenolic content, antioxidant activity, and analytical phytochemical composition (hydroxycinnamic and hydroxybenzoic acids, flavanols, flavonols, and anthocyanins) were assessed in lyophilized wild bilberries from Romania. Secondly, this study evaluated bilberries' effects on reducing ochratoxin A (OTA) bioaccessibility and cytotoxicity. An in vitro digestion model was developed and applied to four different types of bread: Control, VM (2%), OTA (15.89 ± 0.13 mg/kg), and OTA (16.79 ± 0.55 mg/kg)-VM (2%). The results indicated that VM decreased OTA bioaccessibility by 15% at the intestinal level. OTA-VM digests showed improved Caco-2 cell viability in comparison to OTA digests across different exposure times. Regarding the alterations in Jurkat cell line cell cycle phases and apoptosis/necrosis, significant increases in cell death were observed using OTA digests (11%), while VM addition demonstrated a protective effect (1%). Reactive oxygen species (ROS) analysis confirmed these findings, with OTA-VM digests showing significantly lower ROS levels compared to OTA digests, resulting in a 3.7-fold decrease. Thus, bilberries exhibit high potential as a functional ingredient, demonstrating protection in OTA mitigation effects.

LYP regulates SLP76 and other adaptor proteins in T cells.

BACKGROUND: The LYP tyrosine phosphatase presents a SNP (1858C > T) that increases the risk of developing autoimmune diseases such as type I diabetes and arthritis. It remains unclear how this SNP affects LYP function and promotes the development of these diseases. The scarce information about LYP substrates is in part responsible for the poor understanding of LYP function. RESULTS: In this study, we identify in T lymphocytes several adaptor proteins as potential substrates targeted by LYP, including FYB, SLP-76, HS-1, Vav, SKAP1 and SKAP2. We also show that LYP co-localizes with SLP76 in microclusters, upon TCR engagement. CONCLUSIONS: These data indicate that LYP may modulate T cell activation by dephosphorylating several adaptor proteins, such as FYB, SLP-76, HS-1, Vav, SKAP1 and SKAP2 upon TCR engagement.

A pleiotropic recurrent dominant ITPR3 variant causes a complex multisystemic disease.

Inositol 1,4,5-trisphosphate (IP3) receptor type 1 (ITPR1), 2 (ITPR2), and 3 (ITPR3) encode the IP3 receptor (IP3R), a key player in intracellular calcium release. In four unrelated patients, we report that an identical ITPR3 de novo variant-NM_002224.3:c.7570C>T, p.Arg2524Cys-causes, through a dominant-negative effect, a complex multisystemic disorder with immunodeficiency. This leads to defective calcium homeostasis, mitochondrial malfunction, CD4+ lymphopenia, a quasi-absence of naïve CD4+ and CD8+ cells, an increase in memory cells, and a distinct TCR repertoire. The calcium defect was recapitulated in Jurkat knock-in. Site-directed mutagenesis displayed the exquisite sensitivity of Arg2524 to any amino acid change. Despite the fact that all patients had severe immunodeficiency, they also displayed variable multisystemic involvements, including ectodermal dysplasia, Charcot-Marie-Tooth disease, short stature, and bone marrow failure. In conclusion, unlike previously reported ITPR1-3 deficiencies leading to narrow, mainly neurological phenotypes, a recurrent dominant ITPR3 variant leads to a multisystemic disease, defining a unique role for IP3R3 in the tetrameric IP3R complex.

Anti-Inflammatory and Anti-(Lymph)angiogenic Properties of an ABCB5+ Limbal Mesenchymal Stem Cell Population.

Corneal transparency and avascularity are essential for vision. The avascular cornea transitions into the vascularized conjunctiva at the limbus. Here, we explore a limbal stromal cell sub-population that expresses ABCB5 and has mesenchymal stem cell characteristics. Human primary corneal stromal cells were enriched for ABCB5 by using FACS sorting. ABCB5+ cells expressed the MSC markers CD90, CD73, and CD105. ABCB5+ but not ABCB5- cells from the same donor displayed evidence of pluripotency with a significantly higher colony-forming efficiency and the ability of trilineage differentiation (osteogenic, adipogenic, and chondrogenic). The ABCB5+ cell secretome demonstrated lower levels of the pro-inflammatory protein MIF (macrophage migration inhibitory factor) as well as of the pro-(lymph)angiogenic growth factors VEGFA and VEGFC, which correlated with reduced proliferation of Jurkat cells co-cultured with ABCB5+ cells and decreased proliferation of blood and lymphatic endothelial cells cultured in ABCB5+ cell-conditioned media. These data support the hypothesis that ABCB5+ limbal stromal cells are a putative MSC population with potential anti-inflammatory and anti-(lymph)angiogenic effects. The therapeutic modulation of ABCB5+ limbal stromal cells may prevent cornea neovascularization and inflammation and, if transplanted to other sites in the body, provide similar protective properties to other tissues.

The n-Butanol Extract Obtained from the Inner Bark of Tabebuia rosea (Bertol.) DC, Specioside, and Catalposide Induce Leukemia Cell Apoptosis in the Presence of Apicidin.

The cell signaling pathways involved in the antiproliferative activities of T. rosea inner bark remain unexplored. This study evaluated the apoptotic effects of two iridoids from the inner bark of T. rosea and apicidin on THP-1 cells. The cytotoxic effects of the extract and the pure compounds on THP-1 and Jurkat cells were also evaluated using the MTT assay. The apoptotic effect was determined by measuring the mitochondrial membrane potential. The expression of mRNA and MAPK kinase, Bax, and Bcl-2 proteins was detected by Western blotting and RT-qPCR, respectively. The extract and the compounds evaluated increased the percentage of apoptotic cells. Depolarization of the mitochondrial membrane was observed, and the number of cells in the G0/G1 phase increased. Catalposide and specioside significantly increased p38 protein expression, mostly in cells pretreated with apicidin. The p38 MAPK signaling pathway is at least one of the pathways by which the n-butanol extract obtained from Tabebuia rosea, catalposide, and specioside exerts its apoptotic effect on THP-1 cells, and this effect generates a response in the G0/G1 phase and subsequent cell death. In addition, there was depolarization of the mitochondrial membrane, an effect that was related to the participation of the proapoptotic protein Bax.

Cell-Penetrating Peptides Translocate across the Plasma Membrane by Inducing Vesicle Budding and Collapse.

Cell-penetrating peptides (CPPs) enter the cell by two different mechanisms-endocytosis followed by endosomal escape and direct translocation at the plasma membrane. The mechanism of direct translocation remains unresolved. In this work, the direct translocation of nonaarginine (R9) and two cyclic CPPs (CPP12 and CPP17) into Jurkat cells was monitored by time-lapse confocal microscopy. Our results provide direct evidence that all three CPPs translocate across the plasma membrane by a recently discovered vesicle budding-and-collapse (VBC) mechanism. Membrane translocation is preceded by the formation of nucleation zones. Up to four different types of nucleation zones and three variations of the VBC mechanism were observed. The VBC mechanism reconciles the enigmatic and conflicting observations in the literature.

A pilot study of microRNAs expression profile in plasma of patients with hydatid disease: potential immunomodulation of hydatid disease.

Echinococcosis is a zoonotic disease, which seriously endangers human health. The immune game between parasite and host is not fully understood. Exosomes are thought to be one of the ways of information communication between parasite and host. In this study, we attempted to explore the communication between Echinococcus granulosus and its host through the medium of exosomes. We collected plasma from E. granulosus patients (CE-EXO) and healthy donors (HD-EXO) and extracted exosomes from the plasma. The expression profile of miRNA in plasma was determined by second generation sequencing. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to annotate the function of target genes of differential miRNAs. Meanwhile, we co-cultured plasma exosomes from healthy donors and plasma exosomes from E. granulosus patients with Jurkat T cells with or without phytohaemagglutinin (PHA) stimulation. The expression of CD69 on Jurkat T cells was detected by flow cytometry. The results showed that the miRNA of exosomes between healthy donors and E. granulosus patients was significantly different. GO and KEGG were used to annotate the function of target genes of differential miRNAs. The results indicate that many important pathways are involved in inflammation, metabolism, and immune response after parasite infection, such as p53 signaling pathway, PI3K-Akt signaling pathway, and glycolysis/gluconeogenesis. Flow cytometry showed that CE-EXO reduced the expression of CD69 + on Jurkat T cells. Our present results suggest that these differentially expressed miRNAs may be important regulators of parasite-host interactions. Meanwhile, functional prediction of its target genes provides valuable information for understanding the mechanism of host-parasite interactions. These results provide clues for future studies on E. granulosus escape from host immune attack, which could help control E. granulosus infection.