Epigenetic landscapes reveal transcription factors that regulate CD8+ T cell differentiation.

Dynamic changes in the expression of transcription factors (TFs) can influence the specification of distinct CD8+ T cell fates, but the observation of equivalent expression of TFs among differentially fated precursor cells suggests additional underlying mechanisms. Here we profiled the genome-wide histone modifications, open chromatin and gene expression of naive, terminal-effector, memory-precursor and memory CD8+ T cell populations induced during the in vivo response to bacterial infection. Integration of these data suggested that the expression and binding of TFs contributed to the establishment of subset-specific enhancers during differentiation. We developed a new bioinformatics method using the PageRank algorithm to reveal key TFs that influence the generation of effector and memory populations. The TFs YY1 and Nr3c1, both constitutively expressed during CD8+ T cell differentiation, regulated the formation of terminal-effector cell fates and memory-precursor cell fates, respectively. Our data define the epigenetic landscape of differentiation intermediates and facilitate the identification of TFs with previously unappreciated roles in CD8+ T cell differentiation.

In vivo RNA interference screens identify regulators of antiviral CD4(+) and CD8(+) T cell differentiation.

Classical genetic approaches to examine the requirements of genes for T cell differentiation during infection are time consuming. Here we developed a pooled approach to screen 30-100+ genes individually in separate antigen-specific T cells during infection using short hairpin RNAs in a microRNA context (shRNAmir). Independent screens using T cell receptor (TCR)-transgenic CD4(+) and CD8(+) T cells responding to lymphocytic choriomeningitis virus (LCMV) identified multiple genes that regulated development of follicular helper (Tfh) and T helper 1 (Th1) cells, and short-lived effector and memory precursor cytotoxic T lymphocytes (CTLs). Both screens revealed roles for the positive transcription elongation factor (P-TEFb) component Cyclin T1 (Ccnt1). Inhibiting expression of Cyclin T1, or its catalytic partner Cdk9, impaired development of Th1 cells and protective short-lived effector CTL and enhanced Tfh cell and memory precursor CTL formation in vivo. This pooled shRNA screening approach should have utility in numerous immunological studies.

MicroRNA-directed program of cytotoxic CD8+ T-cell differentiation.

Acquisition of effector properties is a key step in the generation of cytotoxic T lymphocytes (CTLs). Here we show that inflammatory signals regulate Dicer expression in CTLs, and that deletion or depletion of Dicer in mouse or human activated CD8(+) T cells causes up-regulation of perforin, granzymes, and effector cytokines. Genome-wide analysis of microRNA (miR, miRNA) changes induced by exposure of differentiating CTLs to IL-2 and inflammatory signals identifies miR-139 and miR-150 as components of an miRNA network that controls perforin, eomesodermin, and IL-2Rα expression in differentiating CTLs and whose activity is modulated by IL-2, inflammation, and antigenic stimulation. Overall, our data show that strong IL-2R and inflammatory signals act through Dicer and miRNAs to control the cytolytic program and other aspects of effector CTL differentiation.

Kopsileuconines A-D: Bisindole alkaloids with cytotoxic activity from Kopsia hainanensis.

Kopsileuconines A-D (1-4), four monoterpenoid bisindole alkaloids with unprecedented skeletons, along with their biosynthetically related precursors (5-8) were isolated from the roots of Kopsia hainanensis. Compound 1 possessed an undescribed C-6-C-5' dimerization pattern of aspidofractinine-type alkaloids. Compounds 2-4 were rhazinilam-kopsine (2) and rhazinilam-aspidofractinine type (3 and 4) bisindole alkaloids with undescribed skeletons, respectively. Their structures with absolute configurations were fully accomplished by extensive spectroscopic analysis, quantum-chemical calculations, and X-ray crystallography. A plausible biosynthetic pathway for 1-4 was proposed. Compound 2 exhibited a significant inhibitory effect against human lung cancer cell lines PC9 (EGFR mutant), with an IC50 value of 15.07 ± 1.19 µM.

Eugenilones A-N: sesquiterpenoids from the fruits of Eugenia uniflora.

(+) and (-)-Eugenilones A-K, 11 pairs of undescribed enantiomeric sesquiterpenoids, together with three undescribed biogenetically related members eugenilones L-N, were discovered from the fruits of Eugenia uniflora Linn. (Myrtaceae). Structurally, eugenilones A-D were four caged sesquiterpenoids featuring 9,10-dioxatricyclo [6.2.2.02,7]dodecane, 11-oxatricyclo [5.3.1.03,8]undecane, and tricyclo [4.4.0.02,8]decane cores, respectively. Eugenilones E-K were eudesmane-type sesquiterpenoids, while eugenilones L-N were epoxy germacrane-type sesquiterpenoids. Notably, eugenilones A-K were efficiently resolved by chiral HPLC to give 11 pairs of optically pure enantiomers. The structures and absolute configurations of eugenilones A-N were determined through spectroscopic analyses, X-ray crystallography, and ECD calculations. The putative biosynthetic pathways for these undescribed isolates were proposed. Moreover, eugenilones A and E exhibited significant anti-inflammatory effects by inhibiting LPS-stimulated NO overproduction in RAW264.7 cells (IC50 values of 4.89 ± 0.37 µM and 20.89 ± 1.49 µM, respectively) and TNF-α-induced NF-κB activation in HEK293 cells (IC50 values of 10.97 ± 1.03 µM and 28.63 ± 1.59 µM, respectively).

Antibody-based binding domain fused to TCRγ chain facilitates T cell cytotoxicity for potent anti-tumor response.

Chimeric antigen receptor T-cell (CAR-T) therapy has demonstrated potent clinical efficacy in the treatment of hematopoietic malignancies. However, the application of CAR-T in solid tumors has been limited due in part to the expression of inhibitory molecules in the tumor microenvironment, leading to T-cell exhaustion. To overcome this limitation, we have developed a synthetic T-cell receptor (TCR) that targets programmed death-ligand 1 (PD-L1), a molecule that is widely expressed in various solid tumors and plays a pivotal role in T-cell exhaustion. Our novel TCR platform is based on antibody-based binding domain, which is typically a single-chain variable fragment (scFv), fused to the γδ TCRs (TCRγδ). We have utilized the T-cell receptor alpha constant (TRAC) locus editing approach to express cell surface scFv of anti-PD-L1, which is fused to the constant region of the TCRγ or TCRδ chain in activated T cells derived from peripheral blood mononuclear cells (PBMCs). Our results indicate that these reconfigured receptors, both γ-TCRγδ and δ-TCRγδ, have the capability to transduce signals, produce inflammatory cytokines, degranulate and exert tumor killing activity upon engagement with PD-L1 antigen in vitro. Additionally, we have also shown that γ-TCRγδ exerted superior efficacy than δ-TCRγδ in in vivo xenograft model.

Delivering an mRNA vaccine using a lymphatic drug delivery device improves humoral and cellular immunity against SARS-CoV-2.

The exploration and identification of safe and effective vaccines for the SARS-CoV-2 pandemic have captured the world's attention and remains an ongoing issue due to concerns of balancing protection against emerging variants of concern while also generating long-lasting immunity. Here, we report the synthesis of a novel messenger ribonucleic acid encoding the spike protein in a lipid nanoparticle formulation (STI-7264) that generates robust humoral and cellular immunity following immunization of C57Bl6 mice. In an effort to improve immunity, a clinically focused lymphatic drug delivery device (MuVaxx) was engineered to modulate immune cells at the injection site (epidermis and dermis) and draining lymph node (LN) and tested to measure adaptive immunity. Using MuVaxx, immune responses were elicited and maintained at a 10-fold dose reduction compared to traditional intramuscular (IM) administration as measured by anti-spike antibodies, cytokine-producing CD8 T cells, neutralizing antibodies against the Washington (wild type) strain and South African (Beta) variants, and LN-resident spike-specific memory B cells. Remarkably, a 4-fold-elevated T cell response was observed in MuVaxx-administered vaccination compared to that of IM-administered vaccination. Thus, these data support further investigation into STI-7264 and lymphatic-mediated delivery using MuVaxx for SARS-CoV-2 and VoC vaccines.

Chimeric mRNA-based COVID-19 vaccine induces protective immunity against Omicron and Delta variants.

The emerging SARS-CoV-2 variants of concern (VOCs) exhibit enhanced transmission and immune escape, reducing the effectiveness of currently approved mRNA vaccines. To achieve wider coverage of VOCs, we first constructed a cohort of mRNAs harboring a furin cleavage mutation in the spike (S) protein of predominant VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2). The mutation abolished the cleavage between the S1 and S2 subunits. Systematic evaluation in vaccinated mice discovered that individual VOC mRNAs elicited strong neutralizing activity in a VOC-specific manner. In particular, the neutralizing antibodies (nAb) produced by immunization with Beta-Furin and Washington (WA)-Furin mRNAs showed potent cross-reactivity with other VOCs. However, neither mRNA elicited strong neutralizing activity against the Omicron variant. Hence, we further developed an Omicron-specific mRNA vaccine that restored protection against the original Omicron variant and some sublineages. Finally, to broaden the protection spectrum of the new Omicron mRNA vaccine, we engineered an mRNA-based chimeric immunogen by introducing the receptor-binding domain of Delta variant into the entire S antigen of Omicron. The resultant chimeric mRNA induced potent and broadly nAbs against Omicron and Delta, which paves the way to developing new vaccine candidates to target emerging variants in the future.

RBMX suppresses tumorigenicity and progression of bladder cancer by interacting with the hnRNP A1 protein to regulate PKM alternative splicing.

The prognosis for patients with metastatic bladder cancer (BCa) is poor, and it is not improved by current treatments. RNA-binding motif protein X-linked (RBMX) are involved in the regulation of the malignant progression of various tumors. However, the role of RBMX in BCa tumorigenicity and progression remains unclear. In this study, we found that RBMX was significantly downregulated in BCa tissues, especially in muscle-invasive BCa tissues. RBMX expression was negatively correlated with tumor stage, histological grade and poor patient prognosis. Functional assays demonstrated that RBMX inhibited BCa cell proliferation, colony formation, migration, and invasion in vitro and suppressed tumor growth and metastasis in vivo. Mechanistic investigations revealed that hnRNP A1 was an RBMX-binding protein. RBMX competitively inhibited the combination of the RGG motif in hnRNP A1 and the sequences flanking PKM exon 9, leading to the formation of lower PKM2 and higher PKM1 levels, which attenuated the tumorigenicity and progression of BCa. Moreover, RBMX inhibited aerobic glycolysis through hnRNP A1-dependent PKM alternative splicing and counteracted the PKM2 overexpression-induced aggressive phenotype of the BCa cells. In conclusion, our findings indicate that RBMX suppresses BCa tumorigenicity and progression via an hnRNP A1-mediated PKM alternative splicing mechanism. RBMX may serve as a novel prognostic biomarker for clinical intervention in BCa.

Kinome siRNA screen identifies SMG-1 as a negative regulator of hypoxia-inducible factor-1alpha in hypoxia.

Hypoxia-inducible factor-1 (HIF-1) plays a central role in tumor progression by regulating genes involved in proliferation, glycolysis, angiogenesis, and metastasis. To improve our understanding of HIF-1 regulation by kinome, we screened a kinase-specific small interference RNA library using a hypoxia-response element (HRE) luciferase reporter assay under hypoxic conditions. This screen determined that depletion of cellular SMG-1 kinase most significantly modified cellular HIF-1 activity in hypoxia. SMG-1 is the newest and least studied member of the phosphoinositide 3-kinase-related kinase family, which consists of ATM, ATR, DNA-PKcs, mTOR, and SMG-1. We individually depleted members of the phosphoinositide 3-kinase-related kinase family, and only SMG-1 deficiency significantly augmented HIF-1 activity in hypoxia. We subsequently discovered that SMG-1 kinase activity was activated by hypoxia, and depletion of SMG-1 up-regulated MAPK activity under low oxygen. Suppressing cellular MAPK by silencing ERK1/2 or by treatment with U0126, a MAPK inhibitor, partially blocked the escalation of HIF-1 activity resulting from SMG-1 deficiency in hypoxic cells. Increased expression of SMG-1 but not kinase-dead SMG-1 effectively inhibited the activity of HIF-1alpha. In addition, cellular SMG-1 deficiency increased secretion of the HIF-1alpha-regulated angiogenic factor, vascular epidermal growth factor, and survival factor, carbonic anhydrase IX (CA9), as well as promoted the hypoxic cell motility. Taken together, we discovered that SMG-1 negatively regulated HIF-1alpha activity in hypoxia, in part through blocking MAPK activation.

Evidence that the transcription elongation function of Rpb9 is involved in transcription-coupled DNA repair in Saccharomyces cerevisiae.

Rpb9, a small nonessential subunit of RNA polymerase II, has been shown to have multiple transcription-related functions in Saccharomyces cerevisiae. These functions include promoting transcription elongation and mediating a subpathway of transcription-coupled repair (TCR) that is independent of Rad26, the homologue of human Cockayne syndrome complementation group B protein. Rpb9 is composed of three distinct domains: the N-terminal Zn1, the C-terminal Zn2, and the central linker. Here we show that the Zn1 and linker domains are essential, whereas the Zn2 domain is almost dispensable, for both transcription elongation and TCR functions. Impairment of transcription elongation, which does not dramatically compromise Rad26-mediated TCR, completely abolishes Rpb9-mediated TCR. Furthermore, Rpb9 appears to be dispensable for TCR if its transcription elongation function is compensated for by removing a transcription repression/elongation factor. Our data suggest that the transcription elongation function of Rpb9 is involved in TCR.

A unique group of inactive serine protease homologues from snake venom.

A number of inactive serine protease homologues (SPHs), which have poorly understood functions, have been identified in invertebrates and vertebrates. Recently, several SPH transcripts have been reported from snake venom glands, which provide potential new tools for the study of the functions of SPHs. Herein we report for the first time a snake venom serine protease homologue (svSPH) protein, designated as TjsvSPH, isolated from the venom of Trimeresurus jerdonii. Despite its high sequence similarity to snake venom serine proteases (SVSPs), TjsvSPH is devoid of arginine esterase and proteolytic activity. This is probably due to the replacement of Arg-43 by His-43 in the catalytic triad. TjsvSPH did not influence the coagulation time of human plasma, induce human platelet aggregation, inhibit adenosine diphosphate/thrombin-induced human platelet aggregation or increase capillary permeability. Phylogenetic analysis showed that svSPHs were separated from SVSPs and formed an independent group. Structural analysis revealed that the structures of svSPHs are quite different from those of SPHs previously reported. These results indicate that snake venoms contain a unique group of svSPH proteins.

[Expression of DKKL1 in spermatozoa of men with asthenospermia].

OBJECTIVE: To compare the expression of DKKL1 in ejaculated spermatozoa of normal fertile men and men with asthenospermia and investigate the role of DKKL1 in the pathogenesis of asthenospermia. METHODS: The characteristics of semen samples collected from normal fertile men and men with asthenospermia were analyzed using computer-assisted sperm analysis according to WHO criteria. The ejaculated sperms were isolated by Percoll discontinuous density gradients to detect the expression of DKKL1 mRNA and protein using real-time PCR and Western blotting. RESULTS: The expression of DKKL1 mRNA was significantly down-regulated by 11.1 times in asthenospermic men as compared with that in normal fertile men (P<0.01). Western blotting showed that the expression of DKKL1 protein was down-regulated by 2.4 times in asthenospermic men compared to normal fertile men. CONCLUSION: The expression of DKKL1, which may play an important role in sperm motility,is significantly decreased in ejaculated spermatozoa of men with asthenospermia.

Characterization and molecular cloning of dabocetin, a potent antiplatelet C-type lectin-like protein from Daboia russellii siamensis venom.

A novel C-type lectin-like protein, dabocetin, was purified from Daboia russellii siamensis venom. On SDS-polyacrylamide gel electrophoresis, it showed a single band with an apparent molecular weight of 28 kDa and two distinct bands with the apparent molecular weights of 15.0 kDa and 14.5 kDa under non-reducing and reducing conditions, respectively. cDNA clones containing the coding sequences for dabocetin alpha and beta subunits were isolated and sequenced. The deduced protein sequences of both subunits were confirmed by N-terminal amino acid sequencing and trypsin-digested peptide mass fingerprinting. Dabocetin did not induce platelet aggregation in platelet-rich plasma. It also had little effect on the platelet aggregation induced by ADP, TMVA or stejnulxin. Whereas, dabocetin inhibited ristocetin-induced platelet agglutination in platelet-rich plasma in a dose-dependent manner with an IC50 value of 0.35 microM. Flow cytometry analysis showed that dabocetin significantly inhibited mAb SZ2 binding to platelet membrane glycoprotein Ib alpha, indicating that platelet membrane glycoprotein Ib is involved in the inhibitory effect of dabocetin on ristocetin-induced platelet agglutination.

Expression profile of SPACA5/Spaca5 in spermatogenesis and transitional cell carcinoma of the bladder.

The majority of bladder cancer-associated mortalities are due to transitional cell carcinoma (TCC), which is the most prevalent and chemoresistant malignancy of the bladder. Sperm acrosome associated 5 (SPACA5)/Spaca5 is a sperm acrosome-associated, c-type lysozyme-like protein that has been recently identified, and has been designated as an attractive candidate antigen for cancer testis. In the present study, the expression profile of SPACA5/Spaca5 was analyzed in spermatogenesis and TCC of the bladder using diverse molecular and cellular biology methods. Using reverse transcription-polymerase chain reaction (RT-PCR) to analyze the multi-tissue distribution and temporal expression of SPACA5/Spaca5, the SPACA5/Spaca5 gene was determined to be generally not expressed in normal tissue, with the exception of the testis, and it could be detected at a low level on day 20 after birth in mouse testes and at a higher level on day 28. Immunohistochemistry staining revealed that the SPACA5/Spaca5 protein was exclusively observed in the elongated spermatid of the normal testes, and was ectopically expressed in the cytoplasm of TCC, while it was not expressed in normal bladder tissues. The frequency of SPACA5 messenger RNA was detected in 45% of TCC (9/20) by RT-quantitative PCR. Furthermore, SPACA5 protein was more frequently detected in high-grade than in low-grade tumors (61.54 vs. 30.00%, P=0.035). Accordingly, high SPACA5 staining scores were observed to be significantly associated with high-grade tumors (n=65, R=0.279, P=0.027). Collectively, our findings indicated that SPACA5/Spaca5 may be important in male spermatogenesis and may be used as a potential target for specific immunotherapy in patients suffering from TCC.

Molecular characterization of a weak fibrinogen-clotting enzyme from Trimeresurus jerdonii venom.

A fibrinogen-clotting enzyme designed as jerdonobin-II was isolated from the venom of Trimeresurus jerdonii. It differed in molecular weight and N-terminal sequence with the previously isolated jerdonobin, a thrombin-like enzyme from the same venom. The enzyme consists of a single polypeptide chain with molecular weights of 30,000 and 32,000 under non-reducing and reducing conditions, respectively. Jerdonobin-II showed weak fibrinogen clotting activity and its activity unit on fibrinogen was calculated to be less than one unit using human thrombin as standard. The precursor protein sequence of jerodonobin-II was deduced from cloned cDNA sequence. The sequence shows high similarity (identity=89%) to TSV-PA, a specific plasminogen activator from venom of T. stejnegeri. Despite of the sequence similarity, jerdonobin-II was found devoid of plasminogen activating effect. Sequence alignment analysis suggested that the replacement of Lys239 in TSV-PA to Gln239 in jerdonobin-II might play an important role on their plasminogen activating activity difference.

Purification, cloning and biological characterization of a novel disintegrin from Trimeresurus jerdonii venom.

A novel disintegrin, jerdonin, was purified from the Trimeresurus jerdonii venom by means of gel filtration and reverse phase high pressure liquid chromatography. Its coding cDNA was also isolated from the venom gland. The jerdonin coding cDNA is part of a precursor composed of proprotein, metalloproteinase, and disintegrin domains. From the deduced amino acid sequence, jerdonin is composed of 71 amino acid residues including 12 cysteines and the tripeptide sequence Arg-Gly-Asp (RGD), a well-known characteristic of the disintegrin family. Molecular mass of jerdonin was determined to be 7483Da by matrix-assisted laser desorption ionization time of flight mass spectrometry. Jerdonin inhibited ADP- and collagen-induced human platelet aggregation with IC(50) of 220 and 240 nM, respectively. In vivo, jerdonin inhibited the growth of subcutaneously inoculated B16 solid tumor in C57BL/6 mice and improved the survival time of the tumor-bearing mice.

A novel high molecular weight metalloproteinase cleaves fragment F1 of activated human prothrombin.

A hemorrhagic proteinase, jerdohagin, was purified from Trimeresurus jerdonii venom by gel filtration and ion-exchange chromatographies. It was a single chain polypeptide with an apparent molecular weight of 96 kDa as estimated by SDS-PAGE under the non-reducing and reducing conditions. Internal peptide sequencing indicated that it consisted of metalloproteinase, disintegrin-like and cysteine-rich domains and belonged to the class III snake venom metalloproteinases (class P-III SVMPs). Like other typical metalloproteinases, hemorrhagic activities of jerdohagin were completely inhibited by EDTA, but not by PMSF. Jerdohagin preferentially degraded alpha-chain of human fibrinogen. Interestingly, jerdohagin did not activate human prothrombin, whereas it cleaved human prothrombin and fragment F1 of activated human prothrombin.

A novel disintegrin, jerdonatin, inhibits platelet aggregation and sperm-egg binding.

A novel disintegrin, jerdonatin, was purified to homogeneity from Trimeresurus jerdonii venom by gel filtration and reversed-phase high-pressure liquid chromatography. We isolated the cDNA encoding jerdonatin from the snake venom gland. Jerdonatin cDNA precursor encoded pre-peptide, metalloprotease and disintegrin domain. Jerdonatin is composed of 72 amino acid residues including 12 cysteines and the tripeptide sequence Arg-Gly-Asp (RGD), a well-known characteristic of the disintegrin family. Molecular mass of jerdonatin was determined to be 8011 Da by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). Jerdonatin inhibited ADP- and collagen-induced human platelet aggregation with IC50 of 123 and 135 nM, respectively. We also investigated the effect of jerdonatin on the binding of B6D2F1 hybrid mice spermatozoa to mice zona-free eggs and their subsequent fusion. Jerdonatin significantly inhibited sperm-egg binding in a concentration-dependent manner, but had no effect on the fusion of sperm-egg. These results indicate that integrins on the egg play a role in mammalian fertilization.